Novel Role For The Transient Receptor Potential Channel Trpm2in Proliferation And Apoptosis Of Human Tongue Carcinoma Cell

ObjectiveTo investigate the expression of Transient Receptor Potential Melastatin type2(TRPM2) in the human tongue squamous cell carcinoma tissues and SCC cell lines(SCC9、SCC25).The effect of activated and down-regulation of TRPM2on SCC cell lines were observed.Methods(1) Immunohistochemical staining was used to survey the expression of TRPM2in human carcinoma of tongue specimens、papilloma of tongue specimens and non-malignant tongue tissues, and the data were analyzed with SPSS11.0software package.(2) Immunofluorescence cell staining was used to survey the expression of TRPM2in cell line SCC9, SCC25and Hela.(3) Western blot and Real time quantitative polymerase chain reaction (Real-time PCR) were used to survey the expression of TRPM2in human carcinoma of tongue specimens、SCC cell lines、breast cancer cell line、 normal tongue epithelial cells and non-malignant tongue tissues.(4) MTT assay、trypan blue staining assay and flow cytometry techniques were used to study the effect of exogenous H2O2on the proliferation、apoptosis and cell viability of SCC cell lines, respectively.(5) Western blot was used to detect the effect of exogenous H2O2on expression of tumor and apoptosis related protein in SCC cell lines.(6) Western blot、Real-time PCR and flow cytometry techniques were used to study the altered expression of TRPM2tumor related protein and cell apoptosis rate in SCC cell lines after treated by shRNA technique.Results(1) Immunohistochemical staining showed TRPM2was strongly positive in 22/23human carcinoma of tongue specimens and was weakly positive in1/23human carcinoma of tongue specimens; TRPM2was strongly positive in0/12papilloma of tongue specimens and weakly positive in6/12papilloma of tongue specimens while negative in the rest of tongue papilloma tissues; TRPM2was weakly positive in1/9non-maligna-nt tongue tissues while negative in8/9non-malignant tongue tissues. The expression of TRPM2was significant difference between human carcinoma of tongue specimens、 papilloma of tongue specimens and non-malignant tongue tissues (p<0.05).(2) Immunofluorescence cell staining showed the expression of TRPM2was positive in cell line SCC9and SCC25while using cervical cancer cell line Hela cell as positive reference.(3) Western blot and Real-time PCR indicated the expression of TRPM2in SCC cell lines and human tongue squamous carcinoma tissues was significantly higher than normal tongue tissues,using cervical cancer cell line Hela cell as positive reference.(4) The proliferation and cell viability of SCC cell lines were significantly inhibited by different concentrations of H2O2(p<0.05)in a dose dependent manner,while apoptosis rate of SCC cell lines was notably increased by different concentrations of H2O2(p<0.05) also in a dose dependent manner.(5) Western blot showed that expression of the tumor and apoptosis related protein including p53、p21、pro-caspase3、pro-caspase9and bcl-2reduced in a dose dependent maner in SCC cell lines by exogenous H2O2.(6) Western blot and Real-time PCR showed that the expression of TRPM2and p53reduced in SCC cell lines, while the expression of p21increased in SCC cell lines after treated by shRNA technique. Flow cytometry techniques indicated the apoptosis rate of SCC cell lines significantly increased(p<0.05).Conclusion (1) The high expression of TRPM2in human carcinoma of tongue specimens and SCC cell lines indicated that the progression of tumor may have something to do with its abnormal expression.(2) Activation of TRPM2channel in SCC cell lines significantly inhibited their proliferation and cell viability while increased cell apoptosis rate,and altered the expression of tumor and apoptosis related protein,which indicated that ionophorous proteins may play an importmant part in the process of the tumour generation and demonstrated a possible downstream mechanism which TRPM2may involved in.(3) Selectively down-regulation of TRPM2in SCC cell lines promoted apoptosis of cell and altered the expression of related protein,indicated that TRPM2may play an important role of dual regulation in progression of tumor and it may provide a new way for tumor gene-targeted therapy.