The Mechanism Of TMEM9 Regulating Wnt/β-catenin Signaling Pathway To Promote The Invasion And Migration Of Triple-negative Breast Cancer Cells

OBJECTIVES: The main treatment modality for TNBC has been chemotherapy,with no significant improvement in efficacy.In recent years,molecular targeting and immunotherapy have achieved significant clinical efficacy in the treatment of malignant tumors.No effective molecular targets with high specificity have been identified in triple-negative breast cancer,so screening genes involved in regulating the proliferation,invasion and metastasis related to triple-negative breast cancer cells is a current hot spot for exploration and research.METHODS: The TCGA database was searched to analyze the expression of TMEM9 in breast cancer tissues and normal breast tissues.Human normal breast epithelial cells MCF10 a and different subtypes of breast cancer cell lines Bcap-37,SK-BR-3,MCF-7,MDA-MB-453 were cultured in vitro to determine the expression of TMEM9 in different breast cancer cell lines.Surgical specimens from triple-negative breast cancer patients were collected and Western Blot assays were performed to detect the difference in TMEM9 expression in cancerous and paraneoplastic tissues.The MDA-MB-453 cell line that interfered with TMEM9 expression was constructed using lentivirus,and the sequence that targeted TMEM9 with the highest interference efficiency was screened by puromycin,and the transfection effect was detected by Western Blot technique.CCK8 proliferation assay was selected to observe the effect of interfering with TMEM9 expression on the proliferation ability of MDA-MB-453 cells;Transwell assay was performed to observe the effect of interfering with TMEM9 expression on the migration and invasion ability of MDA-MB-453 cells;a tumor-bearing mouse model was constructed to verify the cancer-promoting function of TMEM9.western blot was performed to detect the difference between wild-type MDA The expression of Wnt/β-catenin signaling pathway-related molecules in wild-type MDA-453 cell line and MDA-MB-453 cell line with silenced TMEM9 expression was examined by Western Blot.RESULTS: The expression of TMEM9 was significantly higher in triple-negative breast cancer tissues(115 cases)compared with normal breast tissues(11 cases)in the TCGA database,with a statistically significant difference(P < 0.05).the higher the expression of TMEM9 in 176 patients with metastasis-free triple-negative breast cancer in the Kaplan-Meier Plotter database,the shorter the recurrence-free survival,with a statistically significant(P < 0.05).The expression of TMEM9 in surgical specimens from triple-negative breast cancer patients was also significantly higher than that in paracancerous tissues.The expression of TMEM9 in the triple-negative breast cancer cell line MDA-MB-453 was significantly higher than that in other subtypes of breast cancer cell lines and normal breast epithelial cell lines.western Blot results showed that the experimental group(sh TMEM9#1 group)had the lowest expression of TMEM9 in MDA-MB-453 cells and the stable transgenic strain was successfully constructed.the results of CCK8 proliferation assay showed that The results of CCK8 proliferation assay showed that the proliferation ability of sh TMEM9#1 group was lower than that of NC group,and the difference was statistically significant(P < 0.05);the results of Transwell invasion assay showed that the number of invaded cells in sh TMEM9#1 group was less than that of NC group,and the difference was statistically significant(P < 0.05);the results of Transwell migration assay showed that the number of migrated cells in sh TMEM9#1group was less than that of NC group,and the difference was statistically significant.In the mouse model,the tumor volume and weight of sh TMEM9#1 group were smaller than those of NC group,and the difference was statistically significant(P < 0.05);TMEM9silencing could affect the expression of downstream genes c-Jun and Cyclin D1 in Wnt/beta-catenin signaling pathway,and the difference was statistically significant(P <0.05).P < 0.05).CONCLUSIONS: The expression of TMEM9 in triple-negative breast cancer tissues was significantly higher than that in paraneoplastic tissues.the expression level of TMEM9 was closely related to clinical prognosis.The expression of TMEM9 in triple-negative breast cancer cell lines was significantly higher than that in other subtypes of breast cancer cells and normal breast cancer epithelial cells.In this study,after constructing MDA-MB-453 cell line with TMEM9 silent expression and stable amplification,and conducting a series of functional validation and mechanism exploration,we tentatively deduced that TMEM9 could promote the expression of downstream genes c-Jun and Cyclin D1 through affecting Wnt/beta-catenin signaling pathway,and then promote the invasion and metastasis of triple-negative breast cancer.This study provides a new theoretical basis for interfering with TMEM9 gene expression to improve the prognosis of triple-negative breast cancer from an epigenetic perspective.