A Possible Mechanism For Low Expression Of Ca²⁺-activated Clchannels,TMEM16A/ANO1 In Hepatocellular Carcinoma And Its Influence On Cellular Proliferation

Objectives: In this study,we want 1?to explore the protein expression levels of T MEM16 A in hepatocellular carcinoma(HCC)tissues?adjacent non-tumor liver tissu es(ANLT)and benign liver tissues and to explore the m RN A expression levels of TMEM16 A in HCC and ANLT;2?to explore the influence of TMEM16 A on the proliferation?migration or apoptosis in HCC-derived cell line SMMC-7721,with the overexpression plasmid shifted into SMMC-7721;3?to explore methylation lev el of the promoter Cp G island of TMEM16 A gene in HCC and ANLT,and to ex plore the influence of 5-aza-2 –deoxycytidine(5-AC),a demethylation reagent,on the expression level of TMEM16 A in SMMC-7721.Methods: 1?Our study involved in forty pairs of HCC tissues and ANLTs randoml y collected from patients who underwent liver resection and six cases of benign li ver tissues at the Department of Hepatobiliary Surgery,the Affiliated Hospital of Qingdao University.The expression levels of TMEM16 A protein in HCC tissue ?ANLTs and benign liver tissues were studied by way of western blot,and that of TMEM16 Am RNA were studied by real time PCR;2?MTT,wound scratch assay a nd Annexin V & PI apoptosis assay were used to explore the effect of TMEM16 A which was expressed by p EGFP-TMEM16 A and pc DNA3.1-TMEM16 A and their control p EGFP-TMEM16 A and pc DNA3.1-TMEM16 A on such cellular functions a s proliferation,migration and apoptosis in SMMC-7721 cells;3?Bisulfite Sequnce PCR(BSP)and DNA sequence analysis was uesd to reveal the methylation level of TMEM16 A gene promoter in HCC tissues and ANLTs,and SMMC-7721 whic h was in the influence of 5-AC was explored by western blot?q RT-PCR and BSP to study the expression of protein?m RNA and the methylation level of TMEM16 A gene promoter.Results:For the first time,we found that: 1?the protein of TMEM16 A in HCC tissues was dramatically low compared to ANLT(P<0.01)and benign liver tissues(P<0.001),and the m RNA of TMEM16 A in HCC issues was low compared t o ANLT(P<0.001);2?In the SMMC-7721 cells which was expressed by p EGF P-TMEM16 A there were more ab normal cells percentage(P<0.001);3?the over expression of TMEM16 A in SMMC-7721 cells resulted in apoptosis(P<0.05)an d inhibited proliferation(P<0.001),but didn't inhibit migration.;4?the methylat ion level of TMEM16 A gene promoter in HCC tissues was highe r than AN TL(P<0.05),and compared with the control,the application of 5-AC in SMMC-7721 r esulted in increasing the protein and m RN A expression(P<0.05)of TMEM16 A a nd decreasing the methylation level of the TMEM16 A gene promoter(P<0.05).Conclusions: Low expression of TMEM16 A in HCC tissue was associated with hy permethylation of its gene promoter;and TMEM16 A may affect proliferation of H CC-derived SMMC-7721 cel s likely via apoptotic mechanism.