CRISPR/Cas9 In Vivo Screening And Identification Of SDHD As Key Gene For Lung Metastasis In Renal Cell Carcinoma

Research background and objectiveRenal cell carcinoma(RCC),is a common malignant tumor of the urinary system,accounting for 3% of all malignant tumors in adults,ranking 6th and 8th among male and female,respectively.Patients with early stage renal cell carcinoma had no obvious symptoms,and more than 16% of the patients had metastasis at the first diagnosis,of which lung metastasis and bone metastasis were the most common.The prognosis of metastatic renal cell carcinoma is very poor,and the 5-year survival rate is less than 12%.Metastatic renal cell carcinoma is not sensitive to radiotherapy and chemotherapy,and the effect of traditional therapy is not good.The application of TKI targeting drugs,represented by Sunitinib,can improve the overall survival(OS)of metastatic renal cell carcinoma to some extent,but its objective response rate is only about 40%,and drug resistance occurs frequently.In recent years,g RNA-guided CRISPR(cluster regular interval short palindromes)/ Cas9 has recently become a powerful tool for genome research.The editability of CRISPR/Cas9 makes it particularly advantageous in high-throughput genome detection.Simple and effective single-guide RNA(sg RNA)library synthesis enables CRISPR/Cas9 screening to be quickly used to study functional changes after genomic,transcriptional and epigenetic changes.The whole genome screening in vivo can simulate the pathological process of tumor metastasis as far as possible and find out the related genes that play an important role in this process.SDHD(succinate dehydrogenase complex subunit D)is a metabolism-related enzyme in the tricarboxylic acid cycle and is widely expressed in various tissues.Some studies have shown that SDHD is a tumor suppressor gene,which may be related to the occurrence of renal cell carcinoma,but its function in the metastasis of renal cell carcinoma is not clear.Our study will provide a new theoretical basis for the proliferation of renal cell carcinoma and the formation of lung metastasis,and find new potential gene targets for the treatment of renal cell carcinoma.Methods1 ? The in vitro whole-genome CRISPR/Cas9 knockout cell pool model was established by amplifying the human whole-genome knockout library plasmid,preparing the lentivirus library,exploring the screening conditions and establishing the library RCC cell line.2?The screening animal model of lung metastasis of CRISPR/Cas9 renal cell carcinoma was established by xenograft cell transplantation into immunodeficient nude mice,and the tumor formation was observed,tissue samples were collected and high-throughput sequencing was carried out then.3?A series of bioinformatics analysis methods such as MAGe CK(RRA)were used to control and analyze the sequencing data.Combined datas with that from in vitro cell line transcriptome sequencing and bioinformatics database such as TCGA and GEO,the potential key genes of renal cell carcinoma with lung metastasis were screened and identified.4?Functional experiments such as plate cloning,cell proliferation,cell scratching,transwell and tumor formation in mice were carried out to further verify the function of target genes in vitro and in vivo after RCC cell lines were treated with CRISPR/Cas9 knockout and lentivirus transfection.5?Real-time quantitative PCR and Western-blot were used to verify the correlation between the expression level of target genes and clinical efficacy in paired renal cell carcinoma and adjacent tissues.6?The tissue microarray and corresponding clinical data were analyzed,and the value of the target in evaluating the prognosis was analyzed.ResultsThrough the establishment of in vivo genomic CRISPR/Cas9 knockout library model,data bioinformation analysis,functional verification and clinical prognosis analysis,we found that the proliferation,invasion and metastasis ability of renal cancer cell line were improved after SDHD knockout in vitro,but the effect was not obvious after overexpression of the gene.Similarly,in vivo,the ability of tumor formation and metastasis of cells was enhanced after knockout of SDHD,and this effect could be reversed by overexpression.Analysis of TCGA,GEO and other public databases and clinical samples of our center found that in patients with renal cell carcinoma,the lower the expression level of SDHD,the worse the prognosis.ConclusionSDHD shows the characteristics of tumor suppressor gene in renal cell carcinoma,and its inactivation and variation can promote tumor proliferation and metastasis.SDHD may become a new target for gene targeting therapy of renal cell carcinoma and a new molecular marker for predicting the prognosis of renal cell carcinoma.