CN114540490B

CN114540490B - Use of LCDR as therapeutic target for drugs for preventing and/or treating cancer

Info

Publication number: CN114540490B
Application number: CN202110706012.6A
Authority: CN
Inventors: 高山; 文娅; 杨希望; 仝舟
Original assignee: Shanxi Institute Of Higher Innovation
Current assignee: Shanxi Institute Of Higher Innovation
Priority date: 2021-06-24
Filing date: 2021-06-24
Publication date: 2024-01-19
Anticipated expiration: 2041-06-24
Also published as: CN114540490A

Abstract

The invention relates to an application of LCDR as a therapeutic target of a medicament for preventing and/or treating cancers, belonging to the technical field of biology. The invention provides an application of a LCDR, hnRNPK, LCDR and hnRNPK complex and LAPTM5 as a therapeutic target of a medicament for preventing and/or treating cancers; LCDR is highly expressed in cancerous tissues; knocking down LCDR to find that cancer cell proliferation capacity is down-regulated and apoptosis occurs; knocking down LCDR reduces the tumorigenic capacity of the cancer cells; LCDR functions by binding to hnRNPK, knocking down hnRNPK to develop a phenotype consistent with LCDR; LAPTM5 has the function consistent with LCDR/hnRNPK, and the LAPTM5 knockdown appears to have a phenotype consistent with LCDR/hnRNPK; the LCDR/hnRNPK/lapm 5 axis regulates lysosomal mediated cell death pathways.

Description

Use of LCDR as therapeutic target for drugs for preventing and/or treating cancer

Technical Field

The invention relates to an application of LCDR as a therapeutic target of a medicament for preventing and/or treating cancers, belonging to the technical field of biology.

Background

Tumors are diseases in organisms caused by abnormal growth of cells due to various factors, and are classified into malignant tumors and benign tumors according to their characteristics and harm to organisms. Malignant tumor, also called cancer, has metastatic and invasive properties, and benign tumor is characterized by no invasive and metastatic capacity, often has envelope, well-demarcated from normal tissues, slow growth, few recurrence, and small influence on organisms.

Compared to normal cells, tumor cells generally have the following properties: 1. maintaining the proliferation signal; 2. growth inhibition is avoided; 3. resistance to apoptosis; 4. unlimited replication capability; 5. inducing the generation of new blood vessels; 6. activating invasion and metastasis; 7. metabolic reprogramming; 8. escape immunity monitoring; 9. instability and variability of the genome; 10. causing an inflammatory response. At present, cancer has become one of the main factors of death of Chinese diseases, which is mainly related to the molecular characteristics of tumor cells distinguished from normal cells, so that the treatment of tumors is a worldwide difficult problem.

Lung cancer is one of the most deadly and ill malignant tumors worldwide, has a high degree of heterogeneity, forms throughout the respiratory tract, and can undergo malignant expansion and metastasis via blood and lymphatic vessels. Lung cancer is classified into squamous cell carcinoma, lung adenocarcinoma, small cell carcinoma and lung neuroendocrine tumor according to the current latest world health organization (World Health Organization, WHO) pathology and histological classification.

A series of malignant characteristics obtained by lung cancer cells are often caused by intracellular gene mutations. The presence of a large number of gene mutations, chromosomal rearrangements, etc., in cancer cells of lung cancer patients is revealed by whole genome sequencing. For example: epidermal growth factor receptor (Epidermal growth factor receptor, EGFR) mutation, ALK (Anaplastic Lymphoma kinase, ALK) rearrangement, protooncogene 1 tyrosine kinase (ROS 1) 1receptor kinase,c-ROS, BRAF V600E mutation and the like on the surface of a membrane, and the targeting drugs aiming at the mutations have obvious clinical curative effects.

Therefore, finding more molecular mechanisms related to occurrence and development of lung cancer has important clinical application value for lung cancer treatment.

Disclosure of Invention

In order to solve the problems, the invention provides a molecular marker LCDR, and the nucleotide sequence of the molecular marker LCDR is shown as SEQ ID NO. 1.

The invention also provides application of the molecular marker LCDR, hnRNP K (heterogeneous ribonucleoprotein K), a compound of the molecular marker LCDR and hnRNP K and/or LAPTM5 (lysosomal associated transmembrane protein 5 antibody) as a therapeutic target of a medicament for preventing and/or treating cancer.

In one embodiment of the present invention, the amino acid sequence of hnRNP K is shown in SEQ ID NO. 2.

In one embodiment of the present invention, the amino acid sequence of LAPTM5 is shown in SEQ ID NO. 3.

In one embodiment of the invention, the cancer is lung adenocarcinoma.

The invention also provides application of the inhibitor in preparing a medicament for preventing and/or treating cancer, which is characterized in that the inhibitor has at least one of the following purposes:

(a) Inhibiting the expression of the molecular marker LCDR in the cell;

(b) Inhibiting expression of hnRNP K in a cell;

(c) Inhibiting the recombination of the molecular markers LCDR and hnRNP K in cells; and/or the number of the groups of groups,

(d) Inhibiting the expression of LAPTM5 in the cell.

In one embodiment of the invention, the cancer is lung adenocarcinoma; the cells are normal lung gland cells and/or cancerous lung gland cells.

The invention also provides application of the detection reagent for detecting the molecular marker LCDR, hnRNP K, the compound of the molecular marker LCDR and hnRNP K and/or LAPTM5 in preparation of products for diagnosing cancers.

In one embodiment of the invention, the detection reagent for detecting the molecular marker LCDR of claim 1 comprises a specific primer for amplifying the molecular marker LCDR of claim 1;

the detection reagent for detecting hnRNP K comprises a specific primer for amplifying a nucleotide sequence encoding the hnRNP K;

The detection reagent for detecting LAPTM5 comprises a specific primer for amplifying a nucleotide sequence encoding the LAPTM 5.

In one embodiment of the present invention, the nucleotide sequence of the specific primer for amplifying the molecular marker LCDR according to claim 1 is shown in SEQ ID NO. 4 and SEQ ID NO. 5.

In one embodiment of the invention, the product is a detection reagent or a detection kit.

In one embodiment of the invention, the cancer is lung adenocarcinoma.

The technical scheme of the invention has the following advantages:

the invention provides application of a molecular marker LCDR (nucleotide sequence shown as SEQ ID NO: 1), hnRNP K (heterogeneous ribonucleoprotein K), a compound of the molecular marker LCDR and hnRNP K, and/or LAPTM5 (lysosomal associated transmembrane protein 5 antibody) as a therapeutic target of a medicament for preventing and/or treating cancer; studies have shown that: the LCDR has relatively high expression level in lung adenocarcinoma tissues; knocking down LCDR to find that the proliferation capacity of lung adenocarcinoma cells is obviously down-regulated and apoptosis occurs; knocking down LCDR significantly reduces the tumorigenic capacity of lung adenocarcinoma cells; LCDR is combined with hnRNP K through a secondary structure, so that the biological function of the LCDR is exerted, and the hnRNP K is knocked down to have a phenotype consistent with LCDR; LCDR and hnRNP K do not affect the expression quantity and subcellular localization, and can not be saved from each other in function; LAPTM5 is a key downstream gene of LCDR/hnRNP K axis and is regulated and controlled by LCDR/hnRNP K; LAPTM5 has the function consistent with LCDR/hnRNP K, and the knock-down LAPTM5 has the phenotype consistent with LCDR/hnRNP K; knocking down the inhibition effect of LCDR or hnRNP K on lung adenocarcinoma cell proliferation and clone formation can obtain partial rescue through over-expression of LAPTM 5; overexpression of LAPTM5 can partially rescue apoptosis caused by knocking down LCDR or hnRNP K; the LCDR/hnRNP K/LAPTM5 axis regulates lysosomal mediated cell death pathways.

Drawings

Fig. 1: amplification procedure of nested PCR.

Fig. 2: and (3) a reaction system for pre-running glue.

Fig. 3: acetylation regulated gene profiling and pathway enrichment. (A) TSA treatment of a heat map of BESA-2B differentially expressed genes; (B) TSA treatment of a heat map of NCI-H1299 differentially expressed genes; (C) pathway enrichment of TSA-treated BESA-2B differential genes; (D) pathway enrichment of the TSA treated NCI-H1299 differential gene; (E) TSA processed volcanic images of the differential genes of BESA-2B and NCI-H1299 intersection; (F) TSA treated BESA-2B and pathway enrichment of the differential gene of NCI-H1299 intersection.

Fig. 4: screening of lncRNAs for histone acetylation regulation and differential changes in the cancerous process. (A) TSA treatment BEAS-2B, NCI-H1299 and heat maps of 7 cancer species differentially expressed and co-directionally altered lncRNAs; (B) Varying levels of protein of BEAS-2B, NCI-H1299, HCC827 cell line H3K27ac and H4K16ac were treated with different concentrations of TSA; (C) Peak patterns of H3K27ac enrichment on the genomes of LCDR, SLC12A5-AS1, LUCAT1, NKILA, MNX1-AS1, MNTN1-AS1, SNHK 12; (D) TSA treatment BEAS-2B, NCI-H1299 and lncRNAs heat maps of 7 carcinoma species differentially expressed and co-varying and modified with H3K27 ac; (E-G) qRT-PCR detection of changes in LCDR transcript levels in different concentrations of TSA treated BEAS-2B (E), NCI-H1299 (F), HCC827 (G) cell line; (H-J) qRT-PCR assay of enrichment efficiency of the LCDR promoter by different concentrations of TSA treated BEAS-2B (H), NCI-H1299 (I), HCC827 (J) cell line H3K27 ac.

Fig. 5: and (3) detecting the LCDR full-length clone and relative expression quantity. (A) LCDR 3'race and 5' race analysis, PCR product sequencing map (left) and gel electrophoresis map of 5 'and 3' race PCR products (right); (B) Northern blot analysis of LCDR; (C) localization of LCDR on human genome GRCh 38; (D) qRT-PCR (quantitative reverse transcription-polymerase chain reaction) detection of LCDR (LCDR) transcript levels in lung cancer cell lines; (E) qRT-PCR detects the molecular number of LCDR in lung cancer cell lines. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 6: (a) prediction of LCDR coding capability; (B) analysis of LCDR promoter transcriptional activity; (C) LCDR conservation analysis.

Fig. 7: subcellular localization of LCDR molecules. (A) LCDR molecule FISH plot red represents hybridization signal of LCDR, blue represents DAPI dye signal; (B). qRT-PCR detects the location ratio of LCDR in cytoplasm and nucleus. The GAPDH protein and H3 histone were used as positive controls for cytoplasmic and nuclear proteins, respectively, the GAPDH and U1 genes were used as positive controls for cytoplasmic and nuclear genes, respectively, and WCL, cyto, and Nuc represent whole cell, cytoplasmic, and nuclear lysates, respectively. Data are expressed as mean±sem.

Fig. 8: c-Jun regulates transcription of LCDR. (A) qRT-PCR detection knockdown c-Jun LCDR transcriptional level change; (B) qRT-PCR detection shows that the transcription level of the c-Jun LCDR is changed; (C) CHIP sequencing data of GSE92783 dataset, enrichment level of c-Jun on LCDR promoter region in a549 cell line; (D) Biological information predicts the binding site of c-Jun in the LCDR promoter region; (E) CHIP verifies that c-Jun binds to the promoter region of LCDR; (F) schematic illustration of the insertional mutation of the c-Jun and LCDR binding sites; (G) The mutual combination of the c-Jun and the LCDR promoter is verified by a double enzyme report test; (H) The CHIP experiments verify the effect of TSA treatment on the c-Jun enriched LCDR promoter. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 9: and (3) change of LCDR expression quantity. (A) qRT-PCR (quantitative reverse transcription-polymerase chain reaction) detection of the knockdown efficiency of sh-LCDR in NCI-H1299 cell line; (B) qRT-PCR (quantitative reverse transcription-polymerase chain reaction) detection of knocking down efficiency of si-LCDR in NCI-H1299 cell line; (C) qRT-PCR detects the fold-overstable of LCDR in NCI-H1299 cell line. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 10: effect of varying LCDR expression levels on NCI-H1299 proliferation activity. (A) The shRNA system knocks down the expression of LCDR, and influences the proliferation activity of NCI-H1299; (B) The siRNA system knocks down the expression of LCDR, and influences the proliferation activity of NCI-H1299; (C) effects of the overstable LCDR on NCI-H1299 proliferation activity. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 11: effect of LCDR expression level on NCI-H1299 clone formation. (A) The shRNA system knocks down the expression of LCDR and affects the clone formation of NCI-H1299; (B) The siRNA system knocks down the expression of LCDR and affects the clone formation of NCI-H1299; (C) Effect of the pass-Table LCDR on NCI-H1299 clone formation. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 12: knocking down LCDR induces apoptosis in the cell. (A) A mirror representation of the shRNA system knockdown LCDR expression NCI-H1299; (B) A mirror representation of the siRNA system knockdown LCDR expression NCI-H1299; (C) sh-LCDR flow assay NCI-H1299 cells apoptosis representation; (D) sh-LCDR flow detection NCI-H1299 cell apoptosis proportion statistical diagram; (E) si-LCDR flow assay NCI-H1299 cell apoptosis representation; (F) And si-LCDR flow detection NCI-H1299 cell apoptosis proportion statistical graph. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 13: knocking down LCDR induces apoptosis in the cell. (A) The shRNA system knocks down LCDR expression cytochrome C positioning representative diagram; (B) Knocking down LCDR expression cytochrome C positioning representative diagram by the siRNA system; (C) The shRNA system knocks down LCDR to express caspase and PARP protein expression; (D) The siRNA system knocks down the expression of caspase and PARP protein.

Fig. 14: effect of knockdown LCDR on NCI-H1299 tumor formation ability. (A) Knocking down LCDR to inhibit proliferation capacity of NCI-H1299 tumor; (B) knockdown of LCDR inhibits the tumorigenic capacity of NCI-H1299; (C) the LCDR-knockdown NCI-H1299 cells inhibit tumor growth; (D) Knocking down LCDR immunohistochemical staining clear caspase3 representation map; (E) Immunohistochemical clear caspase3 immunohistochemical score statistics. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 15: LCDR binds specifically to hnRNP K. (A) LCDR RNA p.mu.Ll Down silver staining protocol; (B) Western blot verification of specific binding of LCDR and hnRNP K; (C) RIP experiments verify that hnRNP K and LCDR are mutually bound, igG being a negative control; (D) LCDR in situ hybridization and hnRNP K immunofluorescence confirmed that LCDR and hnRNP K coexist in the nucleus. The two-tailed t-test analyzes the differences between groups and the data are expressed as mean±sem.

Fig. 16: identification of hnRNP K binding LCDR sites. (A) The interaction between the full length LCDR and the truncated LCDR and hnRNP K is detected by western blot; (B) prediction of LCDR secondary structure by RNAfold online software; (C) schematic representation of potential binding site mutations of LCDR and hnRNP K; (D) The western blot detects the interaction of LCDR (1-500) and mut-LCDR with hnRNP K; (E) EMSA identifies the mutual binding of LCDR to hnRNP K; (F) RIP-qPCR detects the mutual binding of hnRNP K to LCDR and mut-LCDR. The two-tailed t-test analyzes the differences between groups and the data are expressed as mean±sem.

Fig. 17: identification of LCDR binding hnRNP K domain. (a) schematic representation of hnRNP K truncations; (B) Detecting the protein expression condition of the hnRNP K truncated body by using a western blot; (C) RIP-qPCR detects the enrichment level of hnRNP K for LCDR for full length and truncations. The two-tailed t-test analyzes the differences between groups and the data are expressed as mean±sem.

Fig. 18: and (3) change of hnRNP K expression level. (A) qRT-PCR (quantitative reverse transcription-polymerase chain reaction) detection of hnRNP K knockdown efficiency in NCI-H1299 cell line; (b) qRT-PCR detects the fold-overstable of hnRNP K in NCI-H1299 cell line. The two-tailed t-test analyzes the differences between groups and the data are expressed as mean±sem.

Fig. 19: effect of hnRNP K on cell proliferation. (a) knockdown of hnRNP K effect on NCI-H1299 proliferation activity; (B) Table hnRNP K effect on NCI-H1299 proliferation activity. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 20: effect of hnRNP K on the clonogenic capacity of cells. (A) Knock down of hnRNP K effect on NCI-H1299 clone formation ability; (B) Table hnRNP K affects NCI-H1299 clone-forming ability. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 21: effect of hnRNP K on cell death. (a) knockdown hnRNP K cell line mirror representation; (B) knockdown hnRNP K apoptosis flow-representative panels; (C) knocking down hnRNP K apoptosis flow statistics; (D) localization of hnRNP K immunofluorescence assay Cytochrome C; (E) Knock-down hnRNP K western blot detects caspase, PARP protein expression. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 22: LCDR and hnRNP K are interrelated. (A) Immunofluorescence of LCDR and hnRNP K binding to each other, blue for DAPI signal, green for hnRNP K protein, red for LCDR; (B) qPCR detection of the change condition of the level of the hnRNP K mRNA knocking down LCDR; (C) Detecting the change condition of the level of the knock-down LCDR (liquid crystal display) hnRNP K protein by using a western blot; (D) qPCR detection of hnRNP K knockdown and LCDR transcript change; (E) And (3) detecting the change condition of the hnRNP K protein level by western blot. The two-tailed t-test analyzes the differences between groups and the data are expressed as mean±sem.

Fig. 23: LCDR and hnRNP K functional rescue. (A) Cell proliferation curves of the hnRNP K cell lines knocked down LCDR were crossed; (B) Cell proliferation curves of hnRNP K knockdown by the overstable LCDR cell line. The two-tailed t-test analyzes the differences between groups and the data are expressed as mean±sem.

Fig. 24: effect of LCDR and hnRNP K on expression of apoptosis-related genes. (a-B) qRT-PCR detection of knockdown LCDR (a) and hnRNP K (B) apoptosis-related gene mRNA variation levels; (C-D) western blot detection of knockdown LCDR (C) and changes in hnRNP K (D) apoptosis-related gene protein levels. The two-tailed t-test analyzes the differences between groups and the data are expressed as mean±sem.

Fig. 25: LCDR together with hnRNP K regulate lysosomal pathways. (A-B) volcanic plot shows knockdown of genes differentially expressed by LCDR (A) and hnRNP K (B), red for up-regulated genes and green for down-regulated genes; (C-D) pathway enrichment of genes differentially expressed by knockdown LCDR (C) and hnRNP K (D); (E) knocking down the LCDR, hnRNP K intersection difference gene; (F) Knocking down LCDR, and enriching the channel of hnRNP K intersection difference genes; (G) Knocking down LCDR, and enriching KEGG channels of hnRNP K intersection difference genes; (H-I) knockdown LCDR, hnRNP K differential gene lysosomal pathway heatmap; (J-K) qRT-PCR detection of the variation in mRNA level of knockdown LCDR (J) and hnRNP K (K) lysosomal pathway genes. The two-tailed t-test analyzes the differences between groups and the data are expressed as mean±sem.

Fig. 26: LCDR and hnRNP K regulate expression of lapm 5. (A) Detecting the change of the level of the knock-down LCDR and LAPTM5 protein by western blot; (B) Detecting the change of the level of the protein of the knock-down hnRNP K and LAPTM5 by western blot; changes in protein levels (C-D) across the LCDR, LAPTM5 mRNA; through the table hnRNP K, LAPTM5 mRNA and protein level changes (E-F); (G) LAPTM5 co-localization with LAMP1 immunofluorescence representation; (H) Overexpression of LAPTM5 co-localizes with LAMP1 immunofluorescence.

Fig. 27: changes in the amount of LAPTM5 expressed. (A) qRT-PCR (quantitative reverse transcription-polymerase chain reaction) detection of the knock-down efficiency of LAPTM5 in NCI-H1299 cell line; (B) qRT-PCR detects the fold-overstable of LAPTM5 in NCI-H1299 cell line. The two-tailed t-test analyzes the differences between groups and the data are expressed as mean±sem.

Fig. 28: effect of lapm 5 on cell proliferation. (a) knocking down the effect of lapm 5 on NCI-H1299 proliferation activity; (B) effect of overstable LAPTM5 on NCI-H1299 proliferation activity. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 29: effect of lapm 5 on the clonogenic capacity of cells. (A) Knock down of the effect of LAPTM5 on the ability of NCI-H1299 to clone formation; (B) Influence of overstable LAPTM5 on NCI-H1299 clone formation ability. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 30: effect of lapm 5 on cell death. (a) knockdown lapm 5 cell optic representation; (B) knockdown lapm 5 apoptosis flow-representative maps; (C) knocking down the lapm 5 apoptosis flow chart; (D) localization of knock-down lapm 5 Cytochrome C; (E) And knocking down LAPTM5 western blot to detect caspase and PARP protein expression. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 31: LCDR, hnRNP K and lapm 5 regulate lysosomal membrane permeability. Immunofluorescent staining of (A-B) LysoTracker, LAMP, CTSB represents a map; (C) The integrity of the lysosome membrane was observed by scanning electron microscopy, and the red arrow represents the site of membrane rupture.

Fig. 32: LCDR and hnRNP K regulate cell death via lapm 5. Detection of cell proliferation capacity of cell lines stabilized by Table LAPTM5 knockdown LCDR (A) and hnRNP K (C); evaluation of cell line knockdown LCDR (B) and hnRNP K (D) cell clone forming ability of stabilized table lapm 5; assessment of apoptosis of cell lines stabilized by Table LAPTM5 knockdown LCDR (E-F) and hnRNP K (G-H); assessment of apoptosis in CA-074Me treated cells knockdown LCDR (I-J), hnRNP K (K-L) and LAPTM5 (M-N). The two-tailed t-test analyzes the differences between groups and the data are expressed as mean±sem.

Fig. 33: LCDR and hnRNP K regulate cell death via lapm 5. (A) The cell strain stabilized by the surface LAPTM5 is knocked down to the growth curve of LCDR and hnRNP K tumor; (B) The cell strain stabilized by the surface LAPTM5 is knocked down again to remove LCDR and the subcutaneous tumorigenicity of the hnRNP K mice can be partially saved; (C) Statistical plots of hnRNP K tumor weights were re-knocked down by cell lines stabilized by Table LAPTM 5. The two-tailed t-test analyzes the differences between groups and the data are expressed as mean±sem.

Fig. 34: cell lines stabilized by Table LAPTM5 were re-knocked down with LCDR and hnRNP K immunohistochemistry (A) representative panels and clear caspase 3 score (B) statistical panels. The two-tailed t-test analyzes the differences between groups and the data are expressed as mean±sem.

Fig. 35: LCDR and hnRNP K bind to LAPTM5-3' UTR. (A) hnRNP K RIP seq data analysis of hnRNP K peak distribution on LAPTM5 transcripts; (B) LCDR RNA-seq, hnRNP K RNA-seq, and hnRNP K-eCIP intersection gene Wen map; (C) LCDR RNA-seq, hnRNP K RIP-seq, and hnRNP K-eCIP intersection gene KEGG pathway enrichment; (D) Double enzyme report experiment to detect the mutual combination of hnRNP K and LAPTM5 coding region and 3' UTR; (E-F) schematic representation of LAPTM5-3' UTR with potential binding site (E) to hnRNP K and mutation (F); (G) RNA p.mu.Ll down experiments verify the mutual binding of hnRNP K and LAPTM5-3' UTR; (H) RIP-qPCR detects the mutual binding of hnRNP K to lapm 5-3'utr and lapm 5-3' utr; (I) EMSA identifies the mutual binding of hnRNP K to LAPTM5-3' UTR; (J) RIP-qPCR detects the enrichment level of lacm 5 by full length and truncated hnRNP K. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 36: LCDR and hnRNP K regulate the stability of lapm 5 mRNA. (A) Detecting the stability of the transcription of the knock-down LCDR overexpression hnRNP K and LAPTM 5; (B) Detection of the stability of the LAPTM5 transcript by knocking down the overexpressed LCDR of hnRNP K.

(C) qRT-PCR detection of the change of the level of the hnRNP K-knockdown transcription LCDR and LAPTM 5; (D) qRT-PCR detection of knockdown LCDR changes in the levels of the hnRNP K, LAPTM5 transcripts (E-F) double-enzyme assay identified LCDR (E) and the binding of hnRNP K (F) to LAPTM5-3' UTR. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem. The differences between the groups were analyzed by two-tailed t-test and the data are expressed as mean±sem.

Fig. 37: LCDR and hnRNP K synergistically promote reading of lapm 5. (A) RIP-qPCR detection of hnRNP K-enriched lapm 5 levels in LCDR knockdown cell lines (B) RIP-qPCR detection of hnRNP K-enriched lapm 5 levels in cell lines that have been tabulated LCDR; (C) Detecting the level of LCDR enriched LAPTM5 in the knockdown hnRNP K cell line by P.mu.Ll down; (D) mu.LlDown detected levels of LCDR enriched LAPTM5 in the hnRNP K cell line; (E) qPCR assay of binding levels of biotin-labeled LCDR to lapm 5 before and after treatment of RNA p μl down samples with proteinase K; (F) Schematic representation of hnRNP K and LCDR and LAPTM5 binding site mutations; (G) RIP-qPCR detects levels of hnRNP K enriched lapm 5 in cell lines of double-pass tables LCDR and hnRNP K; (H) Double enzyme report experiments detect the effect of double-pass table LCDR and hnRNP K cotransformation with LAPTM5-3' UTR on enzyme report activity. The two-tailed t-test analyzes the differences between groups and the data are expressed as mean±sem.

Fig. 38: LCDR promotes hnRNP K binding to lapm 5. (A) EMSA experiment detects the influence of incubation of LCDR and LAPTM5-3' UTR with hnRNP K protein on the migration rate of hnRNP K protein; (B-C) EMSA experiments to detect the effect of lapm 5-3' utr on hnRNP K protein and LCDR binding; (D-E) EMSA experiments examined the effect of LCDR on hnRNP K protein and LAPTM5-3' UTR binding. The two-tailed t-test analyzes the differences between groups and the data are expressed as mean±sem.

Fig. 39: LCDR, hnRNP K and lapm 5 were expressed in lung adenocarcinoma tissues. (a) LCDR in situ hybridization cancer and paracancerous expression profiles; (B) an LCDR in situ hybridization expression quantity statistical graph; (C) hnRNP K immunohistochemical cancer and paracancerous expression profile; (D) hnRNP K immunohistochemical expression quantity statistical map; (E) LAPTM5 immunohistochemical carcinoma and paracancerous expression profile; (F) LAPTM5 immunohistochemical expression quantity statistical chart.

Fig. 40: LCDR, hnRNP K and lapm 5 related phases and ROC analysis. (a) LCDR and hnRNP K correlation analysis; (B) LCDR and lapm 5 correlation analysis; (C) hnRNP K and lapm 5 correlation analysis; (D) LCDR, hnRNP K and LAPTM5 ROC.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The following examples do not identify specific experimental procedures or conditions, which may be followed by procedures or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

Example 1: molecular marker

The embodiment provides a molecular marker LCDR, and the nucleotide sequence of the molecular marker LCDR is shown as SEQ ID NO. 1.

Experimental example 1: acquisition of molecular marker LCDR accurate transcript and functional verification of molecular marker LCDR

Experimental materials and methods

1 Gene cloning

1.1PCR

When cloning a gene from cDNA or a plasmid, fast Pfu DNA Polymerase (Transgen) is used to ensure high fidelity of the cloned gene. The reaction system is shown in Table 1.

TABLE 1 PCR reaction System constructed by plasmids

Notice that: 1) The template of the reaction system is not excessive. Taking cDNA as a template to prepare a 50 mu L reaction system, the template amount is preferably 50-100 ng; 2) When the annealing temperature is set for reaction according to the Tm value of the primer, if the mixed band appears, the annealing temperature can be increased to eliminate the mixed band, and the maximum temperature can be up to 72 ℃; if no strip exists, the annealing temperature can be reduced until the strip appears; 3) Note that the appropriate extension reaction time (typically 1 minute can extend 1 kb) was adjusted depending on the size of the product and the amplification rate of the enzyme.

1.2DNA gel electrophoresis

Agarose gel electrophoresis was used. GelRed dye (GelRed nucleic acid dye BioTium41003, dissolved in water, 5. Mu.L GelRed 10,000Xstock solution) 2% (g/100 mL) agarose gel was added to 50mL agarose solution, and electrophoresis was performed at 120V voltage, and the electrophoresis results were directly observed under ultraviolet light.

A common running buffer is TAE, a 50X stock solution is prepared in advance, and deionized water is used for dilution to 1X. A50 XTAE formulation volume of 1L required Tris 242g,0.5M EDTA (pH 8.0) 100mL and glacial acetic acid 57.1mL.

Notice that: 1) According to the required sample amount and application, selecting the volume of the prepared solution, and proper comb and solution concentration; 2) Note that a suitable marker is selected according to the destination stripe size; 3) Note that the direction of DNA movement is from negative to positive.

Electrophoresis: (1) 50mL of 1% agarose gel (weighing 0.5g of agar, adding 50mL of 1 xTAE) was prepared according to the size of the separated DNA molecule by using 1 xTAE-Buffer, and heated in a microwave oven until dissolved; (2) cooling the solution to 50 ℃, adding GelRed nucleic acid dye, fully and uniformly mixing, and pouring into a proper rubber mold in which a comb is inserted; (3) after the gel is completely solidified, the comb is taken away, and the gel is transferred to an electrophoresis tank filled with 1 xTAE buffer solution, so that the buffer solution is soaked on the gel surface; (4) after mixing the DNA sample with 10×loading-buffer, the mixture was added to the sample well and 5. Mu.L of DNA ladder was added; (5) energizing to move the DNA to the anode (red line); (6) after the indicator strip has moved a suitable distance, the current is switched off, the gel is removed and observed under an ultraviolet lamp.

1.3 (cutting glue) recovery

For reaction (PCR or enzyme digestion, etc.) products with specific bands only, the products can be directly recovered; in the case of products in which non-specific bands are present, it is necessary to cut out the gel at the specific band position and then recover the gel.

Note that: 1) The glue cutting is required to be carried out under an ultraviolet lamp, and a shading plate is used for protection and safety; the cut strips are removed from the dead zone as much as possible to improve recovery efficiency; 2) During recycling, the rubber block can be chopped for accelerating dissolution; the volume of the adsorption column is 800 mu L, and if the volume of the sample is more than 800 mu L, the adsorption column can be added in batches; to increase the elution efficiency, the eluent may be heated to 65-70 ℃.

Gel recovery brief procedure (Tiangen agarose gel recovery kit): (1) adding 500 mu L of balance liquid BL into the adsorption column CB2, centrifuging at 12000rpm for 1min, and pouring out waste liquid in the collecting pipe; (2) adding an equal volume of solution PC (100. Mu.L of PC solution if 0.1g of the gel is 100. Mu.L) into the gel block, and standing in a water bath at 50 ℃ for about 10min until the gel is completely dissolved; (3) adding the obtained solution into an adsorption column CB2, centrifuging at 12000rpm for 1min, and pouring out the waste liquid in the collecting pipe; (4) 600. Mu.L of a rinse PW (check whether absolute ethanol has been added) was added to the column CB2, centrifuged at 12000rpm for 1min, and the waste liquid in the collection tube was poured off; (5) repeating the operation step (4); (6) placing the adsorption column CB2 back into a collecting pipe, centrifuging at 12000rpm for 2min, and air-drying the adsorption column CB2 in an ultra-clean bench for 10min; (7) placing the adsorption column CB2 into a clean centrifuge tube, adding 50 mu L of ddH ₂ O, after being left at room temperature for 2min, was centrifuged at 12000rpm for 2min, and the DNA solution was collected.

Notice that: for PCR reaction product recovery, only steps 2 and 3 differ. The PC solution with the same volume can be directly added, and the mixture is added into an adsorption column CB2 for recycling after being uniformly mixed.

1.4 enzyme digestion

Taking pSIH-H1-puro plasmid as an example, the concentration is assumed to be 1000 ng/. Mu.L, 1. Mu.g is digested, and 20. Mu.L of the system is obtained by:

notice that: 1) The total volume of enzyme should not exceed at most 10% of the total system; 2) For the case where the cut fragment is large, which results in collection of not only the desired fragment but also direct recovery, fast Digest Green Buffer is recommended for the reaction; 3) In view of the reduced efficiency of some enzymes that may be left for too long, it is suggested that the cleavage time be suitably extended to 1.5-2 hours.

1.5 attachment to Carrier

The reaction system is shown in Table 2.

TABLE 2 reaction system

Reagent(s)	Dosage of
		2x T4 DNA ligase buffer	5μL
Vector DNA (digested simultaneously)	50ng
		Inert DNA (digested and cut)	X
T4 DNA ligase	1μL
		H2O	Make up to 10 mu L

Notice that: 1) The amount of Insert DNA is determined by the length and concentration of it and Vector DNA, typically in a molar ratio Insert DNA/Vector DNA of 5-10; the mole number is simply summarized as mass/bp. Taking a reaction system of 10 mu L as an example, the Vector DNA concentration is assumed to be 50 ng/. Mu.L, and the length is 8000bp; the concentration of Insert DNA is 100 ng/. Mu.L, and the length is 1000; the molar ratio Insert DNA/Vector DNA was set to 10, and the volume of Insert DNA required was x, calculated as: (100 x/1000)/(50/8000) =10, then x=5/8 μl;

2) The connection reaction can be completed generally at room temperature for about 2 hours, and in order to increase the connection efficiency, the reaction can be performed at four degrees overnight, so that the effect is better.

1.6 conversion, coating

The liquid LB culture medium used for transformation and the solid LB culture medium used for plating are prepared in advance. 1L liquid LB medium was prepared, and it was dissolved by adding 950mL of deionized water to 10g of tryptone, 5g of yeast extract, 10g of NaCl, and adjusting pH to 7.0 with 5M NaOH, and then, the volume was adjusted to 1L. Then autoclaved at 121℃for 20 min.

The solid culture medium is added with 15-20 g of agar powder. In addition, the temperature of 60 ℃ needs to be set for heat preservation during sterilization so as to avoid solidification. After the mixture is taken out, the temperature is reduced by flowing water until the back of the hand touches the bottom and does not burn, corresponding antibiotics are added into an ultra-clean bench at a ratio of 1:1000, and the mixture is poured into a flat plate after shaking uniformly. 10mL was poured into a plate. After the flat plate is poured into the culture medium, shaking uniformly, opening the cover in an ultra-clean bench, cooling to room temperature, sealing by a sealing film, bagging and storing at 4 ℃ for later use.

The conversion step: (1) taking out competent cells from the refrigerator at-80 ℃, and putting the competent cells on ice for dissolution; (2) each tube contains 100. Mu.L of competence, and 50. Mu.L of reaction is sufficient. The ligation product was added to 50 μl competence and placed on ice for 15min. Then heat-shocking at 42 ℃ for 90s, and placing on ice for 3min; (3) 200 mu L of antibiotic-free LB is added into each tube, and the bacteria are shaken for 30 to 60 minutes at the temperature of 37 ℃ and at 200 rpm.

Notice that: avoiding the hands from touching the competent tube bottom to influence the state.

Coating a flat plate: (1) the head of the glass applicator is soaked in 95% ethanol, sterilized by flame of an alcohol lamp and then placed on a bracket for cooling. Repeating the steps once the coating is finished; (2) taking out the required flat plate from the temperature of 4 ℃, adding a proper amount of the transformed bacterial liquid, and uniformly coating by using an applicator. The mixture was poured into a bacterial incubator at 37℃overnight.

Notice that: 1) The above operations need to be performed in an ultra clean bench; 2) To avoid too many clones or no clones growing in the plates, each transformed strain may be plated on two plates separately: 1/10 of the bacterial liquid is coated on one plate, and the rest is coated on the other plate; 3) Preferably, a negative set containing only Vector DNA (digested) is made at the time of ligation to confirm that the digestion is clean.

1.7, cloning and bacterial liquid PCR:

after the plate culture for 12 hours, the cells were taken out of the bacterial incubator, and the presence or absence of cloning was confirmed.

The steps of selecting clone and bacterial liquid PCR are as follows: (1) and (3) configuring a PCR reaction system according to the number of clones to be selected, and reserving the volume of the template. (2) The sterilized EP tube was removed and 500. Mu.L of LB medium to which the corresponding antibiotics had been added at a ratio of 1:1000 was added; after being burnt and sterilized on the flame of an alcohol lamp, tweezers are slightly cooled, 20 mu L of gun heads are clamped, monoclonal on a flat plate is picked up, dipped in a corresponding PCR tube added with a PCR reaction system for several times, and then placed in a corresponding EP tube. (3) Repeating the operation (2) until cloning is completed. Mixing the PCR reaction evenly, centrifuging, reacting in a PCR instrument, and determining whether the clone is positive or not through electrophoresis photographing; EP tubes were shaken with a bacterial shaker at 37℃at 200 rpm. (4) After positive clones were confirmed, 100. Mu.L was aspirated from the corresponding EP tube to the new EP tube for sequencing.

Notice that: 1) The above operations need to be performed in an ultra clean bench; 2) 4-6 clones were picked from one plate for sequencing.

2 cell culture

2.1 Experimental materials

The cell lines used in this study and the names of the media used are shown in Table 3. All cells were purchased from Shanghai cell bank, national academy of sciences. All media, fetal bovine serum (Fetal bovine serum, FBS), a mixture of Penicillin and Penicillin (Penicillin-Streptomycin Solution, PS) and a 0.25% trypsin solution were purchased from Gibco corporation.

TABLE 3 cell lines and culture Medium

Cell line and culture medium	Source
		BEAS-2B	Shanghai cell bank
NCI-H1299	Shanghai cell bank
		Lenti-X ^TM 293T	Shanghai cell bank
DMEM	gibco
		RPMI-1640	gibco

2.2 Experimental methods

(1) Cell resuscitation

Before preparing to resuscitate the cells, firstly confirming that the temperature of the constant-temperature water bath is 37 ℃, opening a liquid nitrogen tank, taking out the pre-frozen cells, shaking the pre-frozen cells in the water bath to enable the cells to melt rapidly, centrifuging at 1000rpm for 5min, taking out a 10cm dish during centrifuging, adding a culture medium, taking out the cells after centrifuging, taking out the supernatant, adding 1mL of a cell culture medium (complete culture medium) composed of 10vt%FBS, 1vt%PS and the culture medium, resuspending the cells, adding the cell culture dish, shaking the culture dish to enable the cells to be evenly distributed, and placing the cells in a cell culture box for culturing.

(2) Cell passage

When the cells grow to 90% density, removing the cells from the incubator to a super clean bench (30 min is irradiated by ultraviolet in advance), discarding the supernatant, washing the cells twice by 2mL of 1 XPBS, digesting the cells for 2min by 1mL of 0.25% pancreatin, then completely culturing and finally digesting the cells by 2mL, slowly blowing the cells from the culture dish by a liquid mover, removing the cells to a 15mL centrifuge tube, centrifuging at 1000rpm for 5min, taking out the required 10cm dish number during the centrifuging, adding a culture medium, taking out the cells after the centrifuging is finished, taking out the supernatant, adding 1mL of a cell culture medium (complete culture medium) composed of 10vt% FBS, 1vt% PS and the culture medium, re-suspending the cells, adding the cell culture dish, slowly shaking the culture dish, uniformly distributing the cells, and placing the cells in the cell culture incubator for culturing.

(3) Cell cryopreservation

During cell passage centrifugation, cell cryopreservation solution is prepared according to the cell culture medium: FBS: dmso=6:3:1 ratio, after centrifugation, removing supernatant, adding 1mL cell cryopreservation liquid, blowing and mixing, transferring into cell cryopreservation tube, marking cell name, cryopreservation time, passaging times, wrapping with cotton, placing into-80 ℃ for cryopreservation, and transferring into liquid nitrogen tank after one week.

3 plasmid extraction

Plasmid extraction: (1) taking 15mL of bacterial liquid cultured overnight (16 hours) into a 15mL centrifuge tube, centrifuging at 12000rpm for 1 minute, and collecting bacterial agglomerates; (2) adding 500 mu L Buffer P1 into a centrifuge tube with the bacterial precipitate, and fully and uniformly mixing by using a vortex oscillator or a pipetting gun; (3) adding 500 mu L Buffer P2 into the centrifuge tube, gently reversing and uniformly mixing for 11 times, fully cracking thalli, and standing at room temperature for 6 minutes, wherein the melting in the centrifuge tube gradually becomes clear and transparent; (4) 500. Mu.L Buffer E3 was slowly added to the centrifuge tube, followed by mixing by inverting the tube upside down for 11 times, at which time white flocculent precipitate was gradually formed in the centrifuge tube, and the tube was left at room temperature (25 ℃) for 5 minutes. Centrifuging at 12000rpm for 5 min, collecting supernatant, adding the obtained supernatant into a filter column, standing for 1 min, and centrifuging at 12000rpm for 1 min; (5) adding corresponding isopropanol according to the volume of the obtained filtrate, then mixing the mixture upside down, and standing the mixture at room temperature for 10 minutes; (6) adding 200 mu L Buffer PS into the adsorption column, centrifuging at 12000rpm for 2 minutes, pouring out waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe again; (7) adding the mixed solution of the filtrate and isopropanol into an adsorption column with good balance; (8) standing at room temperature for 1 min, centrifuging at 12000rpm for 1 min, and pouring out filtrate in the collecting tube until all liquid passes through the column; (9) adding 750 mu L Buffer PW into the adsorption column, standing at room temperature (25 ℃) for 2 minutes, centrifuging at 12000rpm for 1 minute, pouring out waste liquid in the collecting pipe, and repeatedly rinsing for 2 times; changing the adsorption column into a new 1.5mL centrifuge tube, and placing the centrifuge tube in an ultra-clean bench for 10min by blowing; 100. Mu.L of sterile water was added to the middle portion of the adsorption film, and the mixture was left at room temperature for 5 minutes and centrifuged at 12000rpm for 2 minutes. Adding the obtained solution into an adsorption column again, standing for 5 minutes at room temperature, and centrifuging at 12000rpm for 2 minutes; plasmid concentration was determined using nanodrop and plasmids were stored in a refrigerator at-20 ℃.

4 lentivirus packaging and infection

293T cells were prepared in 10cm dishes and the cell attachment density was 60%. Plasmid transfection systems are shown in tables 4 to 5.

Table 4A tube transfection System

Reagent(s)	Dosage of
		PEI Reagent	25.5 mu L (total plasmid 3 times)
Opti-MEM	up to 100μL

Table 5B tube transfection System

Lentivirus packaging and infection steps: (1) uniformly mixing the prepared A, B two EP pipes, and standing for 15 minutes; (2) taking out 293T, sucking the supernatant, adding 4mL of serum-free DMEM/10cm dish, adding the mixed solution of (1) into 293T, standing for 6h, and replacing the complete culture medium; (3) collecting supernatants (24 h,48 h) at different time points, centrifuging at 4500rpm for 15min, and adding the supernatant into target cells; (4) the infection is repeated twice, and a corresponding screening can be performed to obtain a stable cell line.

5 5',3' RACE technique

The full-length sequence of LCDR is obtained through Mitrans-script website (http:// www.mitranscriptome.org /), but the sequence is often not completely accurate, in order to obtain accurate transcript sequence, the biological function and molecular mechanism of LCDR are studied in detail, and the accurate transcript of LCDR is finally obtained through cDNA terminal rapid amplification (rapid amplification of cDNA ends, RACE) technical experiment, which comprises the following specific steps:

(1) Extracting total RNA of a lung adenocarcinoma cell line NCI-H1299 by using a Trizol extraction method, and reversely transcribing the total RNA into cDNA by using a reverse transcription kit of PrimerScript RT-PCR kit (Japanese Takara) by using oligo dT as a reverse transcription primer;

(2) the cDNA is used as a PCR reaction substrate template, primers 5'-F and 3' -R are designed according to a predicted sequence to carry out PCR amplification, the full length of LCDR is obtained and connected with a T-vector, the correctness of the sequence is verified through sequencing (comparison with the predicted sequence), if the sequencing is correct, the sequence is the accurate full-length cDNA sequence of the LCDR, and if the sequencing is incorrect, the following steps are carried out;

(3) using the cDNA in (1) as a PCR template, designing a primer according to the predicted sequence, and performing a nested PCR amplification reaction (the amplification program is shown in figure 1), wherein the reaction system is as follows:

6 Nuclear mass separation experiment

(1) Cell collection

Taking a 15cm dish of NCI-H1299 cells with the density of 90% and good growth state, sucking and discarding the supernatant of the culture solution, cleaning the cells three times by using PBS (phosphate buffer solution) prepared in advance, scraping the cells by using a cell scraper after adding 2mL of PBS, sucking the cells into a new EP (European patent application) tube by using a pipette, centrifuging at 2000rpm for 6min at 4 ℃, sucking and discarding the supernatant, and slowly adding 2mL of PBS to resuspend the cell sediment at the bottom of the EP tube. The resuspended cell suspension was divided into 2 parts, one of which had a volume of 400. Mu.L for total RNA extraction and the other of 1.6mL for cytoplasmic and nuclear RNA and protein extraction. Centrifuging at 2000rpm for 5min at 4deg.C, removing the whole supernatant as much as possible, adding 50 μl of RIPA strong cell lysate, standing on ice for 30min, centrifuging at 12000rpm at 4deg.C for 15min, and collecting supernatant to obtain total protein and total RNA.

(2) Isolation of cytoplasm

1.6mL of the cell suspension was centrifuged at 1000rpm for 5min, the supernatant was aspirated off, and the cell pellet was repeatedly freeze-thawed twice using liquid nitrogen. Subsequently 400. Mu.L of buffer A (10 mM HEPES, pH 7.9, 1.5mM MgCl) was added to the EP tube ₂ 10mM KCl, 0.1% Nonidet P-40 and 1%Protease Inhibitor Cocktai) was repeatedly blown 30 times with a pipette and left on ice for 10min. The lysed cells were centrifuged at 2000rpm at 4℃for 10min and the supernatant was aspirated for cytoplasmic protein and RNA extraction.

(3) Isolation of nuclei

400. Mu.L of buffer A (1%Protease Inhibitor Cocktai,g/10 mL) was added to the above-described cytoplasmic cell nucleus pellet, and the mixture was thoroughly mixed with a pipette, 300. Mu.L of buffer A containing 1%Protease Inhibitor Cocktai (g/10 mL) was added thereto, and the mixture was again thoroughly mixed and allowed to stand in iceberg for 10 minutes. Centrifuge at 2000rpm at 4deg.C for 10min, adding 500. Mu.L buffer S1 (0.25 mM sucrose, 10mM MgCl) into the supernatant ₂ 1%Protease Inhibitor Cocktai) the nuclear pellet was resuspended and 500. Mu.L buffer S2 (0.35 mM sucrose, 0.5mM MgCl) was slowly added along the tube wall ₂ 1%Protease Inhibitor Cocktai). Centrifuging at 3500rpm for 10min at 4deg.C, discarding supernatant, adding 200 μL of RIPA strong lysate, mixing, standing on ice for 30min, centrifuging at 12000rpm for 15min at 4deg.C, and sucking supernatant for extracting nuclear protein and RNA.

7 cell proliferation and clone formation

7.1 cell proliferation

After cell gene knockout or overexpression, cellTiter-Glo luminecent was used to further investigate the effect of gene expression on cell function ^TM Cell Viability Assay assays (CTG) detection of the relative number of cells in 96 platesThe number of the steps is as follows:

(1) Collecting cells: covering the surface of a cell culture dish with pancreatin, adding a complete culture medium to stop digestion after cell digestion is completed, slowly blowing the cells by using a pipetting gun, transferring the cells into a 15mL centrifuge tube, centrifuging at 1000rpm for 5min, sucking off the supernatant by using a pipetting pump, adding 1mL of complete culture medium to suspend cell sediment, and blowing and mixing uniformly.

(2) Cell count: based on the cell pellet, the corresponding dilutions were performed. The cell suspension from the above step was pipetted 20. Mu.L into a clean sterile EP tube and 180. Mu.L PBS was added and mixed slowly upside down, at which time the cell concentration was diluted to 1/10 of the original. 10. Mu.L of the diluted cell suspension was pipetted slowly into the cell suspension along the wells of the technical plate until all coverage was achieved while avoiding air bubbles as much as possible.

(3) Cell plating: according to the counting result, adding the complete culture medium or the cell suspension, and finally, adding 1000-2000 cells into 100 mu L of the cell suspension, accurately adding the cell suspension into a 96-well plate by using a pipette, and reading by using CTG, namely, obtaining the number of cells on the first day.

(4) Cell reading: 3 sets of data were read once a day at intervals.

7.2 cloning formation

Cloning and forming: (1) absorbing cell proliferation suspension, adding into 6-well plate with cell number of 500-1500, adding complete culture medium 2mL, standing at 37deg.C and 5% (v/v) CO ₂ Culturing in a cell incubator, and observing the cells at regular intervals, wherein the culture period is 2 weeks; (2) when proper cell clones with proper size and number are observed in the 6-well plate, the culture is stopped, the 6-well plate is taken out, PBS is washed 3 times, and 4% (g/100 mL) of paraformaldehyde is added for fixing at room temperature for 10min; (3) absorbing and discarding 4% of paraformaldehyde, washing for 3 times again by using PBS, adding crystal violet, and standing at room temperature for 1h for dyeing; (4) after the dyeing is completed, crystal violet is recovered, and the wall of the 6-pore plate is dyed and slowly washed until the washed water flow becomes clear.

8RNA extraction and reverse transcription

8.1RNA extraction

And (3) RNA extraction:

(1) after cells or tissues are added into Trizol, the cells or tissues are left at room temperature (25 ℃) for 5min to be fully lysed, which can lead to complete separation of nucleic acid protein complexes (a centrifuge tube can be gently turned over and evenly mixed);

(2) centrifuge 12000rpm at 4℃for 10min, after which the supernatant was aspirated into a new Rnase Free EP tube. The EP tube needs to be marked while care is taken to prevent the marks from being attacked by the organic solvent;

(3) Chloroform is added for extraction to remove impurities such as sucrose, protein and the like and promote the separation of the water phase and the organic phase. At least 0.2mL chloroform (1/5 volume) was added per 1mL Trizol. The sample bottle cap is tightly covered, the test tube is shaken by hand for 15s to be fully and uniformly mixed, and the mixture is kept stand for 5min at room temperature;

(4) isopropanol was added to precipitate the aqueous RNA. After the completion of the addition of chloroform and the standing, the mixture was centrifuged at 12000rpm at 4℃for 20 minutes, and the phase separation was three layers. Carefully transfer the upper aqueous phase into a new 1.5mL RNase-free centrifuge tube (about 400-500. Mu.L), add an equal volume of isopropanol (0.5 mL isopropanol/mL Trizol), mix, and then stand at room temperature for 10min or-20deg.C for 1 hour;

(5) centrifuge 12000rpm at 4℃for 10min, carefully discard the supernatant. (RNA precipitation is often not visible before centrifugation, and gel-like precipitates form on the tube side or wall after centrifugation);

(6) the RNA precipitate was washed with 75vt% ethanol to allow the salt ions in the RNA precipitate to be fully dissolved. 1mL 75vt% ethanol was added and the RNA pellet was sprung for rinsing. RNA pellet was washed with at least 1mL 75vt% ethanol per 1mL Trizol;

(7) after washing the RNA pellet, the pellet was centrifuged at 12000rpm for 10min at 4 ℃. Sucking and discarding the supernatant, taking care not to pour out the sediment, centrifuging the residual liquid for a short time, sucking out the residual liquid by using a gun head, taking care not to suck out the sediment;

(8) The EP tube was opened and placed on a clean bench for blow-drying (15 min). RNA precipitation cannot be blown to be too dry, so that the RNA precipitation is difficult to dissolve after being completely dried, but ethanol is sufficiently avoided from affecting subsequent tests;

(9) adding DEPC water (100 mu L) according to the test requirement and the precipitation amount of RNA, repeatedly blowing and mixing uniformly, and fully dissolving RNA;

quantification (Nanodrop): the extracted RNA is detected by an ultraviolet spectrophotometer and passes through an OD _260/280 Detecting the purity and concentration of RNA;

wherein OD _260/280 As a reference value:

OD _260/280 between 1.9 and 2.1, the purity of RNA is considered to be good, and the concentration is: μg/μl=od ₂₆₀ * Dilution factor of 40/1000;

OD _260/280 the value is smaller than 1.8, which indicates that the protein impurities are more;

OD _260/280 a value greater than 2.2 indicates partial degradation of the RNA;

OD _260/280 and if the value is less than 2.0, the guanidine isothiocyanate and the beta-mercaptoethanol method residue are shown in the lysate.

8.2 reverse transcription

Reverse transcription step:

(1) The required RNA amount was calculated: since only 1. Mu.g of RNA is required for the removal of genomic DNA (gDNA) reaction, quantitative calculations of RNA are required. I.e. required amount of RNA = 1000/concentration of RNA extracted.

(2) Genomic DNA was removed (system: 10. Mu.L): the reagent is placed on ice or in an ice box, a reaction liquid main pipe is configured according to a system (the system is only one sample system) of the table 6, and finally, the reagent is packaged in a PCR tube, and an RNA sample is added and marked according to a calculation result. After the completion of the configuration, the mixture was homogenized, and the PCR tube was placed in a PCR instrument and reacted according to the procedure of Table 7.

TABLE 6 PCR System

2.5×gDNA Eraser Buffer	2μL
		1.gDNA Eraser	1μL
RNA	1 μg (RNA required amount=1000/concentration of RNA extracted)
		6.RNase Free dH2O	(h2o=7-1000/extracted RNA concentration) μl

TABLE 7 PCR procedure

42℃	2min
		4℃	Preservation of

(3) Reverse transcription (System: 10. Mu.L): the reaction solution for reverse transcription was prepared in the system of Table 8 (the system was only one sample system) while the gDNA removal reaction was performed on the sample. The process is the same as above, the reagent is placed on ice or ice box, the reaction liquid main pipe is configured, and finally, the reverse transcription reaction liquid is added into the gDNA removed sample. The reaction solutions were added to the tubes, mixed well, and subjected to PCR according to the procedure of Table 9.

TABLE 8 reverse transcription system

4.5×PrimeScript Buffer 2(for Real Time)	4μL
		5.RT Primer Mix	1μL
6.RNase Free dH2O	4μL
		3.PrimeScript RT Enzyme Mix I	1μL

TABLE 9 reverse transcription procedure

37℃	15min
		85℃	5sec
4℃	Preservation of

After the reaction is completed, 20 mu L of total cDNA can be obtained, and the cDNA can be diluted according to the requirement of the subsequent test. The cDNA sample can be stored for a long time at-20 ℃.

9 real-time fluorescent quantitative PCR (qPCR)

Real-time fluorescent quantitative PCR (qPCR) step:

PCR reaction solutions (the reaction solutions were placed on ice) were prepared as shown in Table 10. In view of the draw error, the premix volume is configured to be at least 10% greater than the total volume for all reactions. The qRT-PCR primers are shown in Table 11.

TABLE 10 PCR reaction liquid formulation

TABLE 11 qRT-PCR primers

Extraction of 10 genomic DNA

Extraction of genomic DNA:

(1) Digestive cells and cell counts: cell count is performed after cell digestion and centrifugation to make the cell count not more than 5×10 ⁷ Individual cells. After counting was completed, 300g was centrifuged for 5min, the supernatant was discarded, taking care not to discard the cell pellet;

(2) Adding 200 μl of PBS to the sample to resuspend the cells, adding 20 μ L QIAGEN Protease or proteinase K to the sample;

(3) 200. Mu.L of buffer AL is added to the sample, vortexed for 15s, and in order to ensure high efficiency of lysis, the sample and buffer AL must be thoroughly mixed to produce a homogeneous solution, if the sample volume is greater than 200. Mu.L, QIAGEN Protease (or proteinase K) and buffer AL are added in equal proportion; for example, 400. Mu.L of sample would require 40. Mu. L QIAGEN Protease (or proteinase K) and 400. Mu.L of buffer AL. If a sample size of greater than 400. Mu.L is desired, QIAamp DNABlood Midi or Maxi kit may be used; these can handle up to 2mL or 10mL of sample;

(4) The DNA was incubated in a water bath at 56℃for 10min and at 56℃for 10min, the DNA yield was maximized. However, it is noted that longer incubation times have no effect on the yield or quality of purified DNA;

(5) Centrifuging the centrifuge tube for a short time to enable the liquid on the tube cover and the tube wall to slide to the bottom;

(6) 200. Mu.L of ethanol (96 vt) was added to the sample and vortexed for 15s; after uniform mixing, the mixture is centrifuged for a short time to enable the liquid on the tube cover and the tube wall to slide to the bottom, and if the volume of the sample is more than 200 mu L, the amount of ethanol is increased proportionally; for example, 400. Mu.L of ethanol is required for 400. Mu.L of the sample;

(7) The mixture obtained in (6) was transferred to QIAamp Mini spin column (2 mL collection tube), and 6000g was centrifuged for 1min. QIAamp Mini spin column is then transferred to a new 2mL collection tube, the old collection tube is discarded, the cap of each filtration column is closed, and aerosol formation during centrifugation is avoided;

(8) QIAamp Mini spin column was opened and 500. Mu.L of AW1 buffer was added thereto, and 6000g was centrifuged for 1min. QIAamp Mini spin column was transferred to a new 2mL collection tube (kit provided) and the old collection tube was discarded;

(9) QIAamp Mini spin column was opened and 500. Mu.L of AW2 buffer was added thereto, the lid was closed, and centrifuged at 14000rpm for 3min at full speed;

(10) QIAamp Mini spin column was transferred to a new 2mL collection tube (kit not provided), the old collection tube was discarded, and high speed centrifugation was performed for 1min;

(11) QIAamp Mini spin column is transferred to a new 1.5mL collection tube (kit not provided) and the old collection tube is discarded. The QIAamp Mini spin column lid was carefully opened and 200. Mu.L of buffer AE or ddH was added thereto ₂ O。

11 protein extraction and immunoblotting

Protein extraction and immunoblotting steps:

11.1 protein extraction:

(1) The cells were removed and washed 3 times with PBS;

(2) Scraping the cells down into a 1.5mL centrifuge tube with a cell scraper;

(3) Placing on ice, and lysing cells with lysate for 30min;

(4) Centrifuging at 12000rpm at 4deg.C for 20min, collecting supernatant, and discarding precipitate;

(5) Protein concentration was determined by BCA method;

(6) Adding 5xSDS Loading Buffer to the cell lysate according to the protein volume;

(7) The protein sample was boiled at 100℃for 10 minutes.

11.2 immunoblotting

(1) And (3) glue preparation: the upper layer is 5% (g/100 mL) concentrated gel, and the lower layer is 15% (g/100 mL) separating gel according to the protein size;

table 12 15% Release gel formulation (10 mL)

TABLE 13 5% SDS-PAGE gel formulation

(2) Running glue: loading 30 mug, adjusting loading amount according to protein abundance, running the concentrated gel at constant voltage of 80v, and running the concentrated gel at constant voltage of 120v to the bottom of the separation gel or stopping after the bromophenol blue bottoms out at constant current of 25 milliamp/gel;

(3) Transferring: constant current 200 mA, film transfer time is determined according to the size of target protein, and 1 minute is transferred to 1 kd;

(4) Closing: blocking with blocking solution containing 5vt% skimmed milk or BSA at room temperature (25deg.C) for 1 hr or at 4deg.C for 16 hr (depending on antibody properties, time and temperature);

(5) Incubating primary antibodies: anti-4 ℃ overnight, primary antibody concentration 1:1000, according to the instruction book and experimental results;

(6) Washing the film: TBST elution for 3×10min, tween concentration 0.1% was added to TBS (TBS formulation see table 14);

(7) Domestic secondary antibody 1: 2000-fold dilutions in TBST, secondary antibody incubated for 1 hour at room temperature (antibodies see table 15);

(8) Washing the film: TBST elution 3 x 10 min;

(9) Exposure: and (3) putting the film bubble into a luminous solution (the luminous solution is added from the solution A and the solution B in equal proportion), drying, putting into a machine, automatically exposing, and then manually adjusting the exposure time according to the result.

TBS preparation (buffer, 1L)

10×TBS：20mM Tris-HCl，pH 7.5

Table 14 TBS formulation

Table 15 Gene antibodies

12RNA pμLl down

12.1 preparation of RNA

(1) Preparing a plasmid: obtaining sense and anti-sense, cloning the sense and anti-sense on a PCDNA3.1 vector (the full-length sequence of LCDR is abbreviated as sense chain and the reverse complementary chain of LCDR is abbreviated as anti-sense chain obtained by PCR), constructing the sequences to the vector PCDNA3.1 (+) through enzyme cutting sites EcoR I and Xho I, and extracting plasmids;

(2) Plasmid linearization: confirming a specific enzyme cutting site contained in a 3' -terminal sequence of sense, antisense, and linearizing the plasmid by using a corresponding endonuclease;

(3) Linearized fragment purification, for the reaction (PCR or enzyme cutting etc.) products with specific bands only, can be directly recovered; for products with non-specific bands, the gel at the specific band position needs to be cut off and then recovered;

(4) In vitro RNA transcription (thermo)Kit+thermo AM 8452), the RNA transcription system is as follows:

2μL ATP

2μL CTP

2μL GTP

1.3μL UTP

5μL Biotin UTP

2μL 10X Reaction Buffer

1 μg of linearized DNA (i.e. at least 270 ng/. Mu.L)

2μL Enzyme Mix

Nuclease-free H ₂ O is filled to 20 mu L

Reacting for 4 hours at 37 ℃;

(5) 1. Mu.L of Turbo DNAase (template removed) was added to the transcript. Mixing, and standing at 37deg.C for 15min;

(6) To the above product was added 30. Mu.L of nucleic-free H ₂ O and 30 mu L LiCl are mixed evenly and placed at the temperature of minus 20 ℃ for more than 30 min. Centrifuging at 12000rpm at 4deg.C for 15min, and removing supernatant; 1mL 70vt% enzyme-free ethanol is added for blowing up, and then the mixture is centrifuged, the supernatant is carefully discarded, and a proper amount of enzyme-free water is added for dissolution after the mixture is dried. After split charging, the liquid nitrogen is frozen quickly and stored in a negative eighty refrigerator.

1.2 preparation of Cell lyaset

6 cells were prepared in 10cm dishes at a density of 95%. Washing twice with cold PBS (4deg.C) on ice, and discarding the supernatant; 120. Mu.L of IP lysis (1% inhibitor) was added to each dish and the mixture was lysed on ice for 5min; then scraping by using a cell scraper (treated by RNase scavenger in advance), collecting into a centrifuge tube, and placing on ice for 15min; centrifuge 12000rpm at 4deg.C for 20min; the supernatant was transferred to a fresh tube, and after concentration measurement, the supernatant was dispensed to an EP tube, and the concentration was adjusted so that each tube contained 2mg of lysate (volume 1 mL).

1.3RNA pμLldown

(1) RNA forms a secondary structure: 5pmol of each biotin-labeled RNA was taken and 50. Mu. L RNA structure buffer (10 mM Tris pH 7.0, 0.1M KCl, 10mM MgCl) was added ₂ ) Standing at 95deg.C for 2min, ice for 3min, and standing at room temperature (25deg.C) for 30nmin to form a secondary structure;

(2) RNA binding to protein: step two, taking out the cell lysate from the refrigerator, dissolving the cell lysate on ice, uniformly dividing the cell lysate into two enzyme-free EP pipes (each pipe contains 1mg of protein), respectively adding the same amount of the formed reaction liquid, and rotating the vertical suspension instrument for 1h at room temperature;

(3) Respectively taking 50 mu L Streptavidin agrose beads (life, M280), and washing with IP lysis buffer for 5 times each for 5min; respectively adding washed beads into the reaction liquid, and rotating a vertical suspension instrument for 1h at room temperature;

(4) Washing the beads with IP lysis buffer for 5 times, each time for 5min; adding 40 μL of each of the above materials, and decocting at 1X loading buffer 95 ℃ for 5min;

(5) And (3) carrying out electrophoresis after obtaining the sample, and carrying out corresponding dyeing.

13RNA co-immunoprecipitation (RIP)

(1) Cell lysis: prepare 3 dishes of 10cm dishes of NCI-H1299 cells at 80% density, aspirate the medium, wash 3 times with pre-chilled medium (4 ℃), scrape the cells with a cell scraper and place them in a 15mL centrifuge tube, centrifuge at 1500rpm for 5min at 4℃and collect the cells. The cell pellet was resuspended in PBS and centrifuged at 1500rpm for 5min at 4℃and repeated twice. Adding 200 mu L of cell lysate into the cell sediment, blowing up and down by a pipetting gun, mixing uniformly, and incubating on ice for 10min;

(2) Cleaning magnetic beads: before use, re-suspending the magnetic beads, sucking 50 mu L of the magnetic beads, placing the magnetic beads in a 1.5mL EP tube, adding 500RIP Wash Buffer, blowing and mixing for 15 times by using a pipetting gun, placing the magnetic beads in a magnetic rack, sucking and discarding the supernatant, and repeatedly cleaning for two times;

(3) Magnetic beads bind to antibodies: adding 100 mu L of RIP Wash Buffer to the washed magnetic beads to resuspend the magnetic beads, adding 5 mu g of corresponding antibody, and placing the magnetic beads on a rotator at room temperature for 30min to combine the antibody with the magnetic beads;

(4) After the incubation of the magnetic beads and the antibodies is completed, repeatedly cleaning the magnetic beads for 3 times by using the RIP Wash Buffer, and removing non-specifically bound proteins;

(5) Incubation of magnetic bead-antibody complexes with cell lysates: binding buffer was configured as in table 16; centrifuging the cell lysate at 4 ℃ at 1500rpm for 20min, and then sucking 100 mu L of the cell lysate and 900 mu L of a binding buffer solution at 4 ℃ for overnight incubation by a vertical rotator;

table 16 binding buffer formulations

Reagent name	Dosage (mu L)
		RIP Wash Buffer	860
0.5M EDTA	35
		RNase Inhibitor	5
Total	900

(6) Separating the EP tube, placing on a magnetic rack, sucking and removing supernatant, adding 500 mu L RIP Wash Buffer, mixing up and down for 20 times by using a pipetting gun, and repeatedly washing for 6 times to remove non-specifically bound RNA;

(7) Protease digestion releases RNA: the protein digestion system was configured as in table 17; performing protein digestion on the washed magnetic beads, and placing the magnetic beads on a vibrator at 60 ℃ for digestion for 30min;

Table 17 protein digestive system formulation

Reagent name	Dosage (mu L)
		RIP Wash Buffer	117
10％SDS	15
		Proteinase K	18
Total	150

(8) And (3) separating the EP tube, placing the EP tube on a magnetic rack, and sucking the supernatant to purify and recycle RNA.

14 chromatin co-immunoprecipitation (CHIP)

ChIP experiments were performed using a kit of Magna ChIP HiSens Chromatin IP Kit (Merck Millipore), and the specific procedure is as follows:

(1) planting NCI-H1299 in advance on a 6cm dish, wherein the cell density is 90%;

(2) the medium was aspirated and washed 3 times with pre-chilled PBS (4 ℃);

(3) absorbing and discarding the culture medium, adding a stabilizing buffer A, adding 11.1% (g/100 mL) of fresh formaldehyde, enabling the final concentration of formaldehyde to be 1% (g/100 mL), and shaking on a shaking table for 5min;

(4) the supernatant was aspirated and washed 3 times with pre-chilled PBS (4 ℃ C.), removing residual formaldehyde;

(5) cell scraping is carried out, and the cell scraping is placed in an EP tube with the volume of 1.5mL, and 200g of the cell scraping is centrifuged for 5min at the temperature of 4 ℃;

(6) absorbing and discarding supernatant, adding PBS, mixing up and down for 10 times, centrifuging 200g for 5min at 4 ℃ by using a centrifuge, and cleaning twice;

(7) absorbing and removing PBS, adding cell lysate into the cell sediment, and incubating for 10min at 4 ℃;

(8) centrifuging 1700g of the mixture for 10min at 4 ℃, sucking and removing the supernatant, and collecting complete nuclei;

(9) adding 200 mu L of Wash buffer into the cell nucleus, uniformly mixing by using a pipetting gun, and incubating at 4 ℃ for 10min;

Centrifuging 1700g of the mixture for 5min at 4 ℃ in a centrifuge, collecting cell nuclei, and sucking and discarding supernatant;

shareing buffer D3 is slowly added along the pipe wall of the EP, and 200g is centrifuged for 5min at 4 ℃ by a centrifuge, and the steps are repeated twice;

the supernatant was aspirated, and 130. Mu.L of shareing buffer D3 was added to resuspend the nuclei for sonication;

mixing magnetic beads by using a pipetting gun, sucking 10 mu L of magnetic beads, placing the magnetic beads in an EP tube with 1.5mL, adding magnetic force, sucking and discarding supernatant, adding 50 mu L of SCW buffer spiral for 10.S, and repeatedly cleaning for 3 times;

placing the mixture in a magnetic force, sucking and removing the supernatant, adding 200 mu L of SCW buffer, adding 5 mu g of corresponding antibody, and incubating for 2 hours at room temperature by a vertical rotator;

separating the EP pipe, placing the EP pipe on a magnetic rack, and sucking and discarding supernatant;

adding 500 mu L of SCW buffer containing protease inhibitor by using a pipette, adding 10 mu L of chromatin, mixing uniformly, and placing the mixture at 4 ℃ for overnight incubation by a vertical rotator;

the above overnight incubated EP tube was washed off, the supernatant was aspirated off, and washed three times with SCE buffer containing protease inhibition;

500. Mu.L of pre-chilled Low Stringency IP Wash Buffer (4 ℃) containing protease inhibitors was added to the EP tube, vortexed for 10s, placed in a magnetic rack, and the supernatant was aspirated;

eluting DNA: 50 mu LChIP Elution Buffer and 1 mu L proteinase K were added to the EP tube, incubated at 75℃for 15min, the protein digested, cooled to room temperature, centrifuged briefly, the supernatant was removed, and the DNA was purified.

15 protein gel migration blocking experiment (electrophoretic mobility shift assay EMSA)

(1) Preparing 5% natural non-denaturing agarose gel, and placing in 0.5 XTBE solution, and pre-running gel at 100V for 30-60min;

(2) In the process of pre-running the gel, the configuration of a reaction system is carried out according to the figure 2;

(3) Incubating the probe and purified hnRNP K protein according to the reaction system, adding 5 mu L of loading buffer for 30min at room temperature (25 ℃), and uniformly mixing;

(4) Sucking 20 mu L of the reflected sample, adding the sample into agarose gel for electrophoresis until bromophenol blue migrates to one quarter of the bottom of the gel;

(5) Transferring: cutting a corresponding nylon film, soaking in 0.5TBE for 5min, and using 400mA current to transfer the film for 30min;

(6) Measuring the size of a corresponding nylon membrane after membrane transfer is completed, and performing ultraviolet crosslinking;

(7) Placing the ultraviolet-crosslinked film in Nucleic Acid Detection Blocking Buffer for 15min, and shaking;

(8) Placing the closed nylon membrane into a connecting/blocking buffer for 15min, and shaking;

(9) Transferring the nylon membrane to wash solution buffer for 5min, and repeatedly washing for 4 times;

(10) Placing the washed nylon membrane in Substrate Equilibration Buffer containing 30mL, shaking and incubating for 5min;

(11) The nylon membrane was held up using forceps and placed on dry paper, which was then left to incubate at Substrate Working Solution room temperature for 5min;

(12) Exposure is performed and an image is acquired.

16Northern blot

Northern blot procedure:

(1) preparing a probe: probes for in vitro transcription and labeling of LCDR (the nucleotide sequence of the probes is shown as SEQ ID NO: 42);

(2) carrying out DEPC treatment on all instruments used in the experiment, and washing with DEPC water;

(3) 50mL of 1 xGel Prep/RunningBuffer was prepared, 0.5g of Agarose (Agarose-LE) was weighed, and Agarose Gel was prepared;

(4) sucking prepared 25 mug total RNA, adding an equal volume of Glyoxal Load Dye reagent, and placing in a 50 ℃ oven for incubation for 30min;

(5) soaking the Gel prepared in the step (3) in 1 xGel Prep/RunningBuffer for 5min, adding corresponding RNA into the agarose Gel hole, and running Gel under 5V/cm voltage until bromophenol blue is transferred to the bottom of the Gel;

(6) placing the gel in a siphoning device, and transferring the gel for 2 hours;

(7) measuring the size of a nylon membrane, and performing corresponding crosslinking according to the size;

(8) preheating a reagent mu LTRAhyb at 68 ℃, placing a crosslinked nylon membrane in a 10cm dish, adding the preheated mu LTRAhyb to cover the surface of the membrane, and prehybridizing for 30 minutes at 68 ℃;

(9) Adding LCDR probe with final concentration of 0.2nm to nylon membrane in (8), adding corresponding RNase inhibitor, hybridizing at 4deg.C overnight and incubating;

is placed in 3mL Low stringency washing Solution for washing and is repeated twice for 10min each time; then placing the nylon membrane in a 3mL High stringency washing Solution solution, and washing twice at 68 ℃ for 20min each time;

exposure is performed and an image is acquired.

17 apoptosis flow assay

Apoptosis flow-through detection steps:

(1) Positive cells were prepared: 1 dish of 10cm dish of cells was selected, the dish lid was opened, and the dish was irradiated under an ultraviolet lamp for 2 hours. Or the positive cells are treated in advance by using a Biyun-Tian apoptosis positive control kit;

(2) Collecting cells: the supernatants of the test cells (including negative and positive control cells) were collected into 15mL EP, the cells were rinsed with 2mL PBS, 1mL pancreatin without EDTA was added, and digested in a 37 ℃ incubator until the cell mass was detached from the bottom of the dish. After termination of digestion with the supernatant just collected, transfer to the just 15mL EP tube. Centrifuging at 2000rpm at room temperature for 5min, and discarding the supernatant;

(3) Washing the cells: cells were resuspended in 1mL of room temperature PBS and transferred to 1.5mL EP tubes. Centrifuging at 2000rpm at room temperature for 5min, and discarding the supernatant;

(4) Cell count: cell counting was performed using a cytometer, 2X 10 for each group ⁶ A cell;

(5) Dyeing: cells were resuspended (2X 1) using 200. Mu.L buffer0 ⁶ ) All experimental groups required double staining with negative control and positive control required single staining tubes with two dyes. mu.L of Annexin V dye, 5 mu.L of 7-AAD, and 5 mu.L of Annexin V dye alone or 5 mu.L of 7-AAD were added to the double-dyeing tube, and the name of the single-dyeing dye was clearly shown. The mixture was flicked by hand and dyed in dark place for 15min. The flow tube and filter membrane were prepared during the staining process.

Opening a flow cytometer, and checking the conditions of sheath fluid and waste liquid;

(6) After the completion of staining, 1mL of PBS at 2000rpm was added, and the mixture was centrifuged at room temperature for 5min, and the supernatant was discarded. Adding 100 mu L PBS for resuspension, and filtering;

(7) Setting up: the SSC, FSC, FITC channel under BB515, and 7-AAD channel are opened, and the other unused channels are deleted. The log mode is used to select all the A, H and W indexes. Setting an initial reading value: cancer cell SSC set 200, FSC set 200, FITC voltage set 280,7-AAD set 350;

(8) Voltage and compensation adjustment: looking at the peak diagram of each channel, the voltage is regulated so that the peak tail of the negative peak is between 102 and 103, the voltage is reduced when the peak is right, and the voltage is increased when the peak is left. When an Annexin V single-stained tube is applied, 7AAD-FITC compensation is regulated, X is input, and a two-quadrant of the cross gate is free of cell signals. When the single-dye tube is used for 7AAD, FITC-7AAD compensation is regulated, X is input, and a four-quadrant cross gate has no cell signal. If the experimental time is limited, the compensation is not adjusted well. FITC-7AAD and 7AAD-FITC are both 1% input to preserve the compensation gates, which can be readjusted on the FlowJ software;

(9) After the voltage and compensation are adjusted, they cannot be changed again. If the voltage changes, the compensation should be regulated again, and 30000-50000 cells are collected per tube;

(10) Cleaning the flow cytometer according to the cleaning flow standard, copying data;

(11) The FlowJ adjusts the compensation, opens the FlowJ interface, and drags two single dye tubes into the compensation group. The compensation is automatically adjusted and then applied to all experimental groups;

the cross gate is arranged according to the negative control, so that the cell number in two, four and three quadrants is ensured to be less than 0.4 percent. Negative control loop gates were applied to all experimental groups.

Outputting the cross gate results of each group to Layou, and after reasonable arrangement, adjusting the font size and format, and outputting the Tiff format.

18 digital PCR

Digital PCR step:

(1) Establishing a standard curve: the pcDNA3.1 (+) vector was used to construct LCDR recombinant plasmid, which was transferred into E.coli DH-5a for amplification culture, and then the plasmid concentration was measured using Nanodorp 2000. The plasmid concentration was then converted to plasmid copy number by empirical formula: copy number (copies/. Mu.L) = [ plasmid concentration (ng/. Mu.L). Times.1. Mu.L.times.6.02.1023.times.10 ] ^-9 ]/(vector length+target gene length) ×2X 324.5]；

(2) Serially diluting the plasmid into 7 gradients according to a 10-fold gradient dilution method, detecting the ct value of each gradient sample by fluorescence quantitative PCR (three technical repetitions respectively), and then establishing a linear relation between log10 (copy number) and the ct value to obtain a standard curve;

(3) Absolute quantification of single cell copy number: after cell counting, 100 ten thousand cells are taken for RNA extraction, 100 mu L of DEPC water is used for dissolving RNA, then 1 mu L of RNA is taken for reverse transcription into cDNA in a 20 mu L system, after dilution to 40 mu L, 1 mu L is taken as a template for fluorescent quantitative PCR to obtain a sample ct value, then the copy number of a target gene in the 1 mu L template is calculated according to a standard curve, and finally the copy number of single cells is obtained after division by the cell number.

19 double luciferase reporter assay

The procedure of the dual luciferase report experiment:

(1) Usingin vitro DNA&The siRNA transfection reagent reagent transfects plasmids. According to the reagent specifications, a proper transfection system is selected, a 24-well plate is taken as an example in the experiment, and a specific system is shown in Table 18; after preparing a transfection system according to the table, swirling for 10s, centrifuging briefly, and standing for 10min;

TABLE 18 transfection System

Reagent(s)	Dosage of
		jetPRIME buffer	50μL
Total amount of plasmid DNA	500ng
		jetPRIME	1μL

(2) The cells were passaged, 293T cells with good growth state were collected, the supernatant of the culture solution was carefully aspirated and washed twice with 1xPBS, and then digested with pancreatin for 2min, and 2mL of complete medium was added to terminate the reaction at 1000rpm/5min. Gently resuspended with 1mL of complete medium;

(3) Adding 450 mu L of fresh DMEM complete culture medium and a proper amount of cell suspension into each hole in a 24-hole plate to enable the density of the fresh DMEM complete culture medium to reach about 60% in the next day, adding prepared reaction liquid into each hole, slightly shaking a culture dish to enable the liquid to be uniformly mixed, and putting the mixture into a cell culture box for culturing for 72 hours;

(4) Using Dual-purpose-Reporter Assay System kit for dual luciferase reporter gene detection, discarding supernatant, washing twice with PBS;

(5) 100 mu L of PLB lysate is added into each hole, and the mixture is placed in a shaking table at 120rpm for 10min at room temperature for cracking; after the liquid in the holes is blown uniformly by using a liquid transfer device, respectively sucking 20 mu L of the liquid into a white totally opaque 96-hole ELISA plate;

(6) Adding 80 mu L of LAR II diluted by 4 times in volume with PBS into each hole in the plate, placing the LAR II in an enzyme-labeled instrument, and detecting the firefly luciferase activity at full wavelength;

(7) After the detection is finished, 80 mu L of 1 xStop & GLO Reagent is immediately added, and after being blown and evenly mixed by a liquid transfer device, the mixture is placed in an enzyme-labeled instrument, and the activity of the Renilla luciferase is detected in full wave;

(8) Taking the read Renilla luciferase activity as an internal reference, and obtaining the luciferase activity of each group by comparing the firefly luciferase activity with the Renilla luciferase activity;

(9) The luciferase activities of the control groups were normalized, and the relative luciferase activities of each experimental group were determined. And counting.

20 Fluorescence In Situ Hybridization (FISH) and immunofluorescence

20.1 fluorescence in situ hybridization

(1) Construction of LCDR specific probes: constructing a 100bp probe (the nucleotide sequence of which is shown as SEQ ID NO: 42) specifically combined with LCDR to PCDNA3.1 (+), carrying out single enzyme digestion on the plasmid, carrying out in vitro transcription by using a T7 promoter of the PCDNA3.1 (+), and carrying out digoxin marking;

(2) Uniformly spreading target cells into a confocal small dish in advance, wherein the density is 60% in the next day;

(3) Removing the culture medium, gently cleaning the confocal dish for 3 times by using PBS (phosphate buffer solution) prepared by DEPC (DEPC) water, avoiding blowing up cells, adding 4% paraformaldehyde, and fixing for 20min at room temperature;

(4) Removing formaldehyde, adding PBS prepared by DEPC water, and washing for 10min in a shaking table for 3 times;

(5) The medium was aspirated off, the confocal dish was dried slightly at room temperature, and then the bottom of Jiao Xiao dish was covered with pancreatin diluted 1:9 for digestion;

(6) Absorbing and discarding pancreatin, adding PBS prepared by DEPC water, and washing with shaking table for 3 times each for 5min;

(7) Adding 0.5% (g/100 mL) Triton-100 (containing 1% RNase inhibitor, g/100 mL), standing on ice for 10min, removing Triton-100, adding DEPC water-formulated PBS, and washing with shaking table for 3 times each for 5min;

(8) Absorbing and discarding PBS, slightly airing at room temperature, adding a prehybridization solution to cover the bottom of a Jiao Xiao dish, and placing the dish in a very constant temperature box at 52 ℃ for 2 hours;

(9) Adding 100pmol of LCDR specific probe into the prehybridization solution, and hybridizing for 18h;

(10) After hybridization is completed, absorbing prehybridization liquid, adding 2 XSSC preheated at 52 ℃ in advance, washing for 3 times, and 5 minutes each time;

(11) Adding deionized formamide in an equal volume into 4 XSSC, preheating at 52 ℃ for 3 times, and 15 minutes each time;

(12) Adding 1% PBST for washing 5 times for 5min each time;

(13) PBST is absorbed, 10% goat serum is added for sealing for 1 hour at room temperature, alexa Fluor 647-conjugated IgG Fraction Monoclonal Mouse anti-digoxin anti-ibody secondary antibody which is diluted 500 times by the goat serum is added, and the mixture is placed in a refrigerator at 4 ℃ for overnight incubation;

(14) Absorbing and discarding the secondary antibody, adding PBS (phosphate buffer solution) prepared by DEPC (DEPC) water, and placing the mixture in a shaking table for washing for 3 times, wherein each time is 5min;

(15) 4',6-diamidino-2-phenylindole (DAPI) of PBS prepared by adding DEPC water is covered at the bottom of Jiao Xiao dishes for copolymerization, the mixture is left at room temperature for 5min, DAPI is sucked and removed, and the mixture is washed for 3 times each for 5min;

(16) Observations were made using a confocal microscope, with corresponding results obtained.

20.2 immunofluorescence

(1) PBS soaks the cell climbing sheet for 3 times, each time for 5min;

(2) Sucking off PBS, and fixing 4% paraformaldehyde at room temperature (or using cold methanol-20deg.C or cold acetone with permeation effect at the same time) for 15min;

(3) Sucking the fixative, soaking and washing with PBS for 3 times and 5min each time;

(4) Sucking PBS,0.5%Triton X-100 (PBS) to permeate for 20min at room temperature;

(5) Absorbing the permeabilizing agent, soaking and washing for 3 times with PBS for 5min each time;

(6) Sucking the PBS, dripping a sealing buffer solution on the glass slide, and sealing for 30min at room temperature;

(7) Diluting the primary antibody with an antibody dilution buffer according to instructions of the instructions before the end of the blocking;

(8) Sucking off the blocking solution, not washing, dripping enough diluted primary antibody into each slide, and placing into a wet box for incubation at 4 ℃ for overnight;

(9) Taking out the wet box from the temperature of 4 ℃, and rewarming for 15min at room temperature;

(10) The primary antibody was blotted off and PBST (PBS containing 1% Tween 20) was washed 3 times for 5min each;

(11) The water absorption paper absorbs the excessive liquid on the climbing sheet, the diluted fluorescent secondary antibody is dripped, and the water absorption paper is incubated in a wet box for 1h at 37 ℃;

(12) PBST is soaked and washed for 3 times, each time for 5min;

(13) Counterstaining and checking: dripping DAPI, incubating for 5min in dark place, and carrying out nuclear staining on the specimen;

(14) PBST is soaked and washed for 3 times, each time for 5min;

(15) A small drop of sealing liquid containing anti-fluorescence quenching agent is dropped on the glass slide, the climbing sheet is taken out from the hole, the water absorbing paper absorbs the redundant liquid, and the surface of the water absorbing paper is covered on the sealing liquid downwards so as to be contacted with the glass slide;

(16) The slide was left in the dark for 5min to air dry, observed under a fluorescence microscope and the image was collected.

21 Immunohistochemistry (IHC)

(1) The chip is purchased in the Shanghai core, the chip is placed in a 65 ℃ oven for processing for about 1h, the chip is observed to be fixed on the chip, the wax begins to fall off, and the corresponding information of the chip is shown in a table 19;

TABLE 19 chip information

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(2) Soaking the chip in xylene for 10min, and repeating soaking for three times until all wax is completely removed;

(3) Rehydrating the dewaxed chip: soaking the chip in 100vt% ethanol, 95vt% ethanol, 75vt% ethanol at room temperature (25deg.C) for 10min;

(4) Soaking in PBS for 5min, and repeating for 3 times;

(5) Antigen retrieval: preparing 500mL sodium citrate repairing liquid, and injecting the chip into the sodium citrate repairing liquid in a boiling state for 30min;

(6) When boiling is completed, naturally cooling the mixture at room temperature for 3 hours;

(7) Soaking in PBS for 5min, and repeating for 3 times;

(8) Using an immunohistochemical pen to encircle the tissue site, 3% hydrogen peroxide (hydrogen peroxide, H) was added dropwise ₂ O ₂ ) Blocking endogenous peroxidase, standing at room temperature for 10min, and washing with PBS for 3 times each for 5min;

(9) Closing: adding 10vt% goat serum to cover the surface of the tissue, and placing the tissue in a refrigerator at 4 ℃ for overnight;

(10) Absorbing and discarding the blocking solution, washing for 5min by PBS, adding primary antibodies of hnRNP K and LAPTM5 diluted in a ratio of 1:200, and placing in a refrigerator at 4 ℃ for overnight;

(11) Absorbing and discarding the primary antibody, rewarming at room temperature for 5-10min, washing with PBS for 3 times and 5min each time, adding the corresponding secondary antibody, washing with PBS for 45min at room temperature and 3 times and 5min each time;

(12) Preparing DAB color developing agent, dripping the DAB color developing agent on a chip, and observing the color change of the chip;

(13) After the color development is completed, placing the chip in PBS, and stopping the color development;

(14) Adding hematoxylin for counterstaining for about 1min, placing in PBS for stopping dyeing, adding ammonia water for blue-turning, and airing at room temperature;

(15) The neutral resin seals were observed under a fluorescence microscope and images were collected.

22 statistical analysis

Three independent experimental replicates were performed for each set of data, data expressed as mean±standard deviation (Standard Deviation, SD) or standard error (Standard Error of Mean, SEM) ±mean (Mean). The comparison between the control group and the experimental group was performed using Mann-Whitney test or Student's t; correlation analysis of the expression levels of the two molecules was performed using the Spearman method. Experimental data were analyzed using GraphPad or R language software, p <0.05 indicated that differences were statistically significant, and ns (no significant) indicated that p <0.05, p <0.01, p <0.001 were used when differences between groups were not statistically significant.

(II) results of experiments

Gene expression profile of histone acetylation control

To study the gene expression profile of histone acetylation regulation, BEAS-2B and NCI-H1299 cells were treated with koji Gu Junsu (Trichostatin A, TSA) and dimethyl sulfoxide (dimethyl s. Mu. Lfoxide, DMSO), respectively (TSA and DMSO were added to the culture medium of BEAS-2B and NCI-H1299 cells, respectively, at a final concentration of 300nM,37 ℃, 5vt% CO ₂ Incubator for 24 h), then the treated cells were digested, added Trizol to extract RNA, and transcriptome sequenced. BEAS-2B and NCI-H1299 sequencing data for TSA treatment was obtained by thresholding: p is p<0.05 and |log2FC|gtoreq.0.58, 10971 and 5107 differentially expressed genes were screened, respectively (FIGS. 3A-3B). Subsequent pathway enrichment of the differential genes of BEAS-2B and NCI-H1299 by the kyoto and genomic encyclopedia (Kyoto Encyclopedia of Genes and Genomes, KEGG) was performed (FIGS. 3C-3D), while the genes differentially expressed by BEAS-2B and NCI-H1299 were crossed, which showed 3095 crossed differentially expressed genes (FIG. 3E), 1877 co-up-regulated genes, 745 co-down-regulated genes, accounting for 23.8% of all differentially expressed genes, and database analysis of KEGG showed significant enrichment of these crossed differentially expressed genes in the "transcriptional dysregulation" pathway, which just supported the role of histone acetylation in transcriptional regulation of gene expression (FIG. 3F).

2 histone acetylation modulation and screening of lncRNAs differentially expressed during cancerous processes

In order to identify the histone acetylation regulation and simultaneously differentially express lncRNAs between cancers and beside cancers in the process of canceration, the differential expression lncRNAs after the TSA treatment and the differential expression lncRNAs downloaded by TCGA databases are subjected to systematic analysis (searching for the lncRNAs which are co-up and down regulated) for lung cancer, renal clear cell carcinoma, cholangiocarcinoma, liver cancer, gastric cancer and squamous cell carcinoma of head and neck gland, 24 lncRNAs which are differentially expressed and are co-directionally changed are screened (figure 4A), and H4K16ac and H3K27ac are known to be regulatory gene transgenes The important factors recorded, but experiments found that TSA inhibitors were only able to increase the expression of H3K27ac in the lung cell line, but not H4K16ac (fig. 4B), so that the differentially expressed and co-directionally altered lncRNAs and NCI-H1299H 3K27ac CHIP seq data described above were further integrated (intersection of the differentially expressed and co-directionally altered lncRNAs with NCI-H1299H 3K27ac CHIP seq enriched genes) (fig. 4C), and AS a result showed that only 7 lncRNAs could be significantly enriched by H3K27ac (fig. 4D), by further analysis of these genes, where the functions of MATN1-AS1, SNHG12, cat1, NKILA, MNX1-AS1 have been annotated, and finally focused on the role of LCDR in lung cancer. Furthermore, BEAS-2B, NCI-H1299 and HCC827 were treated with different concentrations of TSA (final concentrations of 0nM and 300 nM) (TSA was added to the medium of BEAS-2B, NCI-H1299 and HCC827 cells, 37 ℃, 5vt% CO ₂ Incubator incubation for 24H) showed significant increases in LCDR transcript levels (fig. 4E-4G) and H3K27ac levels at LCDR promoter enrichment (fig. 4H-4J) following TSA treatment.

Molecular characterization of 3LCDR

To further understand the biological function of LCDR, the full length nucleic acid sequence of LCDR with nucleotide sequence shown in SEQ ID NO. 1 (FIG. 5A) was first amplified by 3 'RACE and 5' RACE techniques, and LCDR was found to be a full length 2013bp with only one exon lncRNA, the size of which was further confirmed by Northern blot experiments (FIG. 5B). The sequence of LCDR was then entered into website Human BLAT Search (http:// genome. Ucsc. Edu/cgi-bin/hgBlat) and LCDR was found to be on chromosome 20 (FIG. 5C). By designing specific primers through the identified full-length sequences, the relative expression amount of LCDR in different lung cancer cell lines and paracancerous tissues is detected through qRT-PCR, and the qRT-PCR result shows that the expression amount of LCDR in the cancerous tissues is relatively high (FIG. 5D), and the result is further confirmed by absolute quantitative PCR (FIG. 5E).

To understand the coding potential of LCDR, the sequence of LCDR was input to website Coding Potential Calc. Mu. Lator (http:// cpc. Cbi. Edu. Cn /) for prediction by using a comparative genomics approach to distinguish non-coding and protein coding regions, while PCSEAT, HOTAIR long-chain non-coding RNA, which has been reported, was used as positive control, GAPDH, ACTB coding genes were used as negative controls, and LCDR coding capacity of-1.17724, PCSEAT coding capacity of-0.786637, HOTAI coding capacity of-0.534273, GAPDH coding capacity of 2.0942, ACTB coding capacity of 2.95081 were found to be essentially absent from LCDR (FIG. 6A). Modification of the LCDR promoter regions H3K4me1, H3K4me2, H3K4me3, H3K9ac, H3K9me3, H3K27ac, and H3K36me3 were obtained via the Encyclopedia pf DNA Elements (code) database (fig. 6B). Analysis by the UCSC Genome Browser database showed that LCDR was relatively poorly conserved across species (fig. 6C).

The localization of lncRNA may be indicative of its function, thus further understanding of LCDR subcellular localization. Firstly, a specific probe (the nucleotide sequence is shown as SEQ ID NO: 42) is designed according to the prediction of a website, the LCDR is mainly located in the nucleus in an NCI-H1299 cell line through secondary antibody development, meanwhile, along with the reduction of the expression quantity of the LCDR, the fluorescence signal of the probe is correspondingly weakened (figure 7A), then, through a nucleoplasm separation experiment, the GAPDH is mainly located in the cytoplasm, the Histone H3 is mainly located in the nucleus, and meanwhile, the LCDR of a target gene is mainly located in the nucleus (figure 7B), so that the experimental result of in-situ hybridization is further verified.

4c-Jun regulates transcription of LCDR

To further elucidate the molecular mechanisms driving LCDR expression in lung cancer cells, significant c-Jun enrichment of the LCDR promoter region was found by ENCODE database prediction (fig. 8A). To verify the predicted results of the website, the change in LCDR expression level was detected using lentiviral infection NCI-H1299 knock-down C-Jun (FIG. 8B), or overstable C-Jun (FIG. 8C). The results show that knockdown of c-Jun inhibited LCDR expression, whereas table c-Jun promoted LCDR expression. Next, by analyzing GSE92783 sequencing data, two potential binding sites BS1: GAGGCGGAAGTG and BS2: GGTTTCCGGGGT were predicted (FIG. 8D). Subsequent validation by CHIP experiments showed that c-Jun could significantly enrich the LCDR promoter compared to control IgG (fig. 8E). Meanwhile, according to the predicted binding sites of the website, namely-454 bps to-465 bps and-500 bps to-511 bps, the two regions are subjected to the picture-inserting mutation (the mutation sequence is shown in fig. 8F), and the c-Jun over-expression can enhance the double luciferase activity of the wild type and mut2 LCDR promoter regions without affecting the double luciferase activity of the mut1 LCDR promoter region (fig. 8G). Furthermore, the results of CHIP-qPCR showed that c-Jun could be more enriched in the promoter region of LCDR after TSA treatment (FIG. 8H), activating transcription of LCDR. Taken together, these data indicate that c-Jun regulates transcription of LCDR.

5-knockdown LCDR induces cell death

The above experimental results revealed that LCDR was highly expressed in lung cancer, possibly a potential oncogene, and specific shRNA and siRNA were designed for LCDR (primer sequences see table 20) in order to further verify molecular function of LCDR in lung cancer, while constructing LCDR on plvx-IRES-NEO vector, qRT-PCR showed knockdown efficiency of LCDR (fig. 9A-9B) and overstable fold of LCDR (fig. 9C).

Knocking down LCDR expression resulted in a significant down-regulation of the proliferative capacity of lung cancer cell line NCI-H1299 (fig. 10A), and over-expression of LCDR could promote proliferation of lung cancer cell line NCI-H1299 (fig. 10B).

Knocking down LCDR expression resulted in significant downregulation of the clonogenic capacity of lung cancer cell line NCI-H1299 (fig. 11A), and over-expression of LCDR could significantly enhance the clonogenic capacity of lung cancer cell line NCI-H1299 (fig. 11B).

In addition, knockdown LCDR cells were found to exhibit morphological changes and apoptosis-like features such as cell shrinkage, rounding, blebbing and shedding (fig. 12A-12B). To accurately assess the rate of apoptosis, staining was performed by FITC-Annexin V, which specifically binds to phospholipides that evert early in apoptosis, and 7-AAD, which specifically binds to nuclear nucleic acids in late in apoptosis. Flow cytometry results showed: the proportion of knockdown LCDR cells apoptosis was significantly increased compared to the control group.

To further illustrate the effect of LCDR on apoptosis, subcellular localization of cytochrome C was examined by immunofluorescence, and the results showed that lentiviral packaging shRNA knockdown LCDR, cytochrome C released from mitochondria to whole cytoplasm (fig. 13A-13B), and subsequently expression of apoptosis-critical protein caspase3 and PARP was examined, and western blot results showed that knockdown LCDR, cleaned caspase3 and cleaned PARP were all detected (fig. 13C-13D). Taken together, these data indicate that knockdown of LCDR results in apoptosis of lung cancer cells.

To further investigate the effect of LCDR expression on lung adenocarcinoma cell tumor formation ability, LCDR was knocked down by shRNA system to obtain a low LCDR-expressing NCI-H1299 cell line, and the above control group and the low LCDR-stable NCI-H1299 cell line were injected subcutaneously (into the armpit) with the same number of cells, and as a result, it was shown that lowering LCDR significantly reduced lung adenocarcinoma cell NCI-H1299 tumor formation ability (fig. 14A-14C). Meanwhile, tumors taken out from the armpit of the mouse are sliced, embedded and immunohistochemical experimental results show that: the protein levels of clear caspase3 were significantly up-regulated compared to control groups (FIGS. 14D-14E). Taken together, these data indicate that LCDR promotes survival of lung cancer cells.

TABLE 20 qRT-PCR primers

6LCDR specific binding to hnRNP K

A number of documents suggest that lncRNA itself is essentially nonfunctional and it exerts its biological functions by forming secondary structures that bind to proteins, DNA and RNA. To find proteins to which LCDR might bind, LCDR and the reverse complement of LCDR were constructed on PCDNA3.1 vectors, transcribed in vitro using the T7 promoter, and then labeled with biotin. The prepared reverse complement was incubated with NCI-H1299 protein lysate overnight at a final concentration of 3pmol, and the incubated complex was separated by magnetic beads and eluted. By protein gel electrophoresis and silver staining techniques, a specific band was found to exist on the sense strand of LCDR around 60KD relative to the antisense strand (fig. 15A). Protein mass spectrometry technology identified the amino acids of a specific band, and finally identified that the protein was likely hnRNP K by comparing the size of the protein, the number of peptide fragments matched by the protein and the number of specific peptide fragments, see Table 21. The result of western blot verification of LCDR pull down samples showed that this band was hnRNPK and an LCDR specific binding protein molecule (fig. 15B). To further verify the experimental results of RNA pμl down, reverse validation was performed by RIP experiments, which showed that hnRNP K was able to specifically bind LCDR (fig. 15C). LCDR in situ hybridization and hnRNP K immunofluorescence showed that LCDR and hnRNP K coexist in the nucleus, further supporting the interaction therebetween (fig. 15D).

To further identify the binding site of hnRNP K to LCDR, a truncated LCDR was first constructed: the first segment is 1-500bp; the second segment is 500-1000bp; the third section is 1000-1500bp; fourth 1500-2013bp. Truncated LCDR was constructed into PCDNA3.1 vector, labeled with biotin by T7 promoter external transcription, incubated with NCI-H1299 protein lysate, and hnRNP K was shown to bind to 1-500bp LCDR by western blot (FIG. 16A). To further identify the specific base binding to hnRNP K, the secondary structure of LCDR was predicted by Mfold and RNAfold sites, and a potential nucleic acid sequence for binding hnRNP K to LCDR was selected: CCCCCCACC (fig. 16B). Assuming that this poly (C) site is essential for hnRNP K binding, the binding site was mutated (mutation site see FIG. 16C) and verified in reverse by RNA pμLlDown experiments, which showed a significant down-regulation of the binding capacity of mutant LCDR to hnRNP K compared to the control (FIG. 16D). Consistent with RNA pμl down results, gel migration blocking experiments (electrophoretic mobility shift assay, EMSA) showed that the binding capacity of mutant LCDR probes to hnRNP K protein was significantly down-regulated compared to the control group (fig. 16E). And finally, carrying out reverse verification through RIP experiments, wherein the result shows that: the cross-table LCDR enhanced the inter-binding to hnRNP K compared to the control group, and the mutant LCDR attenuated the inter-binding to hnRNP K (fig. 16F).

hnRNP K mainly comprises 3 domains, in order to identify the domain of LCDR binding to hnRNP K, the hnRNP K protein was truncated (truncation process see FIG. 17A), and tag protein Flag was attached at the end of the truncate, and truncated hnRNP K was expressed by lentiviral packaging (FIG. 17B). Incubation with Flag beads with truncated protein lysates by RIP experiments showed that LCDR binds to KH1 of hnRNP K (fig. 17C). Taken together, these data indicate that LCDR and hnRNP K bind to each other.

Table 21 protein mass spectrometry identification of LCDR potential binding proteins

7-knockdown hnRNP K-induced cell death

Because of the interaction between LCDR and hnRNP K, it was speculated that hnRNP K may have a consistent effect on cancer cell survival, two specific siRNA were designed (siRNA sequences see table 22), while hnRNP K was constructed on plvx-IRES-NEO overexpression vectors, qRT-PCR showed knockdown efficiency of hnRNP K (fig. 18A) and fold over-expression of hnRNP K (fig. 18B).

Knocking down hnRNP K resulted in a significant down-regulation of proliferation capacity of lung cancer cell line NCI-H1299 (FIG. 19A), and overexpression of hnRNP K promoted proliferation of lung cancer cell line NCI-H1299 (FIG. 19B).

Knocking down hnRNP K resulted in a significant down-regulation of the clonogenic capacity of lung cancer cell line NCI-H1299 (FIG. 20A), and over-expression of hnRNP K promoted the clonogenic capacity of lung cancer cell line NCI-H1299 (FIG. 20B).

The knockdown of hnRNP K resulted in the appearance of a distinct apoptosis-like feature in NCI-H1299 cells under the light microscope (FIG. 21A), the flow cytometry detection results showed a significant increase in the proportion of apoptotic cells compared to the control group (FIGS. 21B-21C), the immunofluorescence results showed that knockdown of hnRNP K, cytochrome C released from mitochondria to cytoplasm (FIG. 21D), and the western blot results showed that knockdown of hnRNP K, clear caspase3 and clear PARP were all detected (FIG. 21E). Taken together, these data indicate that hnRNP K is essential for survival of lung cancer cells.

8LCDR and hnRNP K are not mutually influenced in expression, positioning and function

Knocking down LCDR and hnRNP K both significantly induced cell death, LCDR functions as lncRNA through hnRNP K? To further investigate the correlation between LCDR and hnRNP K, it was first determined whether their expression levels and localization interacted. Through immunofluorescence technology, LCDR and hnRNP K are combined and positioned in cell nucleus (blue represents the expression and positioning of hnRNP K, red represents the expression and positioning of LCDR, and the LCDR can be seen to be positioned in cell nucleus), so that the expression quantity of LCDR is reduced, and the fluorescence intensity and positioning of hnRNP K are not changed; the decrease in hnRNP K expression did not change the fluorescence intensity and localization of LCDR (FIG. 22A). The levels of transcripts of hnRNP K and protein were unchanged by knocking down LCDR (FIGS. 22B-22C), and also were unchanged by knocking down hnRNP K and LCDR (FIGS. 22D-22E).

Next, a rescue experiment was performed to explore the effect of LCDR/hnRNP K axis on lung cancer cell proliferation capacity. Knocking down LCDR in the hnRNP K stabilized cell line (constructing a cell line stably and highly expressing hnRNP K by using a lentiviral infection system, and then knocking down the expression of LCDR by using siRNA), and the result shows that the effect of cell proliferation caused by knocking down LCDR cannot be saved (FIG. 23A); the results of knocking down hnRNP K in the stable cell lines of the overstable LCDR (construction of stable high-expression LCDR cell lines using lentiviral infection system, followed by knocking down the expression of hnRNP K using siRNA) showed that the effect of cell proliferation caused by knocking down hnRNP K could not be saved (FIG. 23B). Taken together, these results suggest that LCDR and hnRNP K may synergistically regulate proliferation of lung cancer cells.

Table 22 qRT-PCR primers

9LCDR and hnRNP K do not affect expression of apoptosis-related genes

Numerous literature reports that apoptosis is primarily regulated by Bcl2 family related genes. Thus, the knockdown of LCDR and hnRNP K (48 h of transfected cells with siRNA, knockdown of LCDR and hnRNP K expression, respectively) was examined for changes in transcript and protein levels of apoptosis-related genes, and the results showed that none of the knockdown of LCDR, hnRNP K had significantly changed in Bax, bak, bid, mcl1, bcl2 and Bcl-xl transcripts and protein levels compared to the control group (fig. 24A-D), indicating that the LCDR/hnRNP K axis may regulate apoptosis of cells through other signaling pathways.

10LCDR and hnRNP K together regulate lysosomal pathway

Given that the LCDR/hnRNP K axes coordinate to regulate cell death, it is assumed that LCDR and hnRNP K bind to each other to regulate expression of part of the gene together. To verify this hypothesis, RNA sequencing was performed on NCI-H1299 cells knocked down for LCDR and hnRNPK (NCI-H1299 cells were transfected with siRNA for 48H, followed by harvesting of the cells, RNA extraction and transcriptome sequencing), p <0.05 was defined as a differentially expressed gene for the sequencing data knocked down for LCDR and hnRNP K, resulting in a total of 9527 differentially expressed genes knockdown for LCDR (FIG. 25A), 9901 differentially expressed genes knockdown for hnRNP K (FIG. 25B), and KEGG performed pathway enrichment analysis to find that LCDR differentially expressed genes were mainly enriched in proteolytic (focal addition), adhesion (focal addition), variable cleavage (splice), endocytosis (endocytosis), lysosomes (lysome) and other signaling pathways (FIG. 25C); the hnRNP K differentially expressed gene was mainly concentrated in purine metabolism (purine metabolism), actin cytoskeletal regulation (reg. Mu. Lation of actin cytoskeleton), MAPK signaling pathway (MAPK signaling pathway), insulin signaling pathway (ins. Mu. Lin signaling pathway), lysosome (lysoname) and other signaling pathways (FIG. 25D). Next, the sequencing data of LCDR and hnRNP K were intersected to find that there were 4066 genes which were differentially expressed together in total (FIG. 25E-F), and the KEGG pathway was enriched for these genes, and the analysis found that these genes were mainly enriched in p53 signaling pathway (p53_signaling_pathway), small cell lung cancer (small cell lung cancer), cell cycle (cell cycle), lysosome (lysoname), actin cytoskeleton (reg. Mu. Lation of actin cytoskeleton) and other signaling pathways (FIG. 25G). Through comprehensive analysis of the above pathway enrichment, 9 co-regulated signal pathways exist between LCDR and hnRNPK, which indicates that functional interaction exists between LCDR and hnRNP K. Lysosomal cell death, also known as lysosomal dependent cell death, is a regulated cell death mediated by hydrolytic enzymes released into the cytoplasm after changes in lysosomal membrane permeability. We speculate that LCDR/hnRNP K may co-regulate the lysosomal signaling death pathway. Through further analysis of the lysosomal signaling pathway, 6 genes with the largest variation in the lysosomal pathway compared to the control group were selected as candidate genes: ARSB, ASAH1, HNSGAT, LAPTM5, ACP2, GLB1 (FIG. 25H-I). To further verify the sequencing data, the LCDR and hnRNP K knockdown was examined for changes in transcripts of candidate genes (FIG. 25J-K), and qRT-PCR results showed that 5 of the 6 candidate genes knockdown LCDR and hnRNP K could be verified.

Notably, lapm 5 is expressed on lysosomal membranes. Consistent with transcript levels, knock-down of LCDR, hnRNP K resulted in down-regulation of protein levels of lapm 5 (fig. 26A-B), and corresponding overstable LCDR, hnRNP K resulted in up-regulation of transcript levels of lapm 5 (fig. 26C-D) and protein levels (fig. 26E-F). In addition, immunofluorescence showed that both endogenous and overexpressed LAPTM5 could co-localize with LAMP1 (FIG. 26G-H), indicating that LAPTM5 was expressed on lysosomal membranes and regulated by the LCDR/hnRNPK axis.

11-knockdown LAPTM5 induced cell death

The above results indicate that LAPTM5 expression is regulated by LCDR/hnRNP K axis, presumably LAPTM5 may have a consistent effect with LCDR/hnRNP K, thus two specific siRNA sequences were designed (siRNA sequences see Table 23) while LAPTM5 was constructed on plvx-IRES-NEO overexpression vectors, qRT-PCR showed the knock-down efficiency of LAPTM5 (FIG. 27A) and the fold over-expression of LAPTM5 (FIG. 27B).

Knocking down LAPTM5 resulted in a significant down-regulation of proliferation capacity of lung cancer cell line NCI-H1299 (FIG. 28A), and over-expression of LAPTM5 could promote proliferation of lung cancer cell line NCI-H1299 (FIG. 28B) (transfection of NCI-1299 48H with siRNA targeting LAPTM5, knocking down expression of LAPTM 5; construction of full length LAPTM5 sequence to plvx-IRES-NEO followed by lentiviral packaging and infection, over-expression of LAPTM 5).

Knocking down lapm 5 resulted in a significant down-regulation of the clonogenic capacity of lung cancer cell line NCI-H1299 (fig. 29A), and overexpression of lapm 5 promoted the clonogenic capacity of lung cancer cell line NCI-H1299 (fig. 29B).

The knockdown of LAPTM5 resulted in a similar apoptosis-like morphology change of NCI-H1299 cells under the light microscope as LCDR and hnRNP K (FIG. 30A), flow cytometry results showed a significantly increased proportion of apoptotic cells compared to control (FIG. 30B-C), immunofluorescence results showed release of knockdown LAPTM5, cytochrome C from mitochondria to cytoplasm (FIG. 30D), and western blot results showed that knockdown LAPTM5, clear caspase3 and clear PARP were all detected (FIG. 30E).

TABLE 23 qRT-PCR primers

12LCDR, hnRNP K and LAPTM5 together maintain lysosomal membrane integrity

Based on the consistent effect of the LCDR/hnRNP K/LAPTM5 axis on cell survival, it was speculated that the LCDR/hnRNP K/LAPTM5 axis might regulate cell survival by co-maintaining lysosomal membrane integrity. Thus, the integrity of lysosomal membranes was assessed by immunofluorescent staining of lysosomal dye (Lyso Tracker) (fig. 31A), key protein LAMP1 of lysosomal membrane, and CTSB in lysosomal (fig. 31B), showing that knocking down LCDR, hnRNP K, and lap 5 all resulted in Lyso Tracker, LAMP1 and CTSB becoming diffuse from aggregation, while co-localization of LAMP1 and CTSB was significantly reduced compared to control; consistent with immunofluorescence results, scanning electron microscopy showed that the membrane integrity of knockdown LCDR, hnRNP K and lapm 5 lysosomes was compromised, with significant fragmentation occurring (fig. 31C). Taken together, these data indicate that the LCDR/hnRNP K/LAPTM5 axes collectively regulate lysosomal membrane integrity.

LCDR and hnRNP K regulate cell death by LAPTM5

In view of the above findings, it became critical to determine whether LAPTM5 is a critical downstream effector of the LCDR/hnRNP K axis for this study. First, a rescue experiment of cell proliferation and clonogenic capacity was performed, and the results showed that the inhibition of lung adenocarcinoma cell proliferation and clonogenic inhibition by knockdown LCDR or hnRNP K could both be partially rescued by passing through table lapm 5 (fig. 32A-D). Consistent with the results of cell proliferation and colony formation, flow cytometry showed that overexpression of LAPTM5 could partially rescue apoptosis caused by knockdown of LCDR or hnRNP K (FIGS. 32E-H). Studies have shown that cathepsins play a major role in promoting lysosomal cell death, and blocking cathepsin activity can inhibit lysosomal cell death (Tang, kang, berghe, vandenabeele, & Kroemer, 2019). CA-074Me is a highly selective CTSB (Cathepsin BCTSB) inhibitor (Bogyo et al, 2000) and thus the blocking effect of CTSB inhibitors on cell death following depletion of LCDR/hnRNP K/LAPTM5 was investigated, and the results showed that CA-074Me could partially rescue apoptosis caused by knockdown of LCDR, hnRNP K and LAPTM5 (FIGS. 32I-N).

To further investigate the effect of LCDR/hnRNP K/lapm 5 axis on the tumor formation capacity of lung adenocarcinoma cells, rescue experiments of subcutaneous tumors in mice were performed, and the results showed that knockdown of LCDR or hnRNP K inhibited lung adenocarcinoma cell tumor formation, both could achieve partial rescue by passing through table lapm 5 (fig. 33A-C).

Meanwhile, the taken tumor is sliced, embedded and the result of immunohistochemical experiments shows that: compared with the control group, the knock-down of the expression level of clear caspase3 in the cytoplasm of LCDR was up-regulated, and the overstable LAPTM5 promoted a significant return of the expression level of clear caspase3 (FIGS. 34A-B). Taken together, these data indicate that LCDR and hnRNP K promote cell survival through lapm 5.

14LCDR and hnRNP K together regulate and control stability of LAPTM5

HnRNP K acts primarily through binding to target RNA, and to determine if HnRNP K binds to lapm 5, genes of LCDR RNAseq, hnRNP K RNA seq, hnRNP K RIP-seq, and HnRNP K-eCLIP were subsequently subjected to intersection analysis, together obtaining 398 co-regulated genes (fig. 35A), shown by KEGG for pathway enrichment: 398 genes could be efficiently enriched for signal pathways such as lysosomes (lysoname) (FIG. 35B). Meanwhile, hnRNP K RIP-seq and hnRNP K-eCLIP data analysis shows that hnRNP K can be effectively enriched with LAPTM5 (FIG. 35C), in order to further identify the binding sites of LCDR and hnRNP K and LAPTM5, LCDR and hnRNP K are respectively co-transfected with a coding sequence (CDS) and a3 'untranslated region (3-untranslated region,3' UTR) of LAPTM5 by a double enzyme report experiment (LCDR and hnRNP K are respectively co-transfected with plasmids of the coding region and the 3 'untranslated region of LAPTM5 for 48h, cells are collected and then luciferase activity is detected), and the result shows that the activity of double enzyme report can be obviously improved by cotransfecting LCDR and hnRNP K with the 3' UTR of LAPTM5 (FIG. 35D). Subsequent analysis of the 3'UTR base sequence revealed that cytosine was repeated at the 91-107 (FIG. 35E) bases of the 3' UTR, indicating that it was likely a potential binding site for hnRNP K. The above binding sites were mutated (binding sites and mutations are shown in FIG. 35F) and constructed into PCDNA3.1 (+) vector, labeled by in vitro transcription and subjected to p.mu.Ll down experiments, which showed that the 3'UTR of LAPTM5 could bind effectively to hnRNP K and that the ability of the mutated LAPTM5-3' UTR to bind to hnRNP K was significantly reduced (FIG. 35G). Simultaneously, LAPTM5-3' UTR and mut LAPTM5-3' UTR probes (probe sequences shown as SEQ ID NO:59 and SEQ ID NO: 60) were incubated with purified hnRNP K protein, and the results of EMSA experiments showed that only LAPTM5-3' UTR could efficiently bind to hnRNP K protein and slow down the migration rate of hnRNP K protein (FIG. 35H). In addition, reverse verification is carried out through RIP experiments, and the results show that: the cross-table lapm 5-3'utr significantly enhanced the inter-binding to hnRNP K compared to the control group, and the binding capacity of mut lapm 5-3' utr to hnRNP K was significantly down-regulated (fig. 35I). Finally, LAPTM5 binding to hnRNP K was also explored using KH domain mutants, and the results of RIP-qPCR showed that the KH3 domain of hnRNP K could be efficiently enriched for LAPTM5 (FIG. 35J). Taken together, these data suggest that the poly (C) site in the 3' utr of lapm 5 can directly interact with the KH3 domain of hnRNP K.

hnRNP K was overexpressed at the knock-down LCDR level of the NCI-H1299 cell line, while LCDR was overexpressed at the knock-down hnRNP K level, and cells were then treated with 5. Mu.M final concentration of Actinomycin D solution (Actinomycin D) after plating in 6 well plates and cells were harvested at 3 different time points (0H, 12H, 24H) for detection of LAPTM5 transcript levels. The above results show that knockdown LCDR and hnRNP K significantly reduced the stability of lapm 5, and that overstable LCDR and hnRNP K significantly improved the stability of lapm 5, but knockdown LCDR overstable hnRNP K, and knockdown hnRNP K overstable LCDR, stability of lapm 5 were not significantly improved (fig. 36A-B). Meanwhile, the detection of the expression level of the LAPTM5 transcripts also obtains a consistent result, the knockdown LCDR and the hnRNP K obviously reduce the expression level of the LAPTM5, the overstock LCDR and the hnRNP K obviously improve the expression level of the LAPTM5, but the knockdown LCDR and the understock hnRNP K are overstock LCDR and the expression level of the LAPTM5 are not obviously up-regulated compared with the knockdown group (FIG. 36C-D). Furthermore, LCDR and hnRNP K were further verified to bind to the 3' utr of lapm 5 by a double enzyme assay technique (fig. 36E-F). Taken together, these data indicate that LCDR and hnRNP K bind to each other to co-regulate the stability of lapm 5.

The above studies have shown that either LCDR or hnRNP K can significantly increase the stability and expression level of lapm 5, and subsequently, the expression of lapm 5 transcripts is regulated by hnRNP K. Results from RIP experiments showed that knocking down LCDR resulted in a significant decrease in hnRNP K enrichment for lapm 5 (fig. 37A); overexpression of LCDR using lentiviral infection system promoted enrichment of hnRNP K for lapm 5 (fig. 37B); the results of RNA pull down experiments showed that hnRNP K knockdown significantly down the binding capacity of LCDR to lapm 5 compared to the control group (fig. 37C); hnRNP K was overexpressed using the lentiviral infection system, and the binding capacity of LCDR to LAPTM5 was significantly up-regulated compared to the control group (FIG. 37D). After treating the RNA pull down sample of LCDR with proteinase K, the recovered RNA was washed with magnetic beads and purified, at which time LCDR was significantly reduced in LAPTM5 binding capacity (FIG. 37D), as demonstrated by the results above, LCDR was dependent on hnRNP K binding to LAPTM5 (FIG. 37E). The GXXG motif in the KH domain of hnRNP K is reported to be the key amino acid sequence that interacts with the RNA poly (C) site (Siomi, choi, siomi, nussbaum, & Dreyfuss, 1994), while LCDR and LAPTM5 bind to the KH1 and KH3 domains of hnRNP K, respectively, thus creating mutants of KH1 and KH3, mutating GXXG in KH1 and KH3 to GEEG (FIG. 37F, mutating GXXG in KH1 domain to GEEG on the basis of wild-type hnRNP K plasmid, the resulting plasmid is mut-KH1, mutating GXXG in KH3 domain to GEEG, and the resulting plasmid is mut-KH 3). Subsequent co-transfection of LCDR with hnRNP K, mut-KH1 and mut-KH3, the results of RIP (fig. 37G), and double luciferase reporter experiments (fig. 37H) indicated that binding of LCDR to KH1 of hnRNP K could significantly enhance binding of hnRNP K KH3 to lapm 5.

EMSA experiments showed that LCDR and lapm 5-3' utr could bind to hnRNP K protein respectively, both incubated with purified hnRNP K protein (LCDR and lapm 5-3' utr were incubated with hnRNP K protein respectively for 30min, followed by running, transferring, and developing the incubated samples), further enhancing the binding to hnRNP K protein (fig. 38A), suggesting that LCDR and lapm 5 ' utr might have a synergistic binding effect with hnRNPK. Incubation of LCDR RNA probe with hnRNP K protein, addition of LAPTM5-3' UTR did not further enhance the inter-binding with hnRNP K protein (FIGS. 38B-38C); LAPTM5 RNA probe incubated with hnRNP K protein, LCDR was added to enhance the binding to hnRNP K protein (FIGS. 38D-38E). Taken together, these data indicate that LCDR promotes hnRNP K binding to lapm 5.

Significance of expression of the 15LCDR/hnRNP K axis in lung adenocarcinoma

The above study has shown that LCDR is a lncRNA with important biological functions, and to further study the clinical significance of LCDR, using 82 pairs of paired samples and 16 tissue chips of single lung adenocarcinoma (tissue chips purchased super from Shanghai core for a total of 180 tissue points including 82 pairs of paired cancer and paracancerous tissues and cancerous tissues with only 16 tissue points), results consistent with cell levels and TCGA databases were obtained by in situ hybridization experiments, with significantly higher expression levels of LCDR cancer tissues than paracancerous tissues (fig. 39A-39B). Meanwhile, based on the set of tissue chips (both LCDR hnRNP K and LAPTM5 staining used the set of tissue chips), the immunohistochemical technique showed that hnRNP K (FIGS. 39C-39D) and LAPTM5 (FIGS. 39E-39F) were also expressed in the cancer tissue in the same amount as compared to the paracancerous tissue.

The above experiments confirm that LCDR interacts with hnRNP K to regulate expression of lapm 5, and subsequently explore whether there is a significant correlation between LCDR and hnRNP K expression. Based on the set of tissue chips, the relative expression amounts of each tissue point LCDR, hnRNP K and LAPTM5 are obtained (after in situ hybridization and immunohistochemical experiments, the staining intensity of the tissue chips is evaluated, that is, the relative expression of each tissue point LCDR hnRNPK and LAPTM5 is obtained), and the linear correlation analysis shows that the LCDR has obvious correlation with the hnRNP K and LAPTM5 (fig. 40A-B) in the tissue expression amounts. There is no correlation between hnRNP K and LAPTM5 expression levels (40C), indicating the complexity of hnRNP K regulation of LAPTM 5. At the same time, the subject working curve (receiver operator characteristic curve, ROC) combined analysis showed greater diagnostic value as well as clinical significance (fig. 40D).

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Sequence listing

<110> Shanxi higher Innovative institute

<120> use of LCDR as therapeutic target for drugs for preventing and/or treating cancer

<160> 66

<170> PatentIn version 3.3

<210> 1

<211> 2013

<212> DNA

<213> artificial sequence

<400> 1

gtcgtctcgt aggcttgaaa ggtaggtgcc cgacattagg gactgtcgca gcccggggaa 60

ttgtccccat ctgctggctt accttgtggg cgtggatccc ctctaacttt cctaattccc 120

gggcggcccg cgggcctgcg gcagattcag ctctcaggac tccactttca gtttcttcca 180

cttctggggc aggctgcttg tcctgccact ctcaggcccc ccaccagaca cggcagcaac 240

agctgttcct aagtgttcaa ctcgctcctt cccgcaaatg tagaagctca aatccctata 300

gcaaattcca tattctatgt cattcttaat gcttccgtct ctctggttga atctagattg 360

gatacaatca gtgatgcaca tttaccagac agaggatgtg cagcgtttac tgttgatttc 420

tctcttttcc tccctatcac ctcagtttag ttccaggatc cacactcctc caccatagtc 480

aggagattca gggaaagttg gcagggttca gctttcaagt tgacccttga tgaaccatgg 540

tcaatcctgt taagaggcgt gaaagccaat cccagccaat gagactgggg agaaatcact 600

gaagaacttg tggaaaacag accctctgga taaaagacac gtgtggggaa aaaggggctt 660

tttaatgtta ttctggatgt ttcccaaaag tactgctgct gccttgcctc cctgacagac 720

actggcctta ggatgacatg cggcaggaag gaaggacgga tctggggttt ttcaagactc 780

actgagatgc acaaggaacc aaccctcaag tagccaactc gttcctgttt gcatgtgtcg 840

actgaaaaga atcaaaataa acaaaatata actatttcta agaggttaca acttgcaggc 900

aggaagcagg cctctacctg aggccataaa gcatactttg aaggagaaaa ggtaggtatt 960

tatgctaggt ggagaggtag gatatacgta ttctgtagca tacaggagaa gcctatgaaa 1020

attcatggag gaaactcgtt agagacacgc actgggctta catatgaaga atatccaaaa 1080

gcggatgacc tgtgttcact ctggggtgaa gatttaacat ttaaatgtat tataattagg 1140

ctgtaaatgc aaaaaggtga aacatagggc acaagttcac gatctgtgca atctccagag 1200

accagtctga tctagcctgc agctggcagt catctgtcaa gaaggggctc tttgtagccc 1260

agagtagctg tcaggttgga accataaaac caaaaggagg gcatcggcgg ttaggtgaag 1320

aaaagaagtc ttttggcctt ttttcctctt caagtcagct tttgataaga tctagtaata 1380

ataaggggat gggtgtggct gcaggtggga catgtcggtt ctctgtcctg tcacagctaa 1440

gaactaagaa ctttagagtc tttcagccat agagggtcct gtcatccatt gaagggatca 1500

gtccattggg attttgccag ttttagcatt gatgctgtgt cctgggaaac ccctgaatac 1560

ttatcatagc agaagttcag ccactgtagc cctacctcag gaccgctttt atgggaaaga 1620

ataattcctt taactgcttg agccatttga gttgtggctg cttttggtac ttgtaataaa 1680

ttgaaagcat cttgaatgtt ctgggacagt aaaagaatgt gcctttgttt ctgacagcaa 1740

ttataaaatc aattatatgt ttatattcta aaaaatgttt tgagtaatgc tgacatcatt 1800

ggaaaagtgc atcacttaga aaatgtatct gtgtgtcaat tttgtttgtt caaaatcagt 1860

ttcattaatt tgtgggaaga tacaccttgt acattttacc taattaaaaa aaatattgcc 1920

agttgagtat acttgtgata tattcaaaga taaataaact ttttcaattt tagatattca 1980

taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 2013

<210> 2

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<213> artificial sequence

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Met Glu Thr Glu Gln Pro Glu Glu Thr Phe Pro Asn Thr Glu Thr Asn

1 5 10 15

Gly Glu Phe Gly Lys Arg Pro Ala Glu Asp Met Glu Glu Glu Gln Ala

20 25 30

Phe Lys Arg Ser Arg Asn Thr Asp Glu Met Val Glu Leu Arg Ile Leu

35 40 45

Leu Gln Ser Lys Asn Ala Gly Ala Val Ile Gly Lys Gly Gly Lys Asn

50 55 60

Ile Lys Ala Leu Arg Thr Asp Tyr Asn Ala Ser Val Ser Val Pro Asp

65 70 75 80

Ser Ser Gly Pro Glu Arg Ile Leu Ser Ile Ser Ala Asp Ile Glu Thr

85 90 95

Ile Gly Glu Ile Leu Lys Lys Ile Ile Pro Thr Leu Glu Glu Gly Leu

100 105 110

Gln Leu Pro Ser Pro Thr Ala Thr Ser Gln Leu Pro Leu Glu Ser Asp

115 120 125

Ala Val Glu Cys Leu Asn Tyr Gln His Tyr Lys Gly Ser Asp Phe Asp

130 135 140

Cys Glu Leu Arg Leu Leu Ile His Gln Ser Leu Ala Gly Gly Ile Ile

145 150 155 160

Gly Val Lys Gly Ala Lys Ile Lys Glu Leu Arg Glu Asn Thr Gln Thr

165 170 175

Thr Ile Lys Leu Phe Gln Glu Cys Cys Pro His Ser Thr Asp Arg Val

180 185 190

Val Leu Ile Gly Gly Lys Pro Asp Arg Val Val Glu Cys Ile Lys Ile

195 200 205

Ile Leu Asp Leu Ile Ser Glu Ser Pro Ile Lys Gly Arg Ala Gln Pro

210 215 220

Tyr Asp Pro Asn Phe Tyr Asp Glu Thr Tyr Asp Tyr Gly Gly Phe Thr

225 230 235 240

Met Met Phe Asp Asp Arg Arg Gly Arg Pro Val Gly Phe Pro Met Arg

245 250 255

Gly Arg Gly Gly Phe Asp Arg Met Pro Pro Gly Arg Gly Gly Arg Pro

260 265 270

Met Pro Pro Ser Arg Arg Asp Tyr Asp Asp Met Ser Pro Arg Arg Gly

275 280 285

Pro Pro Pro Pro Pro Pro Gly Arg Gly Gly Arg Gly Gly Ser Arg Ala

290 295 300

Arg Asn Leu Pro Leu Pro Pro Pro Pro Pro Pro Arg Gly Gly Asp Leu

305 310 315 320

Met Ala Tyr Asp Arg Arg Gly Arg Pro Gly Asp Arg Tyr Asp Gly Met

325 330 335

Val Gly Phe Ser Ala Asp Glu Thr Trp Asp Ser Ala Ile Asp Thr Trp

340 345 350

Ser Pro Ser Glu Trp Gln Met Ala Tyr Glu Pro Gln Gly Gly Ser Gly

355 360 365

Tyr Asp Tyr Ser Tyr Ala Gly Gly Arg Gly Ser Tyr Gly Asp Leu Gly

370 375 380

Gly Pro Ile Ile Thr Thr Gln Val Thr Ile Pro Lys Asp Leu Ala Gly

385 390 395 400

Ser Ile Ile Gly Lys Gly Gly Gln Arg Ile Lys Gln Ile Arg His Glu

405 410 415

Ser Gly Ala Ser Ile Lys Ile Asp Glu Pro Leu Glu Gly Ser Glu Asp

420 425 430

Arg Ile Ile Thr Ile Thr Gly Thr Gln Asp Gln Ile Gln Asn Ala Gln

435 440 445

Tyr Leu Leu Gln Asn Ser Val Lys Gln Tyr Ala Asp Val Glu Gly Phe

450 455 460

<210> 3

<211> 262

<212> PRT

<213> artificial sequence

<400> 3

Met Asp Pro Arg Leu Ser Thr Val Arg Gln Thr Cys Cys Cys Phe Asn

1 5 10 15

Val Arg Ile Ala Thr Thr Ala Leu Ala Ile Tyr His Val Ile Met Ser

20 25 30

Val Leu Leu Phe Ile Glu His Ser Val Glu Val Ala His Gly Lys Ala

35 40 45

Ser Cys Lys Leu Ser Gln Met Gly Tyr Leu Arg Ile Ala Asp Leu Ile

50 55 60

Ser Ser Phe Leu Leu Ile Thr Met Leu Phe Ile Ile Ser Leu Ser Leu

65 70 75 80

Leu Ile Gly Val Val Lys Asn Arg Glu Lys Tyr Leu Leu Pro Phe Leu

85 90 95

Ser Leu Gln Ile Met Asp Tyr Leu Leu Cys Leu Leu Thr Leu Leu Gly

100 105 110

Ser Tyr Ile Glu Leu Pro Ala Tyr Leu Lys Leu Ala Ser Arg Ser Arg

115 120 125

Ala Ser Ser Ser Lys Phe Pro Leu Met Thr Leu Gln Leu Leu Asp Phe

130 135 140

Cys Leu Ser Ile Leu Thr Leu Cys Ser Ser Tyr Met Glu Val Pro Thr

145 150 155 160

Tyr Leu Asn Phe Lys Ser Met Asn His Met Asn Tyr Leu Pro Ser Gln

165 170 175

Glu Asp Met Pro His Asn Gln Phe Ile Lys Met Met Ile Ile Phe Ser

180 185 190

Ile Ala Phe Ile Thr Val Leu Ile Phe Lys Val Tyr Met Phe Lys Cys

195 200 205

Val Trp Arg Cys Tyr Arg Leu Ile Lys Cys Met Asn Ser Val Glu Glu

210 215 220

Lys Arg Asn Ser Lys Met Leu Gln Lys Val Val Leu Pro Ser Tyr Glu

225 230 235 240

Glu Ala Leu Ser Leu Pro Ser Lys Thr Pro Glu Gly Gly Pro Ala Pro

245 250 255

Pro Pro Tyr Ser Glu Val

260

<210> 4

<211> 21

<212> DNA

<213> artificial sequence

<400> 4

aaatcaaagc cacacttagc g 21

<210> 5

<211> 20

<212> DNA

<213> artificial sequence

<400> 5

cgcccgggaa ttaggaaagt 20

<210> 6

<211> 20

<212> DNA

<213> artificial sequence

<400> 6

ttcagccata gagggtcctg 20

<210> 7

<211> 21

<212> DNA

<213> artificial sequence

<400> 7

aagcagccac aactcaaatg g 21

<210> 8

<211> 20

<212> DNA

<213> artificial sequence

<400> 8

atcttcctct tcctccacca 20

<210> 9

<211> 20

<212> DNA

<213> artificial sequence

<400> 9

catcagcact gaaaccaacc 20

<210> 10

<211> 21

<212> DNA

<213> artificial sequence

<400> 10

tccaagtgcc gaaaaaggaa g 21

<210> 11

<211> 21

<212> DNA

<213> artificial sequence

<400> 11

cgagttctga gctttcaagg t 21

<210> 12

<211> 20

<212> DNA

<213> artificial sequence

<400> 12

gagttggtcg gggaatctgg 20

<210> 13

<211> 20

<212> DNA

<213> artificial sequence

<400> 13

ataatctcca gcgactgccg 20

<210> 14

<211> 20

<212> DNA

<213> artificial sequence

<400> 14

gccatcaatg gcaacccatc 20

<210> 15

<211> 20

<212> DNA

<213> artificial sequence

<400> 15

ctttactgct gccatgggga 20

<210> 16

<211> 20

<212> DNA

<213> artificial sequence

<400> 16

gaactggggg aggattgtgg 20

<210> 17

<211> 20

<212> DNA

<213> artificial sequence

<400> 17

gccggttcag gtactcagtc 20

<210> 18

<211> 20

<212> DNA

<213> artificial sequence

<400> 18

ccacggcaga gaatgcctat 20

<210> 19

<211> 20

<212> DNA

<213> artificial sequence

<400> 19

atgcagcatg aagtcgacca 20

<210> 20

<211> 20

<212> DNA

<213> artificial sequence

<400> 20

tcatgggctg gacattggac 20

<210> 21

<211> 20

<212> DNA

<213> artificial sequence

<400> 21

gcgtcccaaa gtaggagagg 20

<210> 22

<211> 20

<212> DNA

<213> artificial sequence

<400> 22

ctgcaggcct accctagaga 20

<210> 23

<211> 20

<212> DNA

<213> artificial sequence

<400> 23

cgtaggtgcg taggttctgg 20

<210> 24

<211> 21

<212> DNA

<213> artificial sequence

<400> 24

ggagcgagat ccctccaaaa t 21

<210> 25

<211> 23

<212> DNA

<213> artificial sequence

<400> 25

ggctgttgtc atacttctca tgg 23

<210> 26

<211> 22

<212> DNA

<213> artificial sequence

<400> 26

gggagatacc atgatcacga ag 22

<210> 27

<211> 23

<212> DNA

<213> artificial sequence

<400> 27

ccacaaatta tgcagtcgag ttt 23

<210> 28

<211> 20

<212> DNA

<213> artificial sequence

<400> 28

atgccaactc tggcgaatgt 20

<210> 29

<211> 20

<212> DNA

<213> artificial sequence

<400> 29

atggactggc agctcatcac 20

<210> 30

<211> 20

<212> DNA

<213> artificial sequence

<400> 30

ctctcccaga tgggctacct 20

<210> 31

<211> 20

<212> DNA

<213> artificial sequence

<400> 31

gactacgccg atcagtaggc 20

<210> 32

<211> 20

<212> DNA

<213> artificial sequence

<400> 32

gaaactcctc acgggctacc 20

<210> 33

<211> 20

<212> DNA

<213> artificial sequence

<400> 33

tggtagaact gtaggcggga 20

<210> 34

<211> 22

<212> DNA

<213> artificial sequence

<400> 34

attctgggat ttggcagctt tg 22

<210> 35

<211> 21

<212> DNA

<213> artificial sequence

<400> 35

acctttagca ctggtgcctt g 21

<210> 36

<211> 20

<212> DNA

<213> artificial sequence

<400> 36

caaggctcgg accaaagaca 20

<210> 37

<211> 20

<212> DNA

<213> artificial sequence

<400> 37

accagcagga tgaagaaggc 20

<210> 38

<211> 20

<212> DNA

<213> artificial sequence

<400> 38

gagtctgcgc ttcgttacct 20

<210> 39

<211> 20

<212> DNA

<213> artificial sequence

<400> 39

cccagttccc agtgctgtag 20

<210> 40

<211> 20

<212> DNA

<213> artificial sequence

<400> 40

gctcctccga cccagattac 20

<210> 41

<211> 21

<212> DNA

<213> artificial sequence

<400> 41

aacagaacca catcatcccc c 21

<210> 42

<211> 150

<212> DNA

<213> artificial sequence

<400> 42

accagacacg gcagcaacag ctgttcctaa gtgttcaact cgctccttcc cgcaaatgta 60

gaagctcaaa tccctatagc aaattccata ttctatgtca ttcttaatgc ttccgtctct 120

ctggttgaat ctagattgga tacaatcagt 150

<210> 43

<211> 64

<212> DNA

<213> artificial sequence

<400> 43

gatccgcggt tctctgtcct gtcacagttc aagagactgt gacaggacag agaaccgttt 60

tttg 64

<210> 44

<211> 64

<212> DNA

<213> artificial sequence

<400> 44

aattcaaaaa acggttctct gtcctgtcac agtctcttga actgtgacag gacagagaac 60

cgcg 64

<210> 45

<211> 64

<212> DNA

<213> artificial sequence

<400> 45

gatccgcctt tgtttctgac agcaattttc aagagaaatt gctgtcagaa acaaaggttt 60

tttg 64

<210> 46

<211> 64

<212> DNA

<213> artificial sequence

<400> 46

aattcaaaaa acctttgttt ctgacagcaa tttctcttga aaattgctgt cagaaacaaa 60

ggcg 64

<210> 47

<211> 21

<212> DNA

<213> artificial sequence

<400> 47

gguaggauau acguauucut t 21

<210> 48

<211> 21

<212> DNA

<213> artificial sequence

<400> 48

agaauacgua uauccuacct t 21

<210> 49

<211> 21

<212> DNA

<213> artificial sequence

<400> 49

gugggaagau acaccuugut t 21

<210> 50

<211> 21

<212> DNA

<213> artificial sequence

<400> 50

acaaggugua ucuucccact t 21

<210> 51

<211> 21

<212> DNA

<213> artificial sequence

<400> 51

ggguuguaga gugcauaaat t 21

<210> 52

<211> 21

<212> DNA

<213> artificial sequence

<400> 52

uuuaugcacu cuacaaccct t 21

<210> 53

<211> 21

<212> DNA

<213> artificial sequence

<400> 53

gccuccaucu agaagagaut t 21

<210> 54

<211> 21

<212> DNA

<213> artificial sequence

<400> 54

aucucuucua gauggaggct t 21

<210> 55

<211> 21

<212> DNA

<213> artificial sequence

<400> 55

ccugcaaauc auggacuaut t 21

<210> 56

<211> 21

<212> DNA

<213> artificial sequence

<400> 56

auaguccaug auuugcaggt t 21

<210> 57

<211> 21

<212> DNA

<213> artificial sequence

<400> 57

ccaggaggau augccucaut t 21

<210> 58

<211> 21

<212> DNA

<213> artificial sequence

<400> 58

augaggcaua uccuccuggt t 21

<210> 59

<211> 100

<212> DNA

<213> artificial sequence

<400> 59

ctgcctcata atctgctttt ttgctttggt ggcccctgtg gcctgggtgg gccctcccgc 60

ccctccctgg caggacaatc tgcttgtgtc tccctcgctg 100

<210> 60

<211> 100

<212> DNA

<213> artificial sequence

<400> 60

ctgcctcata atctgctttt ttgctttggt ggcccctgtg gcctgggtgg gaaagaaata 60

aaagaaatgg caggacaatc tgcttgtgtc tccctcgctg 100

<210> 61

<211> 58

<212> DNA

<213> artificial sequence

<400> 61

gatccgcaaa cctcagcaac ttcaactcga gttgaagttg ctgaggtttg cgtttttg 58

<210> 62

<211> 58

<212> DNA

<213> artificial sequence

<400> 62

aattcaaaaa cgcaaacctc agcaacttca actcgagttg aagttgctga ggtttgcg 58

<210> 63

<211> 58

<212> DNA

<213> artificial sequence

<400> 63

gatccctatc gacatggagt ctcagctcga gctgagactc catgtcgata ggtttttg 58

<210> 64

<211> 58

<212> DNA

<213> artificial sequence

<400> 64

aattcaaaaa cctatcgaca tggagtctca gctcgagctg agactccatg tcgatagg 58

<210> 65

<211> 21

<212> DNA

<213> artificial sequence

<400> 65

uucuccgaac gugucacgut t 21

<210> 66

<211> 21

<212> DNA

<213> artificial sequence

<400> 66

acgugacacg uucggagaat t 21

Claims

1. The application of an inhibitor for inhibiting the expression of a molecular marker LCDR in cells in preparing a medicament for preventing and/or treating lung adenocarcinoma is characterized in that the nucleotide sequence of the molecular marker LCDR is shown as SEQ ID NO. 1; the inhibitor is shRNA and/or siRNA of a targeting molecular marker LCDR.

2. The use according to claim 1, wherein the cells are normal lung gland cells and/or cancerous lung gland cells.

3. The application of a detection reagent for detecting a molecular marker LCDR in preparing a product for diagnosing lung adenocarcinoma is characterized in that the nucleotide sequence of the molecular marker LCDR is shown as SEQ ID NO. 1; the detection reagent for detecting the molecular marker LCDR comprises a specific primer for amplifying the molecular marker LCDR.

4. The use according to claim 3, wherein the nucleotide sequence of the specific primer for amplifying the molecular marker LCDR is shown in SEQ ID NO. 6 and SEQ ID NO. 7.

5. The use according to claim 3 or 4, wherein the product is a detection reagent or a detection kit.