The Effect Of Capicua On Proliferation And Metastasis Of Malignant Melanoma

Background and objective: Malignant melanoma is a tumor originating from melanocytes in the skin and other organs,characterized by high metastasis and poor prognosis.Despite recent research leading to the rapid development of new treatments,the 5-year survival rate for patients with metastatic malignant melanoma is still only 20%.Individualized targeted therapy and immunotherapy are important approaches for the treatment of malignant melanoma.Clinically,approximately 70%of patients with BRAF mutations receive long-term benefit from combined targeted therapy by blocking the RAS-MAPK pathway.However,these inhibitors are significantly limited in clinical treatment due to the development of drug resistance.Therefore,exploring the mechanisms of malignant melanoma metastasis and drug resistance has always been a serious challenge for researchers.Recent studies have found that Capicua(CIC),as a high mobility group protein inhibitor,is not only involved in the normal development and homeostasis regulation of mammals,but also abnormally expressed in various tumors such as lung cancer,breast cancer,prostate cancer,and liver cancer.It can promote tumorigenesis and development by controlling effector target genes through the MAPK/ERK signaling pathway.CIC is closely related to tumor proliferation,metastasis and treatment resistance,but it’s still not clear whether CIC affects the occurrence and progression of melanoma.Therefore,the function of CIC in malignant melanoma was analyzed by bioinformatics,and RNA interference technology was used to observe the effect of silencing CIC on melanoma cell proliferation and metastasis to explore CIC in the biological behavior of malignant melanoma.Methods: The online database was used to analyze CIC mutation in malignant melanoma and treatment resistance;skin cutaneous melanoma(SKCM)samples were downloaded from the TCGA database,the differentially expressed genes of CIC mutation and wild groups were screened,and functional enrichment was performed.Small interfering RNA(si RNA)was used to construct melanoma cell lines that silenced CIC gene,and the construction was verified by quantitative real-time PCR.CCK-8 assay,clone formation assay,transwell cell migration assay and wound healing test were used to detect the proliferation and migration ability of CIC si RNA and control group.These experiments explore the effect of CIC gene on the proliferation and migration ability of melanoma cells.Western blot was used to detect the expression levels of metastasis associated proteins such as E-cadherin,N-cadherin,Vimentin and MMP9,and tumor growth was observed by subcutaneous tumor model.In addition,the CDK inhibitor Dinaciclib was used to treat melanoma cell lines,and the CCK-8 and clone formation assay were used to observe the effect of Dinaciclib on cell proliferation to explore the role of CDK inhibitors in malignant melanoma cells that silence CIC gene.Results:(1)CIC has multiple site mutations in SKCM,with a mutation rate of 5%,and CIC mutations are resistant to trametinib and other drugs in pan-cancer.Compared to the wild group,41 m RNAs were down-regulated significantly while the 10 m RNAs were up-regulated in the CIC mutant group.MMP9 was shown to be significantly different between the two groups.KEGG pathway analysis Differentially expressed genes were enriched in Salmonella infection,ubiquitin-mediated proteolysis,endophagy,cell cycle and other pathways.(2)q RT-PCR showed that A375 and Hs294 T cell lines were successfully constructed with CIC si RNA;CCK-8 experiment and clone formation experiment showed that the proliferation ability of CIC si RNA transfected melanoma cells was enhanced(p<0.01),and the clone formation rate was increased(p< 0.01);transwell cell migration assay and wound healing test showed that the number of melanoma cells transfected with CIC si RNA increased in migration(p< 0.01).(3)Western blot assay showed that the expression levels of metastasis-related proteins MMP9,N-cadherin and Vimentin in melanoma cells transfected with CIC si RNA increased,and the expression level of E-cadherin decreased;subcutaneous transplanted tumors assay showed that CIC si RNA transfected melanoma cells Subcutaneous tumor cells formed larger volume and weight(p < 0.01).(4)CCK8 and clone formation assay showed that no matter whether CIC was silenced or not,compared with the DMSO group,the proliferation of malignant melanoma cells was reduced after using Dinaciclib;and with the increase of Dinaciclib concentration,the inhibitory effect was more obvious(p<0.05).Conclusion: CIC gene is widely resistant to the treatment of various tumors,and CDK inhibitors have inhibitory effect on malignant melanoma cells after CIC gene silencing.