Investigating The Role Of KLK1 In Hepatocellular Carcinoma Based On Single Cell Sequencing Technology

Background and ObjectivePrimary liver cancer is the sixth malignant tumor in the world with the incidence rate,and its mortality rate ranks the third in cancer related deaths.As the main subtype of primary liver cancer,hepatocellular carcinoma(HCC)accounts for 75-85%of the total incidence rate.HCC is a highly heterogeneous malignant tumor.Its pathogenesis has not been fully elucidated,and specific biomarkers are still lacking.As a result,most patients with HCC are in the middle and late stages of diagnosis,with limited therapeutic effect,high recurrence rate,short survival time and poor quality of life.Research shows that KLK1 can affect fibrosis and oxidative stress by regulating related signal pathways,and may be used as a biomarker of cancer.This study uses single-cell sequencing technology to explore the biomarkers and molecular mechanism of HCC,and provides certain reference for the early diagnosis and treatment of HCC,which has important clinical significance.Method1.In this study,the single-cell transcriptome of HCC was obtained from the GEO database,and the differential genes of HCC were screened through the single-cell sequencing technology.2.The differential genes screened from the GEO database are clustered by gene consensus with the TCGA database to screen out the core genes of HCC.3.GO and KEGG enrichment and analysis of core genes and construction of PPI network were used to explore the impact of core genes on the pathogenesis of HCC.4.Western blotting was used to verify the expression of core genes in HCC.5.The core gene knockout HCC cell was constructed by siRNA transfection technology and verified by Western blotting.6.The effect of knockout of core genes on the proliferation,migration and invasion of HCC was verified by cell proliferation assay,colonies formation assays,wound healing assay and transwell cell migration and invasion assay.Result1.Through the single-cell sequencing technology,the differential genes of HCC were screened,and the core gene KLK1 of HCC was screened out by gene consensus clustering with TCGA database.2.In the GO enrichment analysis,it can be seen that the biological processes involved in the differential genes are mainly the migration of immune cells,the regulation of inflammatory reaction and the regulation of angiogenesis,while their molecular functions are mainly related to the activity and hydrolysis of serine as well as the activity and binding of cytokines.3.In the KEGG enrichment analysis,the main signal pathways involved in the differential genes are:IL-17 signal pathway,protein processing in the endoplasmic reticulum and the interaction between cytokines and receptors.4.By constructing the protein-protein interaction network,we obtained the genes related to the core gene KLK1 expression:KNG1,SERPNA4,IGF1,IGF2,IGFALS,IGFBP3.5.We verified that the core gene KLK1 is highly expressed in HCC cell through Western blotting.6.Through siRNA transfection technology,we knockdown siRNA-KLK1 on human hepatocellular carcinoma Huh7 cell line,and confirmed the transfection effect through Western blotting.7.In the cell proliferation assay and colonies formation assays,the proliferation ability of KLK1 knockdown cell line was significantly lower than that of the control group.In the wound healing assay and transwell cell migration and invasion assay,the migration and invasion ability of KLK1 knockdown cell line was significantly lower than that of the control group.Conclusion1.KLK1 gene is highly expressed in HCC and is the core gene of HCC;2.The high expression of KLK1 can promote the proliferation,migration and invasion of HCC.