CN113265463A - Application of FAM84B in preparation of esophageal squamous cell carcinoma prognosis evaluation reagent and screening of drugs for targeted therapy of esophageal squamous cell carcinoma - Google Patents
Application of FAM84B in preparation of esophageal squamous cell carcinoma prognosis evaluation reagent and screening of drugs for targeted therapy of esophageal squamous cell carcinoma Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and provides an application of FAM84B in preparation of an esophageal squamous cell carcinoma prognosis evaluation reagent and screening of drugs for targeted therapy of esophageal squamous cell carcinoma, in order to clarify a relation between FAM84B copy number change and prognosis of patients with esophageal squamous cell carcinoma, clarify an action mechanism of FAM84B in occurrence and development of esophageal squamous cell carcinoma and an action of FAM84B in screening of drugs for targeted therapy of esophageal squamous cell carcinoma. The FAM84B gene copy number amplification and high expression are used for preparing an esophageal squamous cell carcinoma prognosis evaluation reagent. FAM84B gene copy number amplification becomes a new index of prognosis of esophageal squamous carcinoma patients. The FAM84B gene copy number amplification leads to the increase of the expression of the FAM84B of the esophageal squamous carcinoma, and the FAM84B high expression can promote the proliferation and the cycle of esophageal squamous carcinoma cells; the low expression of FAM84B can inhibit the proliferation and cycle of esophageal squamous carcinoma cells and block the cells in the G1 stage.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of FAM84B in preparation of an esophageal squamous cell carcinoma prognosis evaluation reagent and screening of drugs for targeted therapy of esophageal squamous cell carcinoma.
Background
Esophageal cancer is one of the common malignant tumors worldwide, 47.8 ten thousand new cases occur each year, and 37.5 ten thousand death cases occur. China is a high-incidence country of esophageal cancer, and the morbidity and mortality of China are respectively the third and fourth of various malignant tumors. Unlike western countries, 90% of the histological types of esophageal cancer in china are squamous cell carcinoma, and most patients are already in the middle and late stages of the disease at the time of visit due to the occult symptoms of early esophageal cancer. At present, the treatment mode of the esophageal cancer is mainly operation, and the five-year survival rate of esophageal cancer patients is still maintained at a low level due to the tolerance of esophageal cancer cells to radiotherapy and chemotherapy. Therefore, the deep exploration of the pathogenesis of esophageal squamous cell carcinoma is urgent for the clinical discovery of molecular markers for early diagnosis and the establishment of effective clinical intervention measures.
Copy Number Variations (CNVs) are a new, important type of genomic structural variation that follows single nucleotide polymorphisms and simple repeats. CNVs are widely distributed in the human genome and can affect more than 10% of the sequences in the human genome. The overlap of CNVs and protein coding regions can cause the structural change of a single gene or a group of adjacent genes and the change of gene expression, thereby having important effects on species evolution, phenotypic variation, disease occurrence and the like.
Researches show that malignant tumors have high copy number variation incidence rate, and are expected to become a new way for researching tumor pathogenic genes and pathogenesis. Meanwhile, CNVs are also an ideal cancer diagnostic marker because they are more stable than gene expression and can be achieved by detecting ctDNA, and therefore, it is possible to provide strong support for the diagnosis of tumors.
Esophageal squamous carcinoma (ESCC) is a malignant tumor with high morbidity and mortality in our country, and the five-year survival rate is still maintained at a low level due to the lack of an effective early diagnosis, prognosis and treatment method. In the early stage, sequencing of whole genes and whole exomes is respectively carried out on 14 ESCC tumors and 90 ESCC paired normal tissues from high incidence areas of the esophageal cancer in China, and cytogenetic variation related to ESCC is revealed. This section of ESCC-related omics sequencing results are published in journal of Cell subsection Am J Hum Genet (selected as "Best of AJHG 2014 to 2015"). Recently, the largest whole genome data of esophageal squamous cell carcinoma in China is published, and copy number variation analysis shows that FAM84B is remarkably amplified in esophageal squamous cell carcinoma patients, but the survival prognosis and the specific action mechanism of the patients are not clear.
The FAM84B gene, also known as LRATD2, is located on chromosome 8q24.21, where susceptibility regions have been identified for various cancer types. FAM84B gene, which encodes 310 amino acid protein, is involved in the formation of gene repair complex, and has significant amplification in esophageal, breast and prostate cancer. It has now been found that expression of FAM84B is increased in breast, prostate, esophageal squamous, epithelial ovarian and colorectal cancers. Furthermore, overexpression of FAM84B in prostate cancer cells and esophageal squamous carcinoma significantly promoted cell invasion in vitro as well as xenograft growth and lung metastasis. In neoadjuvant chemotherapy of esophageal squamous carcinoma, the down-regulation of the expression of serum FAM84B protein is related to Pathological Complete Response (PCR), and the increase of the expression of Lnc FAM84B-AS can reduce the expression of FAM84B in gastric cancer, so that the prognosis of the gastric cancer has obvious correlation.
Disclosure of Invention
The invention defines the relationship between FAM84B copy number change and esophageal squamous cell carcinoma patient prognosis, clarifies the action mechanism of FAM84B in the occurrence and development of esophageal squamous cell carcinoma and the action of FAM84B in the screening of drugs for targeted therapy of esophageal squamous cell carcinoma, and provides the application of FAM84B in the preparation of esophageal squamous cell carcinoma prognosis evaluation reagents and the screening of drugs for targeted therapy of esophageal squamous cell carcinoma.
The invention is realized by the following technical scheme: the FAM84B is applied to the preparation of esophageal squamous cell carcinoma prognosis evaluation medicines, and the FAM84B gene copy number amplification is applied to the preparation of esophageal squamous cell carcinoma prognosis evaluation reagents.
The above-mentionedThe relative risk of the FAM84B gene copy number amplified patient with tumor grading as risk factor is 0.57, FAM84B gene copy number amplified group is FAM84BAmpThe copy number is more than or equal to 0.5; FAM84B, a FAM84B gene copy number non-amplification groupnon-AmpCopy number < 0.5, FAM84BAmpThe patient's risk of death is FAM84Bnon-Amp1.57 times of patients.
The FAM84B gene copy number determination method is DNA sequencing or DNA Fluorescence In Situ Hybridization (FISH).
The invention also provides application of FAM84B in screening of drugs for targeted therapy of esophageal squamous carcinoma, wherein the drugs for targeted therapy of esophageal squamous carcinoma are FAM84B gene inhibitors, FAM84B interacting protein NPM1 gene inhibitors or specific inhibitors of FAM84B downstream target genes.
The FAM84B gene inhibitor inhibits FAM84B from high expression, and blocks esophageal squamous carcinoma cells in the G1 stage.
The FAM84B gene inhibitor has the nucleic acid sequence as follows: FAM84B-shRNA 1: 5'-CACCTAAGTTACAAGGAAGTTCTCGAGAACTTCCTTGTAACTTAGGTG-3', respectively; FAM84B-shRNA 2: 5'-AGTCTAGAGGACCTGATCATGCTCGAGCATGATCAGGTCCTCTAGACT-3' are provided.
The nucleic acid sequence of the FAM84B interaction protein NPM1 gene inhibitor is si-NPM1-RNA 1: 5'-ACTGCTTTATACTTTGTCA-3', respectively; si-NPM1-RNA 2: AATGGCAAATAGTCTTGTA-3';
the interaction sites of the FAM84B interaction protein NPM1 and the FAM84B protein are as follows: NPM1-C terminal domain 189-294 aa.
The FAM84B interaction protein NPM1 gene inhibitor inhibits NPM1 expression, blocks the function of FAM84B, and blocks esophageal squamous cell carcinoma cells in the G1 stage.
The specific inhibitor of the downstream target gene of FAM84B is an inhibitor of the downstream gene of FAM84B and NPM1 complex, and the downstream gene of FAM84B and NPM1 complex is CDK4 and CDK 6.
Further, the specific inhibitor of the downstream target gene of FAM84B is an inhibitor targeted to block CDK4 and CDK6 activities.
The inhibitor for inhibiting the cell proliferation of the over-expression of FAM84B genome is palbociclib.
The invention utilizes a GISTIC method to find that the chromosome region 8q24.13-q24.21 of the FAM84B gene is obviously amplified, then, 507 ESCC tumor tissues and paired normal tissues in the high incidence region of the esophageal cancer in China are subjected to whole genome sequencing, and the expanded queue is subjected to somatic copy number variation analysis, so that the FAM84B gene is obviously amplified, and the FAM84B gene copy number amplification accounts for 21.5%.
The invention discloses a relationship between FAM84B copy number amplification and esophageal squamous cell carcinoma patient prognosis by using Kaplan-Meier survival analysis and COX multifactor regression analysis. Kaplan-Meier survival analysis suggests that FAM84B gene copy number amplification is significantly correlated with patient survival prognosis.
COX multifactorial regression analysis suggested that the deeper the tumor infiltration depth of the FAM84B gene copy number-amplified patient, the higher the mortality risk index. Combining TCGA and 155 cases of RNA-seq data analysis of esophageal squamous carcinoma, the FAM84B gene copy number amplification and FAM84B gene high expression are suggested to have significant correlation.
Western blot is used for detecting the expression levels of FAM84B in different esophageal squamous cell lines, cell lines with FAM84B stable high expression and low expression are respectively constructed, and the change of the esophageal squamous cell line phenotype after over-expression and FAM84B knocking-down is detected through MTT, hard cloning and flow cytometry. The stable low expression FAM84B cell line was inoculated subcutaneously into the abdomen of nude mice, raised under SPF conditions for 4-6 weeks, and the number and volume of tumor formation were observed and recorded.
The high expression of FAM84B can promote the proliferation and the cycle of esophageal squamous carcinoma cells, and the low expression of FAM84B can inhibit the proliferation and the cycle of esophageal squamous carcinoma cells and block the cells in the G1 stage.
A Co-IP combined Mass Spectrometry (MS) experiment is carried out by using the FAM84B antibody, proteins interacting with FAM84B are analyzed, and the cancer promotion mechanism of FAM84B is explored. Co-IP experiments and GST-pull down experiments are used for verifying whether a compound is formed between FAM84B and NPM1, and immunofluorescence experiments are used for verifying whether Co-localization exists between FAM84B and NPM 1.
Mass spectrometry and cell experiments prove that FAM84B regulates the proliferation and the cycle of esophageal squamous carcinoma cells by forming a complex with NPM1, and the biological function of NPM1 in esophageal squamous carcinoma is detected by MTT, hard cloning and flow cytometry experiments; interference with the expression of NPM1 can inhibit the cell phenotype change caused by high expression of FAM 84B.
Combining the FAM84B mass spectrum result and the NPM1 database result, the FAM84B-NPM1 complex is explored to regulate the downstream target gene of the cell cycle of the esophageal squamous carcinoma. And the interaction of CDKN2A and FAM84B-NPM1 complex was verified by Co-IP experiments. The FAM84B and NPM1 complex inhibits CDKN2A from being combined with CCND through being combined with CDKN2A, thereby promoting CDK to be combined with CCND, enabling Rb level to be phosphorylated, promoting E2F transcription factor expression and promoting cell cycle generation.
Detecting the change of the cyclin after the FAM84B is highly expressed and lowly expressed by using qRT-PCR and Western blot experiments; the change of the cyclin in the nude mice is detected by using an immunohistochemical experiment after the low expression of FAM84B in the nude mice. The cell cycle inhibitor Pabociclib is added into the FAM84B stable high-expression esophageal squamous cell carcinoma cell line, and the fact that the FAM84B high-expression esophageal squamous cell carcinoma cell line is more sensitive to Pabociclib drugs is shown.
The FAM84B gene copy number amplification of the invention becomes a new index for the prognosis of esophageal squamous cell carcinoma patients. The FAM84B gene copy number amplification leads to the increase of the expression of the FAM84B of the esophageal squamous carcinoma, and the FAM84B high expression can promote the proliferation and the cycle of esophageal squamous carcinoma cells; the low expression of FAM84B can inhibit the proliferation and cycle of esophageal squamous carcinoma cells and block the cells in the G1 stage.
FAM84B forms a complex with NPM1, competitively binds with CCND1 to CDKN2A, promotes CDK to bind with CCND, phosphorylates Rb level, promotes E2F transcription factor expression, thereby promotes cell cycle, accelerates esophageal squamous cell proliferation, and promotes tumorigenesis.
Drawings
FIG. 1 is a heat map of FAM84B gene copy number amplification in 507 patients with esophageal squamous carcinoma high-incidence areas in China; in the figure: a: the chromosome 8q24.21 region exhibited significant amplification; b: the copy number of FAM84B gene is obviously amplified; FIG. 2 shows that the amplification of FAM84B gene copy number by Kaplan-Meier survival analysis is significantly correlated with the prognosis of 507 patients with esophageal squamous carcinoma; FIG. 3 shows FAM84BAmp and FAM84Bnon-Amp with or without smoking in 507 patients with esophageal squamous carcinoma (B)A) Prediction effect graphs of prognosis in different grades (D) and tumor infiltration depth (C); FIG. 4 is a graph of the predicted effect of FAM84BAmp and FAM84Bnon-Amp on prognosis in 507 patients with esophageal squamous carcinoma in different gender (A), different age (B) and different location (C); FIG. 5 is a multi-factor analysis FAM84B of the Cox proportional hazards regression modelAmpAnd FAM84Bnon-AmpA map of the risk of death for patients with esophageal squamous carcinoma; FIG. 6 is a graph showing the effect of Kaplan-Meier survival analysis on the prediction of the FAM84B gene copy number amplification and tumor infiltration depth on the prognosis of patients with esophageal squamous cell carcinoma; FIG. 7 is a graph showing correlation analysis between FAM84B gene copy number amplification and expression level; FIG. 8 is a diagram showing that overexpression of FAM84B gene promotes proliferation and cycle of esophageal squamous carcinoma cells; in the figure: a: verifying the over-expression efficiency of FAM84B gene in esophageal squamous carcinoma cells; b: MTT experiment detects the influence of FAM84B gene over-expression on KYSE450 cell proliferation; c: hard cloning experiments detect the influence of over-expression of FAM84B gene on KYSE450 cell proliferation; d: flow cytometry tests are used for detecting the influence of FAM84B gene overexpression on KYSE450 cell cycle; p <0.05, p < 0.01; p < 0.001; FIGS. 9A-D are graphs of the inhibition of esophageal squamous carcinoma cell proliferation and cycle by knocking down FAM84B gene; wherein: FIG. 9A: verifying the efficiency of knocking down FAM84B gene in esophageal squamous carcinoma cells; FIG. 9B: MTT experiment detects the influence of knocking down FAM84B gene on KYSE150 and KYSE180 cell proliferation; FIG. 9C: the hard cloning experiment detects the influence of knocking down FAM84B gene on the proliferation of KYSE150 and KYSE180 cells; FIG. 9D: detecting the influence of knocking down FAM84B gene on KYSE150 and KYSE180 cell cycle by flow cytometry; p <0.05, p < 0.01; p < 0.001; FIG. 10 shows the mass spectrometry results for the interaction of NPM1 protein with FAM84B protein; in the figure: a is a Co-IP experiment and a silver staining graph performed by using FAM84B antibody; b is a peptide fragment sequence combined by NPM1 and FAM 84B; FIG. 11 shows the interaction of FAM84B with NPM 1; in the figure: a: the Co-IP experiment verifies the endogenous interaction of FAM84B and NPM1 protein; b: the exogenous interaction of FAM84B and NPM1 protein is verified by a Co-IP experiment; c: GST-pull down experiment verifies that FAM84B directly interacts with NPM1 protein; FIG. 12 shows the search for binding sites between NPM1 protein and FAM84B protein; in the figure: a: schematic diagram of deletion mutation of NPM1 protein domain; d: Co-IP experiment verification of FAM84B protein and NPM1-C terminal domain (189-294 aa); FIG. 13 shows immunofluorescence detection of co-localization of FAM84B and NPM1 proteins in esophageal squamous carcinoma cells; FIG. 14 shows the correlation between the expression of NPM1 and the level of FAM84B protein; in the figure: a: FAM84B is consistent with the expression of NPM1 protein in esophageal squamous carcinoma cells; b: the knockdown and overexpression efficiency of NPM1 in esophageal squamous carcinoma cells; FIG. 15 shows the biological function of NPM1 in esophageal squamous carcinoma; in the figure: A-B: MTT (methyl thiazolyl tetrazolium) experiment detects the influence of knockdown and over-expression of NPM1 on the proliferation of esophageal squamous carcinoma cells; C-D: hard cloning experiments are used for detecting the influence of knocking down and over-expressing NPM1 on the proliferation of esophageal squamous carcinoma cells; E-F: detecting the influence of knocking-down and over-expressing NPM1 on the cell cycle of esophageal squamous carcinoma by a flow cytometry; p <0.05, p < 0.01; p < 0.001; FIG. 16 shows that knockdown of NPM1 inhibits the phenotype of cells caused by high expression of FAM 84B; in the figure: a: western blot experiments verify the knockout efficiency of the NPM1 in FAM84B high-expression cell strains; b: MTT (methyl thiazolyl tetrazolium) experiment detection shows that knocking down NPM1 can inhibit the promotion effect of FAM84B high expression on cell proliferation; c: the knockdown NPM1 can inhibit the promotion effect of FAM84B high expression on clone formation through hard clone experiment detection; d: flow cytometry experiment detection shows that knocking-down of NPM1 can inhibit promotion effect of FAM84B high expression on cell cycle; p <0.05, p < 0.01; p < 0.001; FIG. 17 is a graph of proteins sought to interact with the FAM84B-NPM1 complex; in the figure: a: there were 8 intersection genes in FAM84B mass spectrometry results and in NCBI database with NPM1 interacting protein results; b: a peptide fragment sequence of CDKN2A binding to FAM 84B; FIGS. 18-19 are graphs demonstrating the interaction of CDKN2A with the FAM84B-NPM1 complex; in the figure: FIG. 18-A: the Co-IP experiment verifies the interaction of CDKN2A with FAM84B protein; FIG. 18-B: the interaction of CDKN2A and NPM1 protein is verified by a Co-IP experiment; FIG. 19: the Co-IP experiment verifies the interaction of FAM84B with NPM1 and CDKN 2A; FIGS. 20-21 show that FAM84B participates in the regulation of cell cycle protein of esophageal squamous cell carcinoma in vitro; in the figure: FIGS. 20A-B: detecting the change of cycle-related proteins after over-expression and knockdown of FAM84B by using Western-blot; FIG. 21 qRT-PCR detection of mRNA levels of FAM84B overexpression and cycle-related protein changes after knockdown; FIG. 22: detecting the influence of knocking down FAM84B gene on tumor growth in a nude mouse tumor-bearing experiment; in the figure: a: comparing the tumor volumes of the FAM84B knockout group and the control group; b: FAM84B knock-out group andcomparing the weights of the tumors in the control group; FIG. 23: in vivo experiments detect that FAM84B participates in the regulation of cell cycle protein of esophageal squamous cell carcinoma; in the figure: A-B: detecting the expression of cyclin in a nude mouse tumor body by an immunohistochemical experiment; FIG. 24 is a graph showing the effect of palbociclib drugs on esophageal squamous carcinoma cells overexpressing FAM84B gene.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
First, experimental material
1. Study subjects: 507 cases of esophageal squamous carcinoma tissues and matched tissues beside the esophageal squamous carcinoma tissues used in the invention are collected from Shanxi province tumor hospital in the high incidence area of esophageal carcinoma in China and auxiliary tumor hospitals of Xinjiang medical university, and are subjected to surgical excision for the first time without new adjuvant therapy, chemotherapy or radiotherapy before operation. Whole genome sequencing was performed by shanghai pharma minkangde biotechnology limited. Clinical grading of ESCC reference was made to the 2010 version 7 TNM grading standard specified by the united states commission on cancer and the international union for cancer. All specimens were collected with the consent of the patient himself and his family members and signed with an informed consent. Among the cases, the Chinese 437 people and the Kazakh 70 people are included. The specific information is shown in table 1.
Table 1: clinical pathological characteristics of esophageal squamous carcinoma patient
2. Cell line: human esophageal squamous carcinoma cell lines KYSE180, KYSE450, KYSE150 and TE-1 used in the experiment are all stored at low temperature in key laboratories of the transformation research center and the pathogenesis of esophageal cancer of Shanxi medical university transformation medical center.
The main reagents and materials are shown in table 2.
Table 2: main reagents from the company
3. The common reagents are prepared in the following tables 3 and 4.
Table 3: cell culture related reagent preparation
Table 4: preparation of Western blot related reagent
Second, Experimental methods
1. Cell culture
A. Cell recovery: preparing warm water at 37 ℃, taking out the required cells from a liquid nitrogen tank, immediately putting the cells into the warm water, and quickly melting the cells; centrifuging at 1000 rpm for 3 min, and removing supernatant; slowly resuspending the cell pellet with 4 mL complete medium and transferring to a 6cm petri dish; 37 ℃ and 5% CO2And (5) culturing at constant temperature.
B. Cell passage: taking a 10cm dish as an example, when the cell density reaches 80-90%, passage is required. Firstly, discarding the original culture medium in a culture dish, and slowly washing cells for 2 times by adding 1 XPBS liquid which is balanced at room temperature; then adding 1 mL of trypsin, placing the mixture in a constant-temperature incubator for culture, observing cells under a microscope, and immediately adding 1 mL of complete culture medium to stop digestion after the cells become round and float; finally transferring the cell suspension into a centrifuge tube, and centrifuging for 3 min at 1000 rpm; the supernatant liquid was discarded, 1 mL of complete medium was added to resuspend the cell pellet and gently blow it evenly, and the pellet was transferred to 2-3 new plates on average for further culture.
C. Freezing and storing cells: taking a 10cm dish as an example, the same as the work of the previous stage of cell passage, firstly discarding the original culture medium in the culture dish, and adding 1 XPBS liquid balanced at room temperature to slowly clean the cells for 2 times; then adding 1 mL of trypsin, placing the mixture in a constant-temperature incubator for culture, observing cells under a microscope, and immediately adding 1 mL of complete culture medium to terminate digestion after the cells become round and float; centrifuging at 1000 rpm for 3 min; discarding supernatant liquid, adding 3 mL of cell freezing solution to resuspend cell sediment, slowly blowing and uniformly beating, subpackaging to 3-4 sterile freezing tubes for gradient freezing for 4-30 min; -20 ℃ -2 h; -80 ℃ overnight; liquid nitrogen-long term storage.
D. Cell transfection: taking 24-well plate as an example, the dosage is 2-6 × 10 a day in advance4The transfection is started when the density of the cells reaches 70% -90% after the cells are attached to the wall by inoculating the cells per 500 mu L/hole.
Plasmid dilution: 0.8. mu.g of plasmid was diluted in 50. mu.L of optimum medium;
preparation of plasmid EL complex: 1.6 u L TransInto EL added into the diluted plasmid, gently mixing, room temperature standing for 15-20 min;
adding the plasmid EL compound into a cell culture dish, shaking up gently, and then putting into an incubator for culture;
after transfection for 4-6 h, the culture medium is replaced (optional), and culture is continued for 18-72 h.
2. Lentiviral transfection and screening
Cell plating: 0.3 × 10 a day in advance4The cells were seeded at 200. mu.L/well (KYSE 150, KYSE 450) at 37 ℃ with 5% CO2And (5) culturing at constant temperature.
Lentivirus transfection: firstly, setting a FAM84B lentivirus gradient, setting three gradients which are respectively 5, 10 and 50 according to a complex infection value (MOI), and then calculating the volume of lentivirus stock solution (FAM 84B-NC, FAM84B-OE, FAM84B-SH1 and FAM84N-SH 2) required by transfection according to the virus MOI value. Lentivirus bulk volume = (number of cells at transfection x MOI)/lentivirus gradient. Then, the medium was removed and the control was set up by adding different MOI gradients of lentivirus dilutions at 37 ℃ with 5% CO2Culturing at constant temperature, replacing the culture medium after 12-24 h, continuing culturing, adding puromycin with proper concentration into the culture medium when the cell fusion degree reaches 60%, screening, and finally detecting the transfection efficiency by using Western Blot.
3. Western immunoblotting experiment (Western Blot experiment)
A. Extraction of total cellular protein: in the case of 10cm dish cell count, fresh cell pellets were collected and placed in sterile EP tubes, 200. mu.L of RIPA lysate (containing protease inhibitor) was added, and the cells were lysed on ice for 1h with shaking every 10 min. After lysis was complete 12000 rpm, centrifugation was carried out at 4 ℃ for 20 min and supernatant protein was pipetted into a fresh sterile EP tube.
B. Protein concentration determination by BCA method: firstly, preparing a working solution: and mixing the solution A and the solution B in the kit according to the instruction in a ratio of 50: 1, proportional allocation; secondly, protein standard dilution: diluting the concentration of 2 mg/mL into 0, 0.5, 1, 2, 4, 6, 8 and 10 mg/mL by using a precooled 1 XPBS buffer solution, diluting the concentration of the sample by 20 times, repeating 3 wells for each sample, finally adding 200 mu L of working solution into each well, oscillating for 30 min at constant temperature of 37 ℃, detecting the Optical Density (OD) of 570 nm wavelength by using a microplate reader, drawing a standard curve according to the OD value and the loading amount of the standard protein, and obtaining the protein concentration of the sample to be detected according to the standard curve.
C. Protein denaturation and preparation of protein samples: to 50. mu.g of protein loading, 4 Xprotein loading buffer was added, all samples were made up to the same volume with PBS liquid, and the mixture was boiled at 100 ℃ for 10 min and stored at-80 ℃ until use.
D. Polyacrylamide gel electrophoresis (SDS-PAGE):
preparing glue: cleaning the glass plate, and airing for later use; preparing 10% separation gel with the volume of 7 mL according to the specification, adding the separation gel into a gel tank, and then adding 1 mL of isopropanol to keep the surface of the separation gel horizontal; after the separation gel is solidified, the isopropanol is discarded, 3 mL of 5% concentrated gel with volume is prepared and added into a gel tank, and then a matched tooth comb is inserted for standby after the separation gel is solidified.
Loading and electrophoresis: removing the comb, adding electrophoresis liquid, sequentially loading the prepared protein samples, and adding 5 muL protein Marker into a specific lane, wherein the electrophoresis is divided into two stages, namely 80V, 30 min and 120V, and 1.5 h.
Film transfer and sealing: preparing a PVDF membrane, activating methanol, and balancing with a membrane transferring liquid; meanwhile, soaking filter paper sponge by using the membrane transferring liquid, sequentially placing sponge-filter paper-SDS, gel-PVDF and membrane-filter paper-sponge according to the sequence of a negative electrode and a positive electrode to prepare a sandwich, and paying attention to the fact that bubbles are prevented from existing between every two layers; correctly placing the prepared sandwich into a film transferring groove, and keeping the pressure constant at 100V for 2 h at 4 ℃; sealing with 5% skimmed milk on a horizontal shaker at room temperature for 1h after the membrane conversion is finished;
incubation of antibodies and development: removing the confining liquid, diluting the primary antibody according to the instruction, and incubating overnight at 4 ℃; the next day, washing the membrane with TBST for 3 times, each time for 10 min/120 r; discard TBST, as 1: incubating the secondary antibody diluted by 10000 proportion for 2 h at room temperature on a shaking table, washing the membrane for 4 times by TBST, and rotating 5 min/120 r each time; and finally, developing.
4. Cell function test
MTT test: the cells of the experimental group and the control group are arranged according to 0.5X 104Cells/200. mu.L/well were seeded in 96-well plates at 24 h, 48 h, 72 h, 96 h time-gradients, 5 replicates per group of cells per time point, and viable cell counts were determined. Before detection, 20 mu L of MTT solution (5 mg/mL) is added into each hole, after continuous constant-temperature culture for 4h, the original culture medium in each hole is absorbed, 200 mu L of DMSO solution is added into each hole, incubation is carried out for 15min on a shaking table at room temperature, the absorbance value with the wavelength of 490 nm is detected by a microplate reader, and a growth curve is drawn.
Hard cloning experiments: the cells of the experimental group and the control group are added according to the ratio of 0.1X 104Cells/2 mL/well seeded cells were plated in 6-well plates in 3 replicates per group, incubated at 37 ℃ with 5% CO2The cells are cultured in the incubator for about 10 days, and generally, the total number of the cells is more than 50, so that the cell can be judged as a clone. Observing to form small white spots visible to naked eyes under a microscope, stopping culturing, sucking away the original culture medium in the holes, adding 4% paraformaldehyde, fixing at room temperature for 15min, washing with PBS to remove residual formaldehyde, adding crystal violet solution, dyeing at room temperature for 20 min, and washing with PBS until the clones are clearly dyed, and the background is as clean and transparent as possible.
Flow cytometry experiment: the control and experimental cells were harvested and fixed in 70% glacial ethanol overnight. The fixative was discarded by centrifugation, the cells were filtered (300 μ L nylon mesh membrane) after washing 2 times with PBS, the supernatant was discarded by centrifugation, and rnase and Propidium Iodide (PI) dye were added according to the kit instructions. And (4) performing computer detection after being protected from light for 30 min, and analyzing the cell content of each group of cells in G1 and S, G2/M stages respectively.
5. Tumor-bearing experiment in nude mice: BALB/C immunodeficient mice used in the experiments were purchased from Beijing Wittingerihua, Inc., and were generally 4-5 weeks old mice. The purchased mice were divided into two groups: FAM84B-NC and FAM84B-SH groups, each containing 4 cells, were injected subcutaneously into the left and right upper limbs of mice, each injected with 3X 10 cells6. After five weeks, the cervical vertebrae were dislocated and sacrificed, the tumor body was taken out and the weight and volume thereof were measured, and the results were counted.
6. Fluorescent quantitative PCR
A. Preparation of cDNA template:
(1) the cell pellet was collected in 1.5 mL EP tubes at 5-10X 10 intervals6Adding 1 mL of Trizol into each cell, fully and uniformly mixing the cells by using a pipette, transferring the cells into an EP (Eppendorf) tube with RNase-free, and standing the cells for 5min at room temperature; adding 200 μ L chloroform, mixing with oscillator, standing at room temperature for 5min, centrifuging at 12,000 rpm at 4 deg.C for 10 min; carefully sucking the supernatant, transferring to a new EP tube with RNase-free, adding equal volume of isopropanol, turning the solution over for 10 times, and standing at room temperature for 10 min; centrifuging at 4 deg.C for 10 min at 12,000 rpm, discarding supernatant, adding 1 mL of 70% ethanol, blowing with a pipette, mixing, cleaning, centrifuging at 4 deg.C for 10 min at 12,000 rpm again; removing supernatant as far as possible, opening the EP tube, placing in clean and ventilated place at room temperature, adding 30-50 μ L RNase-free water after ethanol is volatilized, dissolving precipitate, measuring concentration, marking, storing at-20 deg.C for a short time, and storing at-80 deg.C for a long time.
(2) Reverse transcription into cDNA: the reaction system was 20 μ L, 5 × Mix: 4 muL; RNA: 3 microgram; RNase-free water: up to 20 μ L. Reaction conditions are as follows: 37 ℃ of: 15 min; 85 ℃: 5 s; 4 ℃, and the temperature is: and +. varies.
B. Dissolving the primer: the primer freeze-dried powder is centrifuged at 12,000 rpm for 1 min, then the tube cover is carefully opened, TE Buffer with a specified volume is added at the bottom of an EP tube, the tube is flicked by a finger, and dissolved into stock solution of 100 mu M for standby, and the stock solution is diluted by 10 times when in use, and the working concentration is 10 mu M.
The Q-PCR reaction system and reaction procedure are shown in Table 5. Reaction conditions are as follows: pre-denaturation: at 95 ℃ for 10 min; and (3) PCR reaction: 95 ℃: 15 s, 60 ℃: 1 min, total 40 PCR cycles; melt Curve.
TABLE 5
7. Protein co-immunoprecipitation
(1) Cell preparation: 1-2 cells with good growth status on 10cm dishes were prepared in advance, and after the medium was discarded, 10 mL of precooled PBS was added to wash the cells 2 times. 1 mL of cell lysate was added and the cells were collected by cell scraping into 1.5 mL EP tubes and placed on ice until needed.
(2) Turning over at 4 deg.C for 1 hr to make the cells contact with cell lysate completely.
(3) The supernatant was transferred to a new EP tube by centrifugation at 12,000 rpm for 20 min at 4 ℃.
(4) Prepare protein a/G agarose, subtract the head of the gun tip with clean scissors to avoid damaging the gel beads, wash twice with ice PBS, and finally, use PBS to follow a 1: 1 preparing a mixed solution.
(5) To 1 mL of the cell lysate was added 100. mu.L of protein A/G agarose (50%), and the mixture was incubated at 4 ℃ for 30 min with rotation to remove non-specifically bound proteins and reduce the background.
(6) The supernatant was centrifuged at 12,000 rpm for 20 min at 4 ℃ and transferred to a new EP tube to remove protein A/G agarose.
(7) 20 μ L of the protein lysate was taken out in advance as control Input, and then the remaining protein lysate was divided into 2 portions, one portion was added with bait protein antibody a and the other portion was added with IgG homologous to antibody a according to the instructions, and incubated overnight at 4 ℃ with rotation.
(8) The washed beads were added to 50. mu.L each in two EP tubes and incubated at 4 ℃ for 4-6 h with rotation.
(9) The mixture was centrifuged at 12,000 rpm for 5 seconds at 4 ℃ to obtain protein A/G agarose, the supernatant was removed, and the gel beads were washed 2 times with cell lysate, suggesting that a decapitated gun tip was used for resuspension of the gel beads.
(10) After the last washing, the residual cell lysate was aspirated up with 20. mu.L of the tip, then 30. mu.L of the loading buffer was added, and the sample was boiled at 100 ℃ for 5min, followed by SDS-PAGE.
8. Silver staining
Fixing: after the electrophoresis is finished, the gel is carefully taken out, put into about 100 mL of the stationary liquid, and slowly shaken on a shaker at room temperature for 40 min to 1h at the speed of 60 to 70 rpm. Extending the fixation time can reduce the background.
Fixing liquid: 50 mL of ethanol and 10 mL of acetic acid were added in this order. The volume is 100 mL by using 40 mL of triple distilled water.
Washing: the fixative was decanted and the gel was washed with 30% ethanol for 10 min at room temperature at 60-70 rpm.
Water washing: 30% ethanol is poured off, 200 mL of triple distilled water is added, and washing is carried out for 10 min under the same washing conditions as above.
Sensitization: the water was poured off and 100 mL of the prepared silver-staining sensitizer was added at room temperature for 2min at a speed of 60-70 rpm.
Water washing: the sensitizer was decanted off, the gel was washed with 200 mL of triple distilled water at room temperature for 1 min at 60-70 rpm, and the procedure was repeated for a total of two washes.
Silver staining: the water was decanted, and 1X 100 mL of the pre-diluted silver solution was added and the staining was carried out at room temperature for 10 min at a shaker speed of 60-70 rpm. The silver solution was poured off and washed with tri-distilled water at the same rate for 1 min. Note that: the time for water washing does not exceed 1.5 min.
Color development: pouring off water, adding 100 mL of silver staining solution, developing at room temperature for 3-10 min, and shaking at 60-70 rpm until ideal expected protein band appears.
And (4) terminating: when the expected protein band appears, pouring out the silver staining solution, adding 1 Xstopping solution 100 mL, and at room temperature for 10 min, the shaking table speed is 60-70 rpm. Carbon dioxide bubbles are generated during the termination process.
Water washing: after the termination process is finished, pouring out the silver staining termination solution, adding 100 mL of tri-distilled water, and keeping the room temperature for 2-5 min at the shaking table speed of 60-70 rpm. The gel can finally be stored in triple distilled water, waiting for gel cutting for mass spectrometry.
9. Immunofluorescence
Cover glass pretreatment: a circular sterile coverslip suitable for a 12-well plate was separately soaked in 75% alcohol for use.
The cover glass is clamped by a pair of tweezers, then the cover glass passes through an alcohol lamp instantly, the cover glass is far away from flame after being ignited, when residual alcohol is completely burnt, the cover glass is placed in a 12-hole plate (one hole is formed in each hole), the cover glass is shaken and cooled in time, the situation that glass sheets are adhered to the bottoms of the holes due to the fact that culture dishes are melted at high temperature is avoided, then 0.1% sterile Gelatin (Gelatin) solution (500 mu L per hole) is added, and the mixture is placed in an incubator at 37 ℃ for pretreatment for 4 hours.
The Gelatin solution was aspirated, washed twice with PBS, and the cells were plated into the wells and cultured for 15-20 h.
Cell fixation: fixing the cells with 4% paraformaldehyde, 500 μ L per well, and fixing at room temperature for 15 min; then washed twice with ice PBS pre-chilled in advance.
Cell permeabilization: adding 500 μ L of 0.25% Triton X-100 solution prepared with PBS into each well, and permeabilizing at room temperature for 10 min; then, the column was washed with ice PBS for 10 min 3 times.
And (3) sealing: blocking was performed using 1% BSA solution in PBST solution at room temperature for 30 min.
Incubating the primary antibody: using confining liquid to prepare primary antibody according to the use proportion in the specification, and incubating overnight at 4 ℃; then, the column was washed with ice PBS for 5min 3 times.
Incubation of secondary antibody: preparing a secondary antibody by using a confining liquid according to the use proportion in the specification, keeping out of the sun, and incubating at room temperature for 1 h; the column was washed 3 times with ice PBS for 5min each.
Sealing: sealing with sealing agent containing DAPI dye, and storing at 4 deg.C in dark condition, wherein the volume is 2-5 μ L.
10. GST fusion protein sedimentation technology
(1) Early preparation: first, a plasmid carrying the GST-tag GST-FAM84B was constructed, and the vector used in this experiment was named pGEX-5X-1.
(2) Extraction of GST protein: converting recombinant GST plasmid using BL21(DE3) cells; picking a single clone into LB culture medium containing AMP (100 mug/mL), adding 3 mL culture medium into a 15 mL centrifuge tube generally, and placing the centrifuge tube at 37 ℃ for shaking culture at 180 rpm overnight; transferring the cultured bacterial liquid to a 50 mL centrifuge tube, carrying out shaking culture at 37 ℃, adding IPTG inducer when OD600 reaches 0.6, carrying out low-temperature induction (18-22 ℃) overnight, wherein the induction time is at least about 20 h; centrifuging at 4 deg.C for 5,000 g for 10 min to collect bacteria, removing supernatant, and using the thallus for protein extraction or storing at-20 deg.C for about 1-3 days, if long-term storage is required, storing at-80 deg.C; the cells were lysed using bacterial lysate and 15 mL of medium using a 50 mL tube, and the collected cells were lysed using approximately 1 mL of lysate.
Sonication, ultrasonication procedure: working for 5 s, stopping for 8 s, and keeping the total time for 40-60 min until the lysate is clear, wherein the centrifugal tube is always kept in an ice-water mixture in the ultrasonic process, then Triton X-100 is added to 1%, and then the mixture is shaken gently, or PMSF is added to 0.2 mM; centrifuging the lysate: the conditions are 15,000 g, 4 ℃, and the time is 20 min; the supernatant was transferred to a new 1.5 mL EP tube and BCA protein concentration assay was performed.
After the concentration is measured, adding a protein solution into previously washed GSH-Beads, adding 25 muL Beads into each 200 mug of protein, and carrying out rotary incubation at 4 ℃ for about 4 h; cleaning the Beads scheme: centrifuging the supernatant, leaving supernatant with equal volume of precipitate, transferring into 1.5 mL EP tube, washing with bacterial lysate for 2 times, centrifuging at 500 g for 3 min, and keeping at 4 deg.C on ice to minimize protein degradation; eluting protein: the cells were incubated with 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM GSSH, about 200 μ L of eluent at 4 ℃ for about 30 min. Centrifuging at 4 deg.C and 500 g for 5min, taking out supernatant, and transferring into new EP tube to obtain soluble GST fusion protein; preparing a sample, taking the protein, adding sample buffer, continuing SDS-PAGE, incubating with GST antibody, and detecting the expression level of the recombinant protein.
11. Immunohistochemistry: placing fresh tissues into 10% neutral formalin for fixation, and then carrying out dehydrated paraffin embedding; cutting the wax block into slices with the thickness of 2 mu m, fishing the slices by using an adhesive glass slide, and baking the slices at the constant temperature of 70 ℃ overnight;
dewaxing: baking the slices at 70 deg.C for 10 min, sequentially passing through 3 glass jars filled with xylene, each time for 10 min; and rehydrating the slices in gradient alcohol glass jars respectively, wherein the alcohol concentration is 90%, 80% and 70% in sequence every 2 min. The slices were placed in 3% hydrogen peroxide for 15min to inactivate endogenous peroxidase. The slices were rinsed in a container with triple distilled water for 5min and then washed 3 times with PBS for 2min each time.
Antigen exposure (antigen retrieval): the general antigen restoration is carried out by high-pressure thermal restoration with sodium citrate with pH of 6.0. Before repairing, water in an autoclave is boiled, then the tissue slices are completely soaked in a plastic box containing a sodium citrate solution with the pH value of 6.0, and then the autoclave is covered and started. When the pressure valve is jacked up, the temperature in the pot is about 120 ℃, and then the high-pressure thermal repair is carried out by timing. Strictly controlling the time to be 1.8-2.2min, directly stopping the procedure after the repair is finished, deflating and opening the cover, and transferring the plastic box into tap water bath to naturally cool the plastic box; after cooling to room temperature, wash with PBST 3 times for 2min each.
And (3) sealing: blocking was performed with 5% BSA at room temperature for 30 min.
Incubating the primary antibody: according to the instruction and experimental groping, PBST is used for preparing primary antibody, and the primary antibody is incubated overnight at 4 ℃ in a wet box; taking out the wet box on the next day, standing at room temperature, and rewarming for 1 h; the slides were washed three times with PBST for 10 min each.
Incubation of secondary antibody: the ready-to-use secondary antibody is dripped on the tissue section, incubated for 30 min at 37 ℃ and washed with PBST for three times, 5min each time.
DAB: and wiping off residual PBST on the slices by using toilet paper for color development, and then dropwise adding DAB color development liquid. The color development was observed under a microscope, and immediately after the color development was observed, the color development reaction was terminated with tap water.
Hematoxylin counterstaining: firstly, fishing out crystals floating on the liquid surface in a hematoxylin cylinder by using a hard paper sheet; soaking the slices in hematoxylin for 1 min, observing with microscope, and taking out after cell nucleus is blue; soaking the slices in a tap water tank, and slightly rotating to rinse away unbound hematoxylin on the slices; then the slices are put into hydrochloric acid ethanol for differentiation and decoloration for 30 s and then put into a running water tank again; if the color of the cell nucleus is slightly light, the cell nucleus can be anti-blue in ammonia water, and if the color is too light, the hematoxylin counterstaining step can be repeated until a satisfactory effect is achieved.
And (3) dehydrating: after satisfactory counterstaining, the slices were dehydrated according to a gradient of 70% ethanol, 80% ethanol, 90% ethanol, and anhydrous ethanol for 5min each 2 times. Then the mixture was re-passed through the xylene tank 3 times, each time for 10 min.
Sealing: sealing with neutral resin, air drying at room temperature, and storing for a long time.
12. Statistical analysis: correlation analysis of copy number amplification of FAM84B with clinical case factors was tested using a four-grid charter. Kaplan-Meier survival analysis and Log-rank test analysis the correlation of copy number amplification of FAM84B with the prognostic survival of patients with different clinical pathological factors. The Cox proportional hazards regression model performed single and multifactorial survival analyses to analyze the role of copy number amplification of FAM84B in the prognosis prediction of ESCC patients. All statistics and corresponding plots were performed using SPSS 18.0 software.
The total survival time is calculated from the beginning of the operation to the death day or the final follow-up day. All experimental data were from three independent replicates and the results are expressed as mean ± standard deviation. All statistical analyses were performed using SPSS 21.0 software and the graphical display was performed using Prism 5 GraphPad software. Differences between groups were tested using t-test and anova. Correlation of FAM84B copy number amplification to clinical pathology characteristics was analyzed using the chi-square test. p <0.05 is statistically significant for the differences.
Third, experimental results
1. Analysis of clinical relevance of FAM84B gene copy number amplification to esophageal squamous carcinoma patient
FAM84B gene copy number amplification can be used as an esophageal squamous cell carcinoma prognosis index: the whole genome sequencing data of 507 patients in the high incidence region of Chinese esophageal cancer is analyzed by a GISTIC method, and the FAM84B gene copy number amplification heat map of the 507 patients is shown in figure 1; 109 patients were found to exhibit amplification of FAM84B gene copy number, which accounts for 21.5%.
507 patients were divided into two groups according to FAM84B gene copy number amplification: FAM84B gene copy number amplification group (FAM 84B)AmpCopy number not less than 0.5) and FAM84B gene copy number non-amplification group (FAM 84B)non-AmpCopy number < 0.5). The results of the Kaplan-Meier survival and Log-rank test analysis are shown in FIG. 2, which suggests FAM84BAmpPatients in group, had short survival times and poor prognosis (p < 0.01) (FIG. 2).
The correlation between the FAM84B gene copy number amplification condition and the clinical pathological features of ESCC patients is shown in Table 6, and chi-square test analysis suggests that FAM84B gene copy number amplification has significant correlation with clinical stages (p = 0.0003) and survival prognosis (p = 0.0011) of patients.
Table 6: correlation of FAM84B copy number amplification with ESCC patient clinical pathology
The results of using Kaplan-Meier survival analysis are shown in FIGS. 3 and 4, and the results show that: FAM84B in patients with esophageal squamous carcinoma who smoke and do not smokeAmpTotal survival time of patients was lower than FAM84Bnon-AmpPatients, more pronounced in patients without smoking (p = 0.001), this trend is also present in patients who had and who did not have alcohol (p < 0.001). Furthermore, FAM84B was present in clinical indications of age, sex and depth of infiltrationAmpTotal survival time of patients was lower than FAM84Bnon-AmpPatient's findings (p < 0.001).
In summary, the following steps: the FAM84B gene copy number amplification in the esophageal squamous carcinoma can be used as an index of poor prognosis of patients with esophageal squamous carcinoma.
2. FAM84B gene copy number amplification is related to the infiltration depth of esophageal squamous carcinoma
FAM84B gene copy number amplification and age, sex, tumor location, pathology were evaluated using COX regression multifactorial analysisThe relationship between factors such as grading, infiltration depth and clinical stage and the survival time of the patient is shown in FIG. 5, and the result shows FAM84BAmpThe patient mortality risk is FAM84Bnon-Amp1.54 times (HR =0.649, 95% confidence interval =0.482-0.874, p = 0.004) patient, FAM84BAmpThe deeper the tumor infiltration depth of the patient, the higher its mortality risk index (HR =2.301, 95% confidence interval =1.744-3.037, p < 0.01).
Meanwhile, 507 patients were divided into 4 groups according to FAM84B gene copy number amplification and tumor infiltration depth: FAM84BAmp + T（Ⅰ+Ⅱ）、FAM84BAmp + T（Ⅲ）、FAM84Bnon-Amp+ T (I + II) and FAM84Bnon-Amp+ T (III), the results of the Kaplan-Meier survival analysis are shown in FIG. 6, which suggests that the FAM84B gene copy number is amplified and the deeper the infiltration depth, the worse the survival prognosis of the patient. Therefore, the FAM84B gene copy number amplification can be used as an independent predictor for ESCC patient prognosis. The FAM84B gene copy number amplification is suggested to have definite prediction value on the survival time of the esophagus squamous carcinoma patient.
3.FAM84B gene copy number amplification is obviously related to FAM84B gene high expression in esophageal squamous carcinoma
The TCGA database ESCC transcriptome data (n = 95) and the transcriptome data from chinese esophageal cancer high-incidence region (n = 155) were analyzed, and as a result, as shown in fig. 7, it was found that there was a significant correlation between FAM84B gene copy number amplification and the expression level of FAN84B gene.
4. Function of FAM84B as a protooncogene affecting proliferation and cycle of esophageal squamous carcinoma cells
A. High expression of FAM84B can promote proliferation and cycle of esophageal squamous carcinoma cells
Selecting an esophageal squamous carcinoma cell line KYSE450 with low FAM84B expression, constructing a FAM84B stable and high-expression cell line through lentivirus transfection, and performing a cell function experiment, wherein the experiment result is shown in figure 8; MTT and hard clone experiments prove that: compared with a control group, the FAM84B with high expression can obviously promote the proliferation of esophageal squamous carcinoma cells (p is less than 0.001); flow cytometry experiments prove that: compared with a control group, the high expression of FAM84B can remarkably promote the generation of the cell cycle of esophageal squamous cell carcinoma (p is less than 0.001).
B. Knocking down FAM84B can inhibit proliferation and cycle of esophageal squamous carcinoma cells
Selecting high-expression esophageal squamous carcinoma cell lines KYSE150 and KYSE180 of FAM84B, constructing a stable low-expression FAM84B cell strain through lentivirus transfection to perform a cell function experiment, and verifying through MTT and hard clone experiments: compared with a control group, the knockdown FAM84B can obviously inhibit the proliferation of esophageal squamous cell carcinoma cells (p is less than 0.001); flow cytometry experiments prove that: knockdown of FAM84B significantly inhibited the proliferation and cycling of esophageal squamous carcinoma cells and blocked cells in stage G1 (fig. 9A-D), compared to the control group; the tumor-bearing experiment of the nude mice in the animal body proves that: compared with the control group, the experimental group remarkably inhibits the proliferation of the tumor in the nude mice after knocking down FAM84B (FIG. 22).
5. Mechanism of action of FAM84B in promoting esophageal squamous cell carcinoma
A. Searching protein interacting with FAM84B in esophageal squamous carcinoma cells
In order to further and deeply explore the action mechanism of FAM84B as oncogene for promoting esophageal squamous cell carcinogenesis, a Co-IP (Mass Spectrometry) combined Mass Spectrometry (MS) experiment is carried out by using FAM84B antibody, and after silver staining, a difference band is selected for mass spectrometry analysis, so as to try to search for protein interacting with FAM 84B. The results of the experiments are shown in FIG. 10, and the results show that 64 proteins are identified in the band, and the literature refers to the function experiment of the prophase combination, which suggests that nucleolar phosphoprotein NPM1 may be the protein interacting with FAM84B, thereby promoting the cell cycle of esophageal squamous cell carcinoma.
B. Interaction of FAM84B and NPM1 in esophageal squamous carcinoma cells
To further verify the interaction between FAM84B and NPM 1. Firstly, the interaction between FAM84B and NPM1 is confirmed by endogenous and exogenous expression of FAM84B and NPM1 proteins by using Co-IP experiments. Secondly, through expressing FAM84B protein in vitro, GST-pull down experiment proves that the direct interaction exists between FAM84B and NPM1, and the result is shown in FIG. 11; finally, through constructing NPM1 protein domain deletion mutant plasmid and Co-transfecting FAM84B into the esophageal squamous carcinoma cell line, the Co-IP experiment proves that the NPM1-C terminal domain (189-294 aa) is the direct interaction site with FAM84B protein (FIG. 12).
C. Co-localization of FAM84B and NPM1 in esophageal squamous carcinoma cells
To further confirm the interaction between FAM84B and NPM1, immunofluorescence experiments were used to verify the localization of FAM84B and NPM1 in esophageal squamous cell carcinoma cell lines, indirectly suggesting the interaction between FAM84B and NPM1 if their sublocalization in the cells can overlap. The results are shown in fig. 13 and confirmed experimentally: red represents the sub-localization of FAM84B protein in the cell, green represents the sub-localization of NPM1 in the cell, blue represents the nucleus, and yellow represents the position where FAM84B protein and NPM1 protein overlap. Therefore, we confirmed the co-localization of FAM84B and NPM1 in esophageal squamous carcinoma cells, further suggesting that FAM84B and NPM1 form protein complexes to play a role in esophageal squamous carcinoma cells.
D. FAM84B influences the occurrence of esophageal squamous carcinoma through NPM1
To further explore whether FAM84B affects the development of esophageal squamous carcinoma by binding to NPM1, we performed the following experiments: first, it was confirmed by MTT, hard cloning and flow cytometry experiments that: compared with a control group, the NPM1 with high expression can obviously promote the proliferation and the period of esophageal squamous carcinoma cells (p is less than 0.001); knocking down NPM1 can significantly inhibit the proliferation and cycle of esophageal squamous carcinoma cells and block the cells in G1 phase (p < 0.001) (FIGS. 14-15).
Secondly, we found that the expression of FAM84B protein and the expression trend of NPM1 protein are consistent in an esophageal squamous carcinoma cell line, so that the inhibition of the expression of NPM1 in a FAM84B high-expression cell line is confirmed by MTT, hard cloning and flow cytometry experiments: compared with a control group, the knocking-down of NPM1 can obviously reverse the proliferation and the cycle of esophageal squamous carcinoma cells caused by the high expression of FAM84B (p is less than 0.001); from the above results, it was confirmed that FAM84B affects the occurrence of esophageal squamous cell carcinoma via NPM1 (fig. 16).
6. FAM84B-NPM1 complex regulates cell cycle by binding CDKN2A
To further explore the mechanism of action of FAM84B-NPM1 complex in regulating the cell cycle of esophageal squamous cell carcinoma, we followed to search for downstream targets of action. By filtering the FAM84B mass spectrum results (64 proteins) and the proteins interacting with NPM1 (673 proteins) in the NCBI database, 8 proteins were screened after intersection, wherein CDKN2A is a tumor suppressor gene, which can bind to CDK4 protein, inhibit the formation of a complex between CCND and CDK4, thereby inhibiting the phosphorylation of Rb protein, preventing the cell cycle from entering S phase from G1 phase, and finally inhibiting the occurrence of cell cycle (fig. 17).
Therefore, the interaction between CDKN2A and FAM84B and NPM1 was verified using Co-IP experiments, and the results confirmed that: CDKN2A interacted with both FAM84B and NPM1, and as FAM84B expression increased, the amount of bound NPM1 and CDKN2A increased (fig. 18-19).
7. FAM84B is involved in regulation of esophageal squamous carcinoma cell cycle
Next, we detect the influence of the expression change of FAM84B on cell cycle signal pathway related proteins, and use Western blot and qRT-PCR experiments to confirm that: the expression of FAM84B is increased, and the expression of cell cycle signal pathway related proteins CDK4, CCND1, p-RB and E2F1 is increased at the level of transcription and translation; otherwise expression is reduced. In nude mice tumor with low expression of FAM84B, immunohistochemical experiments show that FAM84B is low in expression, NPM1, CDK4 and CCND1 are significantly reduced in expression, and the result suggests that FAM84B is involved in regulation of esophageal squamous cell carcinoma cell cycle (FIGS. 20-23).
8. Pabociclib can significantly inhibit proliferation of FAM 84B-overexpressing genomic cells
In the esophageal squamous carcinoma cell line KYSE450 (450-NC) and the cell line (450-OE) over-expressing FAM84B gene, a cell cycle inhibitor, Pabociclib, was added 24 h after adherence, the drug concentration was set at 0, 4, 8, 16, 31nM, MTT was added 48 h later, and the cell line over-expressing FAM84B gene was more sensitive to Pabociclib than the control group (FIG. 24).
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (9)
- The application of FAM84B in preparing an esophageal squamous carcinoma prognosis evaluation reagent is characterized in that: the FAM84B gene copy number amplification is used for preparing an esophageal squamous carcinoma prognosis evaluation reagent.
- 2. Use according to claim 1, characterized in that: the FAM84B gene copy number amplified patient has tumor grading as risk factor with relative risk of 0.57, FAM84B gene copy number amplified group namely FAM84BAmpThe copy number is more than or equal to 0.5; FAM84B, a FAM84B gene copy number non-amplification groupnon-AmpCopy number < 0.5, FAM84BAmpThe patient's risk of death is FAM84Bnon-Amp1.57 times of patients.
- The application of FAM84B in screening of drugs for targeted therapy of esophageal squamous carcinoma is characterized in that: the medicine for targeted therapy of esophageal squamous carcinoma is an FAM84B gene inhibitor, an inhibitor for inhibiting cell proliferation of an FAM84B gene overexpression group, an inhibitor for FAM84B interacting protein NPM1 gene or a specific inhibitor for FAM84B downstream target genes.
- 4. Use according to claim 3, characterized in that: the FAM84B gene inhibitor inhibits FAM84B from high expression, and blocks esophageal squamous carcinoma cells in the G1 stage.
- 5. Use according to claim 4, characterized in that: the FAM84B gene inhibitor has a nucleic acid sequence as follows:FAM84B-shRNA1：5'-CACCTAAGTTACAAGGAAGTTCTCGAGAACTTCCTTGTAACTTAGGTG-3'；FAM84B-shRNA2：5'-AGTCTAGAGGACCTGATCATGCTCGAGCATGATCAGGTCCTCTAGACT-3'。
- 6. use according to claim 3, characterized in that: the nucleic acid sequence of the FAM84B interaction protein NPM1 gene inhibitor is si-NPM1-RNA 1: 5'-ACTGCTTTATACTTTGTCA-3', respectively; si-NPM1-RNA 2: AATGGCAAATAGTCTTGTA-3'; the interaction sites of the FAM84B interaction protein NPM1 and the FAM84B protein are as follows: NPM1-C terminal domain 189-294 aa.
- 7. Use according to claim 3, characterized in that: the specific inhibitor of the downstream target gene of FAM84B is an inhibitor of the downstream gene of FAM84B and NPM1 complex, and the downstream gene of FAM84B and NPM1 complex is CDK4 and CDK 6.
- 8. Use according to claim 7, characterized in that: the specific inhibitor of the downstream target gene of FAM84B is an inhibitor for targeted blocking of CDK4 and CDK6 activity.
- 9. Use according to claim 3, characterized in that: the inhibitor for inhibiting the cell proliferation of the over-expression of FAM84B genome is palbociclib.
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