CN111500722A - L ncRNA for diagnosis, prognosis and anti-angiogenesis treatment of lung squamous carcinoma - Google Patents
L ncRNA for diagnosis, prognosis and anti-angiogenesis treatment of lung squamous carcinoma Download PDFInfo
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Abstract
The invention proves that L INC00173.V1 is obviously and highly expressed in squamous cell lung carcinoma and can be used as a tool for distinguishing whether the squamous cell lung carcinoma is suffered, and also proves that L INC00173.V1 can be used as a tool for judging the prognosis of the squamous cell lung carcinoma, in addition, a reagent for silencing L INC00173.v1 can be used for preparing an anti-angiogenesis agent, and is particularly suitable for preventing and treating the squamous cell lung carcinoma, and the above results provide a new strategy for diagnosing and treating the squamous cell lung carcinoma.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of L INC00173.V1 in diagnosis, prognosis and anti-angiogenesis treatment of squamous cell lung carcinoma.
Background
According to 2018, global cancer statistics show that lung cancer accounts for 11.6% of the total number of cancer cases, accounting for 18.4% of the total number of cancer deaths, and 5-year survival rates of lung cancer patients are reported to be less than 18%. pathologically, lung cancer has two major histological types, including non-small cell lung cancer (NSC L C) and small cell lung cancer (SC L C). NSC L C can be further divided into squamous cell carcinoma (SQC) and Adenocarcinoma (ADC). recent studies have found that lung squamous cell carcinoma and adenocarcinoma patients have significant differences in clinical pathology, chemotherapeutic response and prognosis.
Long non-coding RNAs (lncrnas) are a class of non-protein coding RNAs of greater than 200 nucleotides in length that are involved in multiple biological processes, including as a scaffold protein between proteins and genes, as well as in binding to decoys and enhancers of proteins to regulate their transcriptional target genes. In addition, lncRNA functions as a competitive endogenous rna (cerna) to act as a "miRNA sponge" to disrupt miRNA-mediated degradation of target mRNA. The deregulation of mirnas has been widely demonstrated to contribute to tumorigenesis, development. And metastasis in a variety of cancer types, including lung cancer. Therefore, finding effective lncRNAs targets is very beneficial for developing new SQC treatment regimens.
Disclosure of Invention
The invention aims to provide L INC00173.V1 application in lung squamous carcinoma diagnosis, prognosis and anti-angiogenesis treatment.
The technical scheme adopted by the invention is as follows:
the application of a reagent for specifically detecting L INC00173.V1 expression level in preparing a diagnostic reagent and/or a prognosis reagent, wherein the diagnostic reagent determines whether a subject suffers from squamous cell lung carcinoma by detecting a sample from the subject, and the prognosis reagent predicts the prognosis risk of the subject suffering from squamous cell lung carcinoma by detecting the sample from the subject suffering from squamous cell lung carcinoma.
In some embodiments, the predicting the prognostic risk of a squamous cell lung carcinoma subject comprises predicting the progression-free survival time or overall survival time of a squamous cell lung carcinoma subject.
In some embodiments, the predicting the prognostic risk of a squamous cell lung carcinoma subject comprises predicting the local recurrence-free survival or distant metastasis-free survival of a squamous cell lung carcinoma subject in stage stagel-III.
In some embodiments, the reagent that specifically detects the expression level of L inc00173.v1 comprises a primer or probe that specifically detects L inc00173.v 1.
Use of an agent that silences L INC00173.V1 for the preparation of an anti-angiogenic agent.
In some embodiments, the anti-angiogenic agent is suitable for squamous cell carcinoma, preferably, the anti-angiogenic agent is suitable for squamous cell carcinoma of the lung.
In some embodiments, the agent that silences L inc00173.v1 is a competitive endogenous RNA of miR-511-5 p.
In some embodiments, the mode of silencing comprises RNAi, antisense oligonucleotide, CRISPRi, or knock-out.
Use of a silencing L INC00173.V1 agent for the manufacture of a medicament for the treatment or prevention of squamous cell lung carcinoma.
In some embodiments, the mode of silencing comprises RNAi, antisense oligonucleotide, CRISPRi, or knock-out.
The invention has the beneficial effects that:
the invention proves that L INC00173.V1 is obviously and highly expressed in squamous cell lung carcinoma and can be used as a tool for distinguishing whether the squamous cell lung carcinoma is suffered, and also proves that L INC00173.V1 can be used as a tool for judging the prognosis of the squamous cell lung carcinoma, in addition, a reagent for silencing L INC00173.v1 can be used for preparing an anti-angiogenesis agent, and is particularly suitable for preventing and treating the squamous cell lung carcinoma, and the above results provide a new strategy for diagnosing and treating the squamous cell lung carcinoma.
Drawings
FIG. 1 is a representative picture of in situ hybridization assay L INC00173.V1 for various subtypes of lung cancer and granuloma pneumophila;
FIG. 2 SI distribution of L INC00173.V1 in benign lung lesions, lung adenocarcinoma, lung squamous carcinoma tissue;
FIG. 3 high expression of L INC00173.v1 predicts poor Progression Free Survival (PFS), Overall Survival (OS) in SQC patients, high expression of L INC00173.v1 predicts poor local recurrence free survival (L RFS) and Distant Metastasis Free Survival (DMFS) in SQC patients with clinical staging stage I-III, in which L indicates low expression of L INC00173.v1 and H indicates high expression of L INC00173.v 1;
FIG. 4 Effect of silencing L INC00173.v1(sh L INC00173.V1# 1, sh L INC00173.V1#2) and its control (vector) on L INC00173.v1 levels in squamous cell lung carcinoma cells;
FIG. 5 Effect of silencing L INC00173.v1(sh L INC00173.v1# 1, sh L INC00173.v1#2) and its control (vector) on the growth curve of squamous cell carcinoma cells;
FIG. 6 Effect of silencing L INC00173.v1(sh L INC00173.v1# 1, sh L INC00173.v1#2) and its control (vector) on the cell cycle of squamous cell carcinoma of the lung;
FIG. 7 Effect of silencing L INC00173.v1(sh L INC00173.v1# 1, sh L INC00173.v1#2) and its control (vector) on colony formation of squamous cell lung carcinoma cells;
FIG. 8 Effect of silencing L INC00173.v1(sh L INC00173.v1# 1, sh L INC00173.v1#2) and its control (vector) on lung squamous cell carcinoma cell migration;
FIG. 9 Effect of silencing L INC00173.v1(sh L INC00173.v1# 1, sh L INC00173.v1#2) and its control (vector) on HUVECs tubulogenesis (upper two panels) and HUVECs migration (lower two panels) in lung squamous carcinoma cells;
FIG. 10 Effect of silencing L INC00173.v1(sh L INC00173.v1# 1, sh L INC00173.v1#2) and its control (vector) on angiogenesis in lung squamous carcinoma cells (CAM experiment);
FIG. 11 Effect of silencing L INC00173.v1(sh L INC00173.V1#1) and its control (vector) on L INC00173.v1 levels in tumor tissue in a mouse model constructed of H520 cells;
FIG. 12 Effect of silencing L INC00173.v1(sh L INC00173.V1#1) and its control (vector) on L INC00173.v1 levels in tumor tissue in a mouse model constructed of H520 cells;
FIG. 12 Effect of silencing L INC00173.v1(sh L INC00173.V1#1) and its control (vector) on tumor appearance (panel A), weight (panel B), volume (panel C) in a mouse model constructed of H520 cells;
FIG. 13 Effect of silencing L INC00173.v1(sh L INC00173.V1#1) and its control (vector) on the level of CD31+ L BV in tumor tissue in a mouse model constructed from H520 cells;
FIG. 14 Effect of silencing L INC00173.v1(sh L INC00173.V1#1) and its control (vector) on lung neoplasia (panel A), lung tumor number per unit area (panel B), lung tumor necrosis area (panel C), cumulative generation rate (panel D) in a mouse model constructed from H520 cells;
FIG. 15 shows the lung squamous carcinoma model of mice constructed by cisplatin (Cis) in combination with L INC00173.v1ASO, bevacizumab (Beva) or control (PBS) treated H520 cells, and examined the effects of lung neoplasia and H & E staining (panel A), lung tumor number per unit area (panel B), and cumulative generation rate (panel C) for each group;
FIG. 16: L INC00173.v1 and its mutants with miR-511-5p site prediction;
FIG. 17 luciferase assay L INC00173.v1 vs. miR-511-5 p;
FIG. 18: effect of antagomir-miR-511-5p and its control (NC) on tubulogenesis (Panel A) and angiogenesis (CAM experiment, Panel B) of HUVECs in squamous cell lung carcinoma cells.
Detailed Description
L NCRNA 00173 is one of L NCRNAs with two L INC00173 transcripts of completely different exon sequences, L INC00173.v1 (L INC00173 transcript variant 1, Sequence Reference NCBIReference Sequence: NR _027345.1) and L INC00173.v2 (L INC00173 transcript variant 2, Sequence Reference NCBI Reference Sequence: NR _027346.1), which, by analysis of a large number of clinical samples in combination with expression profiling, have surprisingly found that L INC00173.v1 is specifically highly expressed in squamous cell lung carcinoma and is closely related to disease prognosis and pathology, more likely, silencing L INC00173.v1 can serve as an effective anti-angiogenic agent with an anti-angiogenic efficiency comparable to or even slightly superior to that of bevacizumab, and in place of bevacizumab, the problem that current anti-angiogenic therapy cannot be applied to lung carcinoma.
In some embodiments, the use of a reagent that specifically detects L INC00173.V1 expression level for the preparation of a diagnostic reagent and/or a prognostic judgment reagent in determining whether a subject has squamous cell carcinoma by detecting a sample from the subject, in some embodiments, the sample from the subject is lung lesion tissue or lung tumor tissue of the subject, in some embodiments, the prognostic judgment reagent predicts a prognostic risk of a squamous cell carcinoma subject by detecting a sample from a squamous cell carcinoma subject, in some embodiments, the sample from the subject is lung tumor tissue of the subject, in some embodiments, the prognosis risk of the lung carcinoma subject comprises predicting a non-progression survival time or a total survival time of a squamous cell carcinoma subject, in some embodiments, the predicting a prognostic risk of a squamous cell carcinoma subject comprises predicting a non-recurrence survival time or a non-distant metastasis survival time of a squamous cell carcinoma subject, in some embodiments, the prognostic risk of a squamous cell carcinoma subject comprises predicting a non-recurrence survival time or a distant metastasis time of a squamous cell carcinoma subject, in which the prognostic index of a squamous cell carcinoma subject is greater than a predetermined threshold value, or a predetermined by comparing the specific detection probe for a predetermined in-situ detection, the predetermined threshold value of a predetermined number of patients, or a predetermined number of patients, or a predetermined number of patients, or a predetermined number of patients, or a predetermined number of patients, when a predetermined number of patients, or a predetermined.
In some embodiments, the anti-angiogenic agent is suitable for squamous cell carcinoma, and in some embodiments, the anti-angiogenic agent is suitable for squamous cell carcinoma in some embodiments, the agent that silences L inc00173.v1 is a competitive endogenous rna of miR-511-5p in some embodiments, the means of silencing includes an RNAi, an antisense oligonucleotide, a CRISPRi, or a knockout in some embodiments, the agent that silences L inc00173.v1 is used for the manufacture of a medicament for treating or preventing squamous cell carcinoma in some embodiments, the means of silencing includes an RNAi, an antisense oligonucleotide, a CRISPRi, or a knockout.
Experimental example 1, L INC00173.V1 is obviously highly expressed in lung squamous cell carcinoma
The tissue samples were obtained from 439 cryosections and lung archive samples collected during surgery or needle biopsy, including 43 lung granulomas (Granuloma, which is a benign lung lesion), 248 lung squamous carcinomas (SQC), 122 lung Adenocarcinomas (ADC) and 26 other subtypes of lung cancer, it is noted that other subtypes of lung cancer refer to lung cancers other than SQC and ADC, such as adenosquamous carcinoma (ASC), large cell neuroendocrine carcinoma (L CNE), undifferentiated carcinoma (UDC), epidermoid carcinoma (EPC), sarcomatoid carcinoma (SARC) and pleomorphic carcinoma (PMC), among others, which were provided by Central hospitals in Jiangmen City.
In brief, frozen sections were pre-hybridized with hybridization denaturation buffer (Exon Biotechnology) at 77 ℃ for 30 minutes, biotinylated L INC00173.v1 probe designed and synthesized by Exon Biotechnology was added to hybridization buffer at 77 ℃ for denaturation 5 minutes, then immediately cooled on ice, each section was covered with 20-50. mu.l diluted probe and incubated in humidified hybridization chamber at 37 ℃ overnight, sections were washed in 2 SSC 32 with 0.1% Tween-20 for 15 minutes at 37 ℃, then chamber was stained with blocking buffer (PBS containing 0.05% Tween-20, 1% PBS) for 30 minutes, and the sections were stained with staining buffer (Invitrogen PBS) for 30 minutes, stained with a staining gradient of SDH-PDH, stained with a staining medium such as Sephadex-PBS) for 30 minutes, stained with staining medium such as Sephadex-PBS, stained with staining medium (Invitrogen) for 30 minutes, stained with staining medium for 5 minutes, and air-dried in vitro staining for 5 minutes, and staining the lung cancer cells with a staining reagent for 5 minutes, and staining the same time after staining the staining with staining the staining of a staining buffer (Invitrogen PBS) for 30 minutes, stained with staining the tissue sample with staining medium for 0.05-3, stained for 5 minutes, stained for a staining the same time, stained with staining the same time, the same time as the stain for example, stained sperm stain for the same.
Further evaluation of Staining Index (SI) of L INC00173.v 1in SQC, ADC, benign lung lesion tissue the evaluation criteria are as follows, averaged from S1 evaluated by two independent investigators, wherein the tumor cell ratio is scored as 0 (no positive tumor cells), 1(< 10% positive tumor cells), 2 (10-35% positive tumor cells), 3 (35-70% positive tumor cells) and 4(> 70% positive tumor cells). the staining intensity is graded according to the criteria of 0 (no staining), 1 (weak staining, light yellow), 2 (medium staining, yellowish brown) and 3 (strong staining, brown). SI is calculated as the product of the staining intensity score and the positive tumor cell ratio.based on this evaluation method, the expression of L INC00173.v 1in lung tumor samples is evaluated by SI, with scores of 0, 1, 2, 3, 4, 6, 8, 9 or 12. based on this evaluation method, it is seen that the staining index in lung tumor samples is significantly higher than in benign lung lesion tissue, when SQC is not higher than SQC 00173. SsSQC 7, the staining index can be judged as a diagnostic reagent for the detection of lung lesion tissue expression of VC0011, if the staining index is found to be equal to be a level of VC7.7.
Experimental example 2, L INC00173.V1 high expression indicates poor prognosis in patients with squamous cell lung carcinoma
The tissue samples of squamous cell lung carcinoma are divided into two groups according to the expression condition of L INC00173.V1, with SI > 4 as a high expression group (H) and SI ≦ 4 as a low expression group (L), and Kaplan-Meier survival analysis is further carried out, and the results are shown in FIG. 3, wherein the high expression of L INC00173.v1 indicates the poor progression-free survival (PFS) and Overall Survival (OS) of SQC patients, while the poor local recurrence-free survival (L RFS) and distant metastasis-free survival (DMFS) of SQC patients clinically staged as stage I-III, therefore, L INC00173.V1 can be used as a prognostic marker of squamous cell carcinoma patients, and can be used for predicting the progression-free survival, overall survival or distant metastasis-free survival of squamous cell carcinoma patients in stage I-III.
Experimental example 3 silencing L INC00173.v1 inhibits angiogenesis of squamous cell lung carcinoma cells
The lung cancer cell lines NCI-H520 (abbreviated as H520 and purchased from Procell) and NCI-H226 (abbreviated as simply-made cell) are adoptedCalled H226, obtained from Shanghai cell institute of Chinese academy of sciences) to construct a cell model of silence L INC00173.v1 the materials and brief methods were as follows, cells were cultured in RPMI-1640 medium (purchased from Gibco) containing 10% fetal bovine serum (purchased from Gibco) at 37 ℃ under 5% CO2Cloning of human L INC00173.v1 short hairpin RNA (shRNA, 1# and 2# designed, respectively) into hU 6-MCS-CBh-gcGFP-IRES-puromycin lentiviral vector (GV493, Genechem), cloning of primers sh-L INC00173.v1# -up: 5'-CCGGCACCTTGCTCCGCTGTTCTTTCTCGAGAAAGAACAGCGGAGCAAGGTGTTTTTG-3', sh-L INC00173.v1-1# -dn: 5'-AATTCAAAAACACCTTGCTCCGCTGTTCTTTCTCGAGAAAGAACAGCGGAGCAAGGTG-3', sh-L INC00173.v1-2# -up: 5'-CCGGTGGGATGTCAGAGGTGTTGATCTCGAGATCAACACCTCTGACATCCCATTTTTG-3', sh-L INC00173.v1-2# -dn: 5'-AATTCAAAAATGGGATGTCAGAGGTGTTGATCTCGAGATCAACACCTCTGACATCCCA-3', according to the instructions, 2 vectors and control vectors constructed with L ipocamamine 3000 reagent (Invitrogen) were transfected into HEK293 cells, respectively, infected with NCI-H520, NCI-H520 cells, purified from cDNA synthesized by 0.5 mg/8 of cDNA (Invitrogen) using the real-time PCR amplification of cDNA amplified from cDNA of cDNA amplified cDNA using the cDNA of the cDNA amplified cDNA of the cDNA of HEK 293K 293, cDNA, the cDNA of the cDNA clone, the cDNA of the^-△△Ct) Results of the method are shown in figure 4, the expression level of L INC00173.v1 is obviously reduced in the lung squamous carcinoma cell model transfected with sh L INC00173.v1 sh # 1 or sh L INC00173.v1 sh # 2.
Further evaluation of the above-constructed squamous cell lung carcinoma cell model cell counting using CCK-8, 500 cells were seeded in a 96-well plate, CCK-8 (Dojindo) was added and incubated at 37 ℃ for 1h, and OD650nm was measured as shown in FIG. 5, silencing L INC00173.v1 did not affect the growth curve of squamous lung carcinoma cells, cell cycle analysis was performed using a flow cytometer, cells were washed twice with precooled PBS and fixed overnight with precooled 75% ethanol, then cells were gently resuspended in precooled PBS and ribonuclease was added to degrade RNA and incubated at 37 ℃ for 30 minutes, then propidium iodide (Dojindo) was used for 20 minutes at room temperature, the summary of silencing results of cell cycle analysis is shown in FIG. 6, L INC00173.v1 did not affect the cell cycle of squamous lung carcinoma cells, colony forming ability of the cell model was measured, 500-1000 cells were seeded in six-well plates and cultured for 7-10 days, colony forming ability of squamous lung carcinoma cells was measured using crystal 10% fixed, colony forming ability was measured using crystal water, colony forming ability of squamous cell growth was measured as shown in 7-633, and cell growth curve of silenced cells was measured using crystal growth test 3683.3.7.3. the cell growth curve of blue-7.7.7.34Individual) were inoculated into the upper compartment of a 24-well Transwell infiltration chamber (purchased from Corning) and the lower compartment of the Transwell was filled with the complete medium to which 10% FBS was added as a chemoattractant after 24 hours of incubation, the cells that had moved to the bottom of the chamber were fixed with a fixing solution methanol: acetic acid ═ 3: 1), stained with crystal violet, and photographed and quantified by counting in 5 random fields of view the results are shown in figure 8, silencing L inc00173.v1 did not affect the migratory capacity of the lung squamous carcinoma cells, the above results show that silencing L inc00173.v1 did not affect the proliferation and migratory capacity of the lung squamous carcinoma cells.
Surprisingly, L INC00173.v1 silenced L A cell inhibiting angiogenesis of squamous cell lung carcinoma cells materials and test methods human umbilical vein endothelial cell line (HUVEC) was purchased from Promocell, HUVEC was cultured in endothelial cell growth medium Bulletkit (purchased from EGM), HUVEC cell tubule formation assay method Pre-cooled Matrigel (purchased from Corning) was added to the wells of 24-well plates and polymerized for 30 minutes at 37 deg.C HUVEC (2 × 10)4) Suspended in 200. mu.l of medium and addedInto each well, 5% CO at 37 deg.C2The tubule images were taken under a 100-fold field microscope and analyzed for tubule number using Image View 3.7 while testing using the aforementioned migration ability assay, with the results shown in FIG. 9, silent L INC00173.v1 squamous cell lung carcinoma cells inhibited Human Umbilical Vein Endothelial Cell (HUVEC) angiogenesis and inhibited HUVEC migration ability, further chick embryo chorioallantoic membrane (CAM) analysis was performed on day eight of fertilized egg, a hole of 1 cm diameter was made on the balloon of the eggshell and the surface of the dermal filter paper on the balloon floor was removed to expose the CAM, a 0.5 cm diameter filter was placed on top of the CAM, 100 μ l fresh medium-containing squamous cell carcinoma cells were carefully added to the center of the paper and sealed with air permeable medical tape, then the egg was incubated for 15 minutes with a fixative solution (methanol: acetone ═ 1: 1) and collected for 60% to 80% humidity for 5 days, an anti-squamous cell growth agent was prepared and collected using a fertilized CD 00173, thus a digital anti-growth agent was prepared and analyzed for a visible vascular growth inhibition by Image using Image of INC VIVEC 0013.7, and anti-squamous cell growth assay, preferably was performed on top of the aforementioned squamous cell 3673, a visual growth-3673, a further shortened anti-growth anti-CAV-growth anti-CAC 7, a 3673 assay was performed on top of chicken embryo chorionic cell analysis was performed on the test, a medium was performed on the test, a medium was performed on day 7, a medium was performed on day eight day 7, a medium was performed on.
Experimental example 4 silencing L INC00173.v1 inhibits tumorigenesis of squamous cell lung carcinoma
Mouse graft tumor models were constructed using H520 cells transfected with L INC00173.v1 sh # 1 or a control plasmid (vector, vectored.) using at least 6 or 8 BA L B/c-nu mice per group (4-6 weeks old; 18-20g) and randomly assigned to each mouse of different weights, resuspension (1-4) × 10 in 100. mu.l PBS6Each of the above cells was inoculated subcutaneously into the groin of nude mice, mice were monitored twice a week and sacrificed by cervical dislocation according to survival time, tumor volume was determined using an external caliper, and the formula (length × width)2) Calculation of/2 tumor resection, weighing and storage in liquid nitrogen tank, and finally paraffin embedding of all tissues, andl INC00173.v 1in tumor tissue was detected in the same manner as in example 1, as shown in FIG. 11, and the tumor tissue of mice inoculated with L INC00173.v1 sh #1/NCI-H520 cells showed a down-regulated L INC00173.v1 level at the end of the experiment, as shown in FIG. 12, and silencing L INC00173.v1 decreased the weight and volume of the tumor, CD31 was determined by immunohistochemistry of the embedded tissue, a brief incubation step in which a slide of formalin-fixed paraffin-embedded sections was prepared by microwave heating antigen in TE (pH 9.0) buffer, blocking with hydrogen peroxide and goat serum, respectively, and diluting with anti-CD 31 antibody (Cell Technology) in a humidity chamber at 4 deg.C, after which the slide was washed in TBS/0.05% Tween-20, coupled with biotin secondary antibody (Proteech, Prointc peroxidase and anti-CD 31 antibody (Cell Technology), respectively, and after incubation with overnight staining of biotin-conjugated tissue with streptavidin (Protecht-3, as shown in FIG. 13, and increasing the density of Biotin-conjugated protein (Proteech, Proteec 3, Biotin+LBV)。
The influence of L INC00173.v1 on the lung tumor-causing capacity of lung squamous cancer cells is further examined, the H520 lung squamous cancer cells constructed above are injected into a mouse body through tail vein injection, the mouse is tracked and killed by adopting the method, and the tissue section is subjected to H & E staining and survival curve analysis, and the result is shown in figure 14, the silencing L INC00173.v1 obviously inhibits the growth of NCI-H520 cells in the lung, reduces the tumor number per unit area of the lung, increases the tumor necrosis area and prolongs the cumulative life cycle.
Experimental example 5, L INC00173.v1 antisense oligonucleotide therapeutic action on Lung squamous carcinoma
The current anti-angiogenesis therapy has large adverse effects on patients with lung squamous carcinoma, for example, Bevacizumab (commercial name Avastin) is one of representatives of anti-angiogenesis therapy, can cause severe hemoptysis of patients with lung squamous carcinoma, and is excluded from being used for lung squamous carcinomaIn clinical treatment of lung cancer, anti-angiogenic therapy is usually combined with chemotherapeutic drugs (e.g., cisplatin), and thus tested using the combined effect of L INC00173.v1 antisense oligonucleotide (ASO) and cisplatin.A murine model of squamous cell lung cancer is constructed by injecting H520 cells into mice by tail vein injection, and 7 days after inoculation, the mice are treated with cisplatin (cis, 5mg/kg) intraperitoneally twice a week, and injected with INC L INC00173.v1ASO (antisense oligonucleotide, antisense L NA) once a weekTMGapmeRs), bevacizumab (Beva), negative control (PBS), during which survival was followed and finally mice were H-plated for lung tissue&E staining and survival analysis, the results are shown in FIG. 15, and L INC00173.v1 antisense oligonucleotide can achieve the equivalent or even better effect on treating lung squamous carcinoma than bevacizumab (Beva), thereby proving that L INC00173.v1 silence is an effective anti-angiogenesis treatment strategy and can effectively treat lung squamous carcinoma.
Experimental example 6, L INC00173.v1as competitive endogenous RNA for miR-511-5p
To initially explore the mechanism of L INC00173.v1as an anti-angiogenic agent, L INC00173.v1 was found by study as a competitive endogenous RNA for miR-511-5p was found to have a potential recognition sequence on L INC00173.v1 by using the miRanda algorithm (FIG. 16). cells (5 × 10) were identified using a dual luciferase reporter assay5) Plated in 60mm cell culture dishes, propagated to 60-80% confluence after 24 hours of culture, then transfected into cells with the reporter construct using L ipofectamine3000, after 36 hours of incubation, the cells were harvested and washed with PBS and lysed with lysis buffer. cell lysates were analyzed using a Synergy 2 microplate system (purchased from BioTek.) luciferase and Renilla luciferase were measured using Renilla-L umi luciferase reporter assay kit (purchased from Beyotime.) luciferase activity of each lysate was normalized to Renilla luciferase activity, relative transcription activity was converted to fold induction above the control value, wherein ago-511-5 p (mir-511-5p mimic),the reporter construct, which amplifies human L INC00173.v1 binding site activity from genomic DNA and clones it into pmirG L O vector (purchased from Promeg), along with the construction of mutant reporter gene, results are shown in FIG. 17, upregulation of miR-511-5p is inhibited, while miR-511-5p silencing enhances the luciferase reporter activity of L INC00173.v1, but does not enhance the luciferase reporter activity of mutant L INC00173.v1, from which it can be seen that L INC00173.v1 acts as a competitive endogenous RNA for miR-511-5 p. further anti-angiogenesis experiments were performed using the antagomir-511-5p, reference example 3, results are shown in FIG. 18, and the antagomir-5-L.v 461 reverses the effect of angiogenesis of the cells on the growth of lung cancer cells via the proliferation of < CHEM > CD 00173.v > cells, and the growth of lung cancer cells is thus shown by the growth promotion of growth of the cells via Cv00173H 5H 73. C9-5 p, miR-511, and miR-5 p-5-inhibition by the growth of these cells.
The above description is only exemplary of the present invention, and the protection scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the protection scope of the present invention.
Claims (10)
1. The application of a reagent for specifically detecting L INC00173.V1 expression level in preparing a diagnostic reagent and/or a prognosis reagent, wherein the diagnostic reagent determines whether a subject suffers from squamous cell lung carcinoma by detecting a sample from the subject, and the prognosis reagent predicts the prognosis risk of the subject suffering from squamous cell lung carcinoma by detecting the sample from the subject suffering from squamous cell lung carcinoma.
2. Use according to claim 1, characterized in that: the predicting prognostic risk for a squamous cell lung carcinoma subject includes predicting progression-free survival or overall survival of a subject with squamous cell lung carcinoma.
3. Use according to claim 1, characterized in that: the predicting prognostic risk for a squamous cell lung carcinoma subject includes predicting local recurrence-free survival or distant metastasis-free survival of a squamous cell lung carcinoma subject in stage stagel-III.
4. The use of claim 1, wherein the reagent for specifically detecting the expression level of L INC00173.V1 comprises a primer or probe for specifically detecting L INC00173. V1.
5. Use of an agent that silences L INC00173.V1 for the preparation of an anti-angiogenic agent.
6. Use according to claim 5, characterized in that: the anti-angiogenic agent is suitable for squamous cell carcinoma, preferably, the anti-angiogenic agent is suitable for squamous cell carcinoma of the lung.
7. The use according to claim 5, wherein the agent silencing L INC00173.V1 is a competitive endogenous RNA of miR-511-5 p.
8. Use according to claim 5, characterized in that: the means of silencing includes RNAi, antisense oligonucleotide, CRISPRi, or knock-out.
9. Use of a silencing L INC00173.V1 agent for the manufacture of a medicament for the treatment or prevention of squamous cell lung carcinoma.
10. Use according to claim 9, characterized in that: the means of silencing includes RNAi, antisense oligonucleotide, CRISPRi, or knock-out.
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