| Background:Cervical cancer is one of the most common malignant tumors that seriously threaten women’s health and is the fourth leading cause of female cancer-related deaths.There are over 600,000 new cases and over 300,000 deaths worldwide.Although effective screening has improved the early detection rate of cervical cancer and multidisciplinary treatment has been continuously developed and improved,the prognosis of patients with intermediate and advanced cervical cancer remains poor,with a five-year survival rate of fewer than 50%.As a result,it is critical to investigate the molecular mechanisms of cervical cancer occurrence and progression,as well as to identify effective tumor markers and therapeutic targets for delaying or avoiding cervical cancer recurrence and improving patient survival.The C2 domain is a calcium-binding domain with about 130 residues that is found in many eukaryotic proteins.Proteins with C2 domains have been shown to play crucial roles in numerous biological processes,including protein interaction,cell signal transduction,tumorigenesis,and tumor development.For example,NEDD4 L with the C2 domain is down-regulated in ovarian cancer and controls the proliferation,apoptosis,invasion,and metastasis of ovarian cancer cells.Smurfs with C2 domains can directly interact with PIPKI kinase domains,regulating lung cancer cell proliferation and migration.Because the tandem C2 protein family’s carboxy-terminal type member TC2 N contains a C2 domain,its structural characteristics suggest that it may play a role in the occurrence and progression of tumors.Currently,research on the TC2N’s effects and molecular mechanisms on lung cancer and breast cancer has been published.We intend to investigate the effects of TC2 N on the proliferation and apoptosis of cervical cancer cells further,to clarify the mechanism of TC2 N in the proliferation and apoptosis of cervical cancer,and to offer a theoretical framework for the discovery of new oncogenes and the hunt for novel effective cervical cancer targets.Methods:1 To detect the TC2 N gene expression in cervical cancer tissues and normal cervical tissues.To analyze the TC2 N m RNA expression between cervical cancer tissues and normal cervical tissues through the Oncomine and TCGA database.To further analyze the TC2 N expression between human cervical cancer tissues and paired adjacent tissues by q RT-PCR,Western blot and immunohistochemistry.2 To detect the m RNA and protein expression of TC2 N gene in cervical cancer cell lines(HeLa,Si Ha)and cervical epithelium cell line(HUCEC).To detect the m RNA and protein expression of TC2 N in cervical cancer cell lines(HeLa and Si Ha)and cervical epithelium cell line(HUCEC)by q RT-PCR and Western blot.3 Correlation analysis of TC2 N gene protein expression level with clinicopathologic features and survival of cervical cancer patients.To evaluate the correlation between the protein expression of TC2 N gene and 89 cervical cancer patients’ premenopausal or postmenopausal status,HPV infection,pathological type,FIGO stage,differentiation degree,tumor size,depth of invasion,lymph node invasion and vascular metastasis.The Kaplan-Meier method was used to investigate the relationship between the TC2 N protein expression and the survival time of cervical cancer patients.4 To evaluate the effects of TC2 N gene on the proliferation and apoptosis of cervical cancer cells.To evaluate the effect of differential expression of TC2 N gene on proliferation and apoptosis of cervical cancer cells in vitro by CCK-8,colony formation assay and apoptosis assay.To analyze the effect of differential expression of TC2 N gene on tumor growth in vivo through implanting stable transfected cervical cancer cells subcutaneously in nude mice.5 To investigate the mechanism of how TC2 N gene regulated cervical cancer cell proliferation and apoptosis.High-throughput sequencing technology was used to investigate the downstream signaling pathway of TC2 N gene that regulated the proliferation and apoptosis of cervical cancer cells.The downstream signaling pathway related proteins were detected by Western blot in stable transfected cervical cancer cells,and further in xenograft tumors from nude mice received subcutaneous injections of HeLa and Si Ha stable transfectants.After the application of pathway inhibitors,it was further verified that TC2 N gene was involved in regulating the downstream pathway and thus affecting the proliferation and apoptosis of cervical cancer cells by Western blot assay,clonal formation assay,CCK-8 assay and cell apoptosis assay.Results:1 The expression of TC2 N in cervical cancer tissues was significantly higher than that in normal cervical tissues.By analyzing cervical cancer datasets in TCGA and Oncomine databases,researchers discovered that TC2 N m RNA expression was considerably greater in cervical cancer tissues than in normal cervical tissues(P <0.05).The m RNA and protein expression of TC2 N were studied further in 89 cervical cancer samples and 89 paired adjacent normal samples.The findings revealed that the m RNA and protein expression of TC2 N were considerably greater in cervical cancer tissues than in normal cervical tissues.2 The m RNA and protein expression of TC2 N gene were significantly higher in cervical cancer cell lines than that in cervical epithelium cell line.The m RNA and protein expression of TC2 N gene in cervical cancer cell lines(HeLa,Si Ha)and cervical epithelium cell line(HUCEC)were detected using q RT-PCR and Western blot.Cervical cancer cell lines(HeLa,Si Ha)had significantly higher m RNA and protein expression of TC2 N gene than cervical epithelium cell line(HUCEC)(P <0.01).3 The TC2 N protein expression was positively associated to FIGO stage,tumor diameter and lymph node invasion of patients with cervical cancer,and negatively correlated with the survival of patients with cervical cancer.The TC2 N protein expression was found to be positively associated to FIGO stage(P <0.05),tumor diameter(P <0.01),and lymph node invasion(P <0.05)in cervical cancer patients after analyzing the protein expression of TC2 N gene and clinicopathological parameters.The Kaplan-Meier method was used to investigate the association between the protein expression of TC2 N gene and cervical cancer patients’ overall survival and disease free survival,and it was found that the protein expression of TC2 N gene was negatively correlated with the survival of patients with cervical cancer(P <0.05).4 Interfering the expression of TC2 N gene can inhibit proliferation and promote apoptosis of cervical cancer cells.To explore the effect of TC2 N gene differential expression on cervical cancer cell proliferation and apoptosis in vitro,the colony formation assay,CCK-8 assay,and apoptosis assay were utilized.It was found that TC2 N gene knockdown inhibited proliferation(P <0.01),colony formation ability(P <0.01),and increased apoptosis(P<0.01)in cervical cancer cells.The tumor formation assay in nude mice was used to investigate the influence of differential expression of TC2 N gene on tumor growth in vivo.It was found that TC2 N gene knockdown inhibited the growth of subcutaneous implantation tumor in nude mice(P <0.01).5 TC2 N gene promoted proliferation and inhibited apoptosis of cervical cancer cells by activating MAPK/ERK pathway.Cervical cancer cells with differentially expressed TC2 N gene were analyzed by high-throughput sequencing technology,and 574 differentially expressed genes were found,and the differentially expressed genes were obviously enriched in MAPK signaling pathway.MAPK/ERK pathway related proteins Raf,ERK,p-ERK,c-Fos,and c-Myc were detected by Western blot in stable transfected cervical cancer cells and xenograft tumors from nude mice subcutaneously injected with stable transfected cervical cancer cells to determine whether TC2 N gene was involved in the regulation of the MAPK/ERK pathway.The results showed that TC2 N gene knockdown significantly inhibited the activity of the MAPK/ERK pathway,as well as the expression of related proteins Raf,p-ERK,c-Fos,and c-Myc.Furthermore,the MAPK pathway inhibitor U0126 was applied to cervical cancer cells overexpressing TC2 N gene,and it was found that U0126 restore the promoted proliferation and decreased apoptosis caused by TC2 N gene overexpression(P <0.01).It was further verified that TC2 N gene participated in the regulation of MAPK/ERK pathway in cervical cancer.Conclusions:1 As a new oncogene of cervical cancer,TC2 N gene was highly expressed in cervical cancer and was positively correlated with FIGO stage,tumor diameter and lymph node invasion while negatively correlated with survival.2 TC2 N gene promoted the cervical cancer proliferation while inhibited apoptosis.3 TC2 N gene promoted cell proliferation while inhibited apoptosis in cervical cancer cells through activating the MAPK/ERK signaling pathway. |
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