The Function And Molecular Mechanism Of PinX1 Involved In The Development Of Melanoma

Background: Melanoma is a highly malignant tumor that is prone to lymphatic and hematogenous metastases in the early stage,is insensitive to radiotherapy and chemotherapy,and has a poor prognosis.There is no effective treatment plan.In recent years,with the discovery of various oncogenes and tumor suppressor genes,various molecular biological methods have been continuously developed and improved.Gene therapy has become a hot spot for the treatment of tumors.Telomerase is activated in most human cancers and is essential for the growth of cancer cells.The transgenic telomerase reverse transcriptase catalytic subunit(TERT)is the core component of telomerase.The PIN2 / TRF1 interaction telomerase inhibitor 1(Pin X1)binds directly to the TERT gene,inhibition of telomerase activity.Essentially,it is an endogenous telomerase inhibitor gene that is considered a potential tumor suppressor gene.The purpose of this study was to analyze the expression of Pin X1 in melanoma tissues,adjacent tissues and normal tissues,and to explore the relationship between Pin X1 and the clinical features of melanoma.At the same time,by adjusting the expression of Pin X1,we investigated the effects of PINX1 on angiogenesis,proliferation,migration and invasion of melanoma cells.Methods: Immunohistochemistry was used to analyze the relationship between the expression of Pin X1 in tissue microarray(TMA)of 208 melanoma patients and the pathological grade and clinical stage of melanoma tissue.The Pin X1-si RNA interference plasmid was constructed and transfected into melanoma cells A375 and MV3 respectively.Western Blot assay was used to detect the interference effect of the two melanoma cells.Angiogenesis experiments were performed to observe the effect of melanoma cell supernatant after silencing Pin X1 on the vascularization ability of HUVECs endothelial cells.CCK8 proliferation assay was used to detect the effect of silencing Pin Xl gene on the proliferation of melanoma cells.Western Blot assay was used to detect the effect of silencing Pin X1 on cell cycle related proteins.Transwell cell migration invasion assay was used to observe the changes of the ability of silencing Pin X1 gene to metastasis and invasion of melanoma cells.Western blot was used to determine the protein expression levels of Pin X1,MMP-9 and TIMP-1.Results: Our tissue microarray(TMA)and immunohistochemical analysis showed that low Pin X1 expression was associated with TNM staging in patients,and that stage I and II Pin X1 staining was significantly lower than stage III-IV(P < 0.001).It was found that the expression of Pin X1 was significantly associated with lymph node metastasis(P < 0.001).Western Blot experiments showed that compared with Control-si RNA group,the expression of Pin X1 protein was significantly decreased in A375 and MV3 melanoma cells.The results of tubule formation experiments showed that the number of tube-like structures formed by HUVEC cultured in the supernatants of both A375 and MV3 melanoma cells after silencing Pin X1 gene was significantly increased compared with the control group(P<0.01).It is suggested that the silencing of Pin X1 gene can enhance the ability of melanoma cells to promote the formation of blood vessel-like structures of endothelial cells.CCK8 cell proliferation experiment results showed that after silencing Pin X1,the two groups of cells were cultured at various time points of 24,48,72,and 96 hours.Proliferation effects were significantly increased.The differences were statistically significant(P<0.01).Western Blot experiments showed that the expression of Cyclin D1 and Cyclin E2 in A375 and MV3 cells increased significantly compared with that in the Control group(P<0.01),and A375 and MV3 cells The expression levels of p27 and p21 were significantly decreased(P<0.01),and the results of Transwell cell migration and invasion assay showed that after silencing Pin X1,the migration ability of the two groups of cells was significantly higher than that of the control group,and the difference was statistically significant(P< 0.01).After silencing Pin X1,the invasive ability of the two groups was significantly higher than that of the control group,and the difference was statistically significant(P<0.01).The expression of TIMP-1 and MMP-9 in melanoma cells after Pin Xl silencing was detected by Western Blot assay.The results showed that compared with the control group(Control-si RNA),the MMPs in A375 and MV3 cells were transfected with Pin X1-si RNA.The protein expression level of-9 was significantly higher(P<0.01).On the contrary,the expression of TIMP-1 protein level was significantly decreased.There was a statistically significant difference compared with the control group(P<0.01).Conclusion: Pin X1 expression was significantly downregulated in melanoma,and its down-regulation was related to the clinical pathological stage and lymph node metastasis of melanoma.The detection of Pin X1 expression level has important clinical significance for evaluating the malignant degree,metastatic potential and prognosis of melanoma.The low expression of Pin X1 in melanoma cells can enhance cell proliferation by positively regulating the cell cycle progression.In addition,silencing Pin X1 gene can also increase the activation of MMP-9 pathway by down-regulating TIMP-1 protein,thereby increasing the melanoma cells.Migration and invasive capabilities.Pin X1 gene may become a new target for targeted therapy of melanoma and is expected to play an important role in the treatment of the development and metastasis of melanoma.