| According to the 2020 global cancer statistics,lung cancer ranks second in incidence and first in mortality among malignant tumors.Lung cancer is also the most common malignant tumor with the highest mortality rate in China.Usually only 16% of patients can be diagnosed at an early stage,and most patients are diagnosed as advanced or metastatic stages.Cancers are caused by the accumulation of a series of genomic alterations.At present,next-generation sequencing has allowed identification of millions of somatic mutations in cancer cells but only a few mutated genes(among all tumor-associated mutations)appear to be important for cancer initiation and progression.In fact,most genetic mutations within cancer are thought to represent "passenger" or "bystander" mutations.Genome editing offers the possibility to link genotypes to phenotypes through experimental manipulation.Currently,functional genomic screening techniques can effectively identify “driver” mutations that drive cancer initiation and progression from a wide range of mutations.At present,many potential oncogenes and tumor suppressor genes(TSGs)have been discovered and their functions have been annotated.However,due to the tumor heterogeneity,many functional genes affecting the occurrence and development of cancer have not been discovered.In many cases,TSGs are inactivated by transcriptional repression(often referred to as epigenetic silencing)rather than by mutational events,and thus would not be identified by conventional genome-sequencing methods.The high-throughput positive screening of the CRISPR-Cas9 knockout library is to exert a certain screening pressure on the cells that have successfully integrated the knockout library,so that only a small number of cells with phenotypes can survive and achieve the purpose of enriching key genes.By transplanting normal cells with integrated CRISPR-Cas9 knockout library into nude mice subcutaneously,a small number of neoplastic cells can survive,thereby enriching TSGs that are key to the neoplastic transformation process of normal cells.Therefore,screening new TSGs is crucial for studying the mechanism of early cancer occurrence,and provides new ideas for early diagnosis of cancer.In addition,non-small-cell lung cancer(NSCLC)is highly heterogeneous and prone to drug resistance,the current clinical treatment effect for NSCLC patients is limited,the 5-year survival rate is still very low,and it is very difficult to restore the function of inactivated TSGs.In fact,TSGs are difficult to be used as drug targets.Therefore,the discovery of new potential drug targets will help to provide a new theoretical basis for the clinical treatment of NSCLC,and the essential genes for cell survival are ideal therapeutic targets.In this study,a human normal lung epithelial BEAS-2B cell line stably expressing Cas9 protein was first constructed,and the cells were infected with a Brunello lentiviral library containing 77,441 single-stranded guide RNAs(sg RNAs)targeting 19,114 human genes,subcutaneously transplanted into nude mice.The whole genome DNA of tumor cells(Tumor)and pre-implantation cells(Input)was extracted,sg RNA sequences were amplified,and a library was constructed for deep sequencing.By analyzing the proportion of sg RNA reads in Tumor and the fold change of sg RNA reads in Tumor and Input,the TSGs that induce neoplastic transformation of BEAS-2B cells after loss of function were screened,and the public data platform was used for preliminary verification.Then,the Bio GRID Open Repository of CRISPR Screens(ORCS)Database Statistics database was used to analyze the data of existing screening database for screening out the candidate essential genes for cell survival.Further,CRISPR lentivirus was used to obtain human lung adenocarcinoma A549 cell line with stable knockout of candidate essential genes for cell survival,and its effects on A549 cell proliferation and clone formation were detected.The main results and conclusions are as follows:1.Genome-wide screening of tumor suppressor genes(TSGs)in neoplastic transformation of human normal lung epithelial cellsThe library infection and subcutaneous tumor formation experiments in nude mice were successfully carried out in BEAS-2B cells stably expressing Cas9 protein.Only cells infected with the library formed tumors at some of the inoculation sites.The sequencing results showed that only a few sg RNAs were detected in Tumor.Here then,38 genes with sg RNA reads accounting for more than 1% of the total reads or that two or more sg RNAs were significantly enriched were selected as effective candidate TSGs for human lung cancer.It includes 5 known TSGs such as NF2 and PTEN,and 33 new candidate TSGs such as AP2M1 and PSENEN that have not been reported in lung cancer.These results mean that the design of the sieve library and the experimental results are real and effective,and subsequent analysis can be carried out.2.Preliminary functional validation of the aforementioned 38 genes in clinical databasesEnrichment analysis found that the aforementioned 38 genes were enriched in key biological pathways involved in carcinogenesis,such as Notch and Hippo signalings.It was confirmed in the clinical NSCLC sample database that all of the aforementioned 38 genes were mutated in NSCLC samples,among which PDCD10 and AP2M1 mutation rates were greater than 15%,and 14 of the 17 genes whose mutation rates were greater than 2% were associated with known highly mutated genes KRAS or TP53 among the co-mutations,3mutations were associated with prognosis;the expression levels of 30 genes in NSCLC samples were significantly different from those in normal adjacent tissues,and the expression levels of 17 genes were closely related to the prognosis of patients.The results preliminarily showed that all of the aforementioned 38 genes actually function as TSGs and have a good correlation with the occurrence and development of clinical NSCLC.3.Selection and functional verification of candidate essential genes for cell survivalEssential genes for cell survival are ideal therapeutic targets.Studies have shown that activation of NF-κB signaling promotes the development and progression of cancer and is related to the maintenance of tumor stem cells.Positive regulatory factors of NF-κB signaling pathway may be potential therapeutic targets.Using the Bio GRID ORCS database,LIN37 that is a positive regulator of NF-κB signaling pathway and essential gene for cell survival in33 different cancer cell lines(including 4 NSCLC cell lines)and PKN3(Protein kinase N3)which is essential gene for cell survival of the human stem cell line HUES6 were selected as new candidate essential gene for cell survival.Then,in cell culture experiments in vitro,lentivirus were infected with lung adenocarcinoma A549 cell line to knock out the candidate essential genes,LIN37 and PKN3,respectively.It showed that both of the proliferation ability of A549 cells decreased.Then,in the 1% methyl cellulose clone formation experiment,the clone formation ability of A549 cells with knockout genes essential for cell survival was significantly reduced,and the number and volume of clones were significantly reduced while compared with the control cells.The results showed that the knockouts of LIN37 or PKN3 could inhibit the proliferation and anchorage-independent growth abilities of A459 cells.To sum up,we first screened out 38 TSGs(including 5 known TSGs and 33 new candidate TSGs)for human lung cancer from human normal lung epithelial BEAS-2B cells by using whole-genome CRISPR/Cas9 technology in vivo,and then preliminarily verified them in the clinical NSCLC sample database.Then,the Bio GRID ORCS database was used to conduct in-depth analysis of the existing screening data,and the candidate essential genes for cell survival,LIN37 and PKN3,were screened out,and it were experimentally verified that their knockout respectively could inhibit the proliferation and clone formation of human lung adenocarcinoma A549 cells,which provides a new target for clinical NSCLC targeted therapy. |
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