Screening And Mechanism Research Of Prostate Cancer Driver Genes Based On CRISPR And Bioinformatics Technology

Background and PurposeAccording to the 2018 cancer statistics report,prostate cancer（PCa）accounts for about one-fifth of newly diagnosed cancer,and the incidence increases with age and is the most common cancer in men（164,690 new cases）^[1].According to the report in the SEER database,although the 5-year survival rate of PCa is very high（98.2%）,for 5 percent of men who have distant metastasis at the time of diagnosis,their 5-year survival rate is still only 30%^[2].Compared with western developed countries,the incidence of PCa in China is at a low level,but with the change of lifestyle,population aging and the popularity of prostate specific antigen（PSA）detection,the incidence of PCa is also rapid.The trend of uplift,and the proportion of patients with advanced disease in the newly diagnosed patients has become one of the important diseases that threaten the health of Chinese men^[3].Malignant tumors are a genomic-level disease.Each person has a unique genome that determines the risk of developing a tumor.Mutations in certain important genes play a key role in tumorigenesis,and once the tumor occurs,it will Further instability of the cancer cell genome will quickly lead to other unique mutations and genomic rearrangements.With the accumulation of a large number of somatic mutations and genomic rearrangements,the tumor continues to evolve,and the risk of drug resistance or metastasis increases.In this process,mutations that play a key role in the development of cancer are called driving mutations.The genes involved are called driving genes,and the remaining mutations are called passenger mutations^[4].In recent years,targeted therapy has become an effective strategy for the treatment of cancer,and it can also be used as a neoadjuvant therapy after surgical resection to reduce the rate of cancer recurrence.However,the utility of targeted therapy relies on the precise identification of specific targets during tumorigenesis and progression.Next Generation Sequencing（NGS）,also known as second-generation sequencing,has become a valuable tool in cancer research,analyzing genomic aberrations with single-base resolution,including deoxynucleotide mutations,splicing isoforms,non-coding RNA,DNA methylation,and protein nucleic acid interactions.However,the genes whose expression levels are significantly altered by the current second-generation sequencing technology do not necessarily play a role in the disease process.There are often no obvious phenotypic changes after interfering with these genes,and it is not efficient in identifying the true driving genes^[5].In order to screen out the driving genes in tumors,Chen S et al.introduced the CRISPR library technology^[6].CRISPR/Cas9 technology is a gene editing technology that has attracted much attention in recent years,except that gene knocking and gene knockout can be performed at specific sites.In addition to basic functions,there are also derivative technologies such as gene interference and gene activation^[7],which are widely used in clinical diseases and applications.By constructing a mouse full-length CRISPR library,Chen S et al.successfully identified genes based on phenotype and found 624 genes that cause lung cancer metastasis in 67,405 genes.However,the screening of whole-genuine CRISPR libraries will undoubtedly consume huge human,material and financial resources.In addition,all genes are included in the study,and there is a certain loss rate,and the actual mutation rate of the selected genes in cancer may be low,making The true tumor-driven genes are ignored.Therefore,this master’s research project mainly combines the second-generation sequencing and CRISPR library screening strategy,and uses the results of high-throughput sequencing of PCa cancer and adjacent tissues to establish a PCa-specific gRNA library,which is screened in the development of PCa.The key role of the driving gene.In addition,we have applied emerging bioinformatics techniques to predict driver genes that may be involved in PCa metastasis and predict compounds with potential anti-PCa activity.Finally,we validated the driver genes in clinical samples and cell lines and confirmed the effectiveness of these drugs.Part I:Screening and identification of prostate cancer driver genes based on CRISPR technologyObjective:To screen PCa-specific driver genes using CRISPR library and to identify their functions.Methods:（1）Construction of a CRISPR lentivirus library（containing blasticidin（BSD）resistance）.（2）Screening of cell transfection and cell positive phenotypes.（3）Ta clone sanger sequencing identification library and enriched genes.（4）siRNA interference drives the gene to reproduce its phenotype.Results:（1）A CRISPR library was constructed using 65 differentially expressed genes in the transcriptome sequencing of PCa cancer and paracancerous tissues.（2）Transfected with RISPE-1,C4-2B,DU 145,LNCap,and PC-3 cells using CRISPR library and obtained stable cell lines.After serial passage,clones enriched in cell proliferation were obtained.（3）The library and the most abundant gene MMS22L were identified by Ta cloning and sanger sequencing.（4）MMS22L reproduces its inhibitory effect on cell proliferation by siRNA interference and EdU assay.Conclusion:Based on the second-generation sequencing results we obtained earlier,we constructed a PCa-related CRISPR/Cas9 library and constructed a stable cell line.Furthermore,the MMS22L gene was cloned and identified by cell proliferation and enrichment,and its inhibition on cell proliferation was reproduced.Part II:Screening of prostate cancer driver genes based on bioinformatics and drug candidate identificationObjective:To screen for PCa-related driver genes and identify potential anti-PCa drugs through a public database.Methods:（1）Weighted gene network analysis（WGCNA）was performed based on the data collected from the metastatic PCa expression profile chip dataset GSE6919 and NCBI-Gene database obtained from the GEO database and 10 drivers were selected.gene.（2）Collect PCa tissue samples with different Gleason scores in our hospital and identify the driver genes in tissues and different cell lines by QRT-PCR.（3）Find candidate anti-PCa drugs that regulate the driving genes from the DrugBank database.Results:（1）A total of 72 PCa patients were collected,including 45 low Gleason scores（Gleason score=6,7,8）and 27 high Gleason scores（Gleason score=9,10）.（2）Among the 10driving genes,the expression levels of 5 genes are related to the malignant degree of different prostate cancer cell lines,and the expression levels of 4 genes in the 5 genes are related to the Gleason score of clinical samples.（3）A total of 211 candidate anti-PCa drugs were obtained from the DrugBank database after searching for compounds that regulate the driving genes.Conclusion:We screened 10 driver genes by WGCNA on the data of metastatic PCa expression profile and NCBI-Gene database.The selected driver genes were well verified in tissues and cells,and passed the DrugBank database.We obtained 211 drugs that may have anti-cancer activity.Part III:Verification of candidate drug activity and identification of specific inhibition of copper oxide on prostate cancer cellsObjective:To verify drug candidate activity and specific inhibition of PCa cells by copper oxide in cell lines.Methods:（1）CCK-8 experiments were performed on drug candidates in RWPE-1 and different PCa cell lines using a compound library to identify drugs with antitumor activity.（2）The half maximal inhibitory concentration（IC50）of copper oxide on PCa cells and RWPE-1 was determined by CCK-8 assay.（3）The effects of copper oxide on tumor cell migration and invasion were determined by Transwell experiment and Invasion experiment.（4）Using PI/Annexin V-FITC double-label flow cytometry to determine the apoptosis-inducing effect of copper oxide on PCa cells.（5）Using QRT-PCR technology to preliminarily identify the correlation between the apoptosis-inducing effect of copper oxide on different PCa cell lines and the expression of CIRBP gene.Results:（1）We selected 36 drugs with Connection>3 to construct a compound library,and identified 17 compounds that can significantly inhibit PCa cells and four compounds that specifically target PCa cells according to cck-8 experiments.（2）Copper oxide inhibited the proliferation of PCa cells significantly stronger than normal prostate epithelial cells.（3）Copper oxide can significantly inhibit the migration and invasion ability of various PCa cell lines.（4）Copper oxide can selectively induce apoptosis of PCa cells.（5）The change in the expression level of CIRBP is consistent with the induction of apoptosis by copper oxide.Conclusion:We have identified 17 compounds that can significantly inhibit PCa cells.Among them,copper oxide can specifically inhibit the proliferation,migration and invasion of PCa cells,and can specifically induce tumor cell apoptosis and drive the change and oxidation of gene CIRBP.The effect of copper on tumor cell apoptosis was consistent,suggesting that CIRBP may play a role in the killing effect of copper oxide on tumor cells.SummaryOur study began with the high-throughput transcriptome sequencing of 65 pairs of PCa and adjacent tissues in the early stage of the laboratory,and then screened the driver gene MMS22L based on CRISPR technology and verified its function.In addition,we screened10 driver genes through a public database combined with bioinformatics technology and screened for drugs that target gene-driven genes,and validated the driving genes in cell and clinical samples to clarify the efficacy of the screening strategy,and then based on drug candidates.The compound library was constructed to verify the antitumor activity of these drugs in cell lines.Finally,we focused on copper oxide and demonstrated its ability to specifically inhibit tumor cell proliferation,migration,and invasion in cell lines.Finally,we initially confirmed that the change of the driving gene CIRBP is consistent with the effect of copper oxide-induced tumor cell apoptosis,suggesting that CIRBP may play a role in the killing effect of copper oxide on tumor cells.Using the results of PCa cancer and paracancerous tissue expression profiling,this study combines bioinformatics,CRISPR library and second-generation sequencing technologies to screen for driving genes that play a key role in the development of PCa,and predict the possibility.The anti-PCa drug provides a basis for prostate cancer diagnosis,molecular typing,prognosis and new drug development,and provides a new strategy for studying the molecular mechanism of prostate cancer.