The Function And Mechanism Of TSPAN12 In The Tumorigenesis Of Non-small-cell Lung Cancer

Background Non-small-cell lung carcinoma(NSCLC)is one of the leading causes of cancer-related mortality worldwide and accounts for about 85% of all lung cancers.The most common types of NSCLC are squamous cell carcinoma,adenocarcinoma,and large-cell carcinoma,but there are several other types that occur less frequently.Aberrant expressions of proto-oncogenes and tumor-suppressive genes are involved in the carcinogenesis and development of NSCLC,which lead to tumor cell growth and tumor metastasis.Compared with small-cell lung carcinoma,NSCLC is relatively insensitive to chemotherapy.Presently,chemotherapy is increasingly being used to treat NSCLC both preoperatively(neoadjuvant chemotherapy)and postoperatively(adjuvant chemotherapy),and surgical resection is still the primary therapy,but the 5-year overall survival rate is just 16% for all stages.Considering the complex nature of NSCLC,it is of great importance to discover molecular mechanisms involved in initiation and progression of NSCLC.Tetraspanin 12(TSPAN12),a member of the phylogenetically ancient tetraspanin family,is linked to impaired vascularization of the eye called familial exudative vitreoretinopathy,while the function and mechanism of TSPAN12 in NSCLC have not been well characterized.Methods and materials Paracarcinoma lung tissues and NSCLC tissues(n=16)were collected from NSCLC patients enrolled in the Affiliated Huai’an No.1 People’s Hospital of Nanjing Medecal University.IHC was performed to detect the expression level of TSPAN12 in NSCLC tissues and normal adjacent tissues.q RT-PCR was performed to detect m RNA levels of TSPAN12.The protein levels of TSPAN12 in the paracarcinoma lung tissue and lung cancer tissue were analyzed by Western blot.TSPAN12 was knocked down by sh RNA-derived lentivirus in A549 and H1299 cells.Western blot analysis and q RT-PCR analysis were conducted to determine the expression levels of TSPAN12 in these cells.MTT and colony formation assays were used to observe impacts of TSPAN12 on cell proliferation and viability in NSCLC cells.Also,flow cytometry was used to examine the cell cycle and apoptosis of NSCLC cells after the silence of TSPAN12.Immunofluorescence stains were performed to assess the level of p53 in A549 and wild-type p53 expression plasid in H1299 cells with TSPAN12 knockdown.Western blot analysis was performed to assess the levels of p21,p27,and p53 in A549 and H1299 cells with TSPAN12 knockdown.Nude mice xenograft model was constructed to analyze tumor growth impact of down-regulation of TSPAN12 in vivo.Results In our study,TSPAN12 was found to regulate the growth of NSCLC cells both in vitro and in vivo.TSPAN12 m RNA level was significantly increased in human NSCLC samples compared with their corresponding paracancerous histologic normal tissues.In addition,TSPAN12 expression,which was frequently upregulated in NSCLC,was inversely correlated with p53 expression.Furthermore,the expression levels of TSPAN12 were also increased in three human NSCLC cell lines compared to human bronchial epithelial(16HBE)cells.Then,we studied the effects of TSPAN12 silencing by sh RNA on NSCLC cell growth in vitro and tumor growth in vivo,along with the effect on p53 expression.Knockdown of TSPAN12 in NSCLC cells inhibited cell proliferation and colony formation.In addition,knockdown of TSPAN12 increased apoptosis in NSCLC cells.In mechanism,TSPAN12 could modulate the expression of p53,p21,and p27 in NSCLC cells.In a tumor xenograft model,TSPAN12 silencing inhibited the tumor growth in vivo.Conclusion Taken together,our results revealed that TSPAN12 played an important role in NSCLC and was a potential biomarker and a promising target in the treatment of NSCLC.