Expression Of TRPM3and Its Significance In Patients With Primary Hepatocellular Carcinoma

Primary hepatocellular carcinoma (HCC) is the most common malignancy of the liver and a leading cause of cancer mortality. Viral infections, mycotoxin aflatoxin and alcohol intake are considered as the major risk factors for HCC. Tumor resection and liver transplantation offer patients with HCC the best chance for long-term survival. Due to late detection and advanced underlying liver diseases, these treatments are unavailable to the majority of the patients. This loss of surgical opportunity emphasizes the value of early detection and accurate staging to improve clinical outcomes in HCC. Due to development of molecular biology, analysis of tumor on the molecular level may improve the screening and treatment of HCC patients.RNA interference (RNAi) is the process of post-transcriptional gene silencing mediated by double-stranded RNA. Small interfering RNAs (siRNA), a class of19-21bp double-stranded RNAs, can be incorporated into an RNA-induced silencing complex (RISC) to regulate the expression of target genes by either cleavage of target mRNAs or repressing translation and/or promoting mRNA decay based on the complementarity between the siRNA and its target. TRPM3is a member of the melastatin TRP subfamily of cation-permeable ion channels. It is expressed most obviously in the kidney and brain, but wider expression is also apparent. TRPM3channel is thought to play an important role in renal Ca2+homeostasis; It can be activated by extracellular neurosteroid pregnenolone sulfate coupled to insulin secretion in pancreaticβcells; TRPM3is also expressed in neurons of dorsal root ganglia, where it serves as a thermosensitive channel implicated in the detection of noxious heat; Functional relevance of TRPM3in contraction and cytokine secretion of vascular smooth muscle cells has been reported. At present, no data concerning the TRPM3channel expression in human tumor tissues are available, and it is still unknown whether changes in TRPM3expression could be associated with the clinical pathological parameters. In this study we investigate the expression and clinical significance of TRPM3proteins in HCC patients, and the effect of designed TRPM3siRNA sequence on the proliferation of hepatocellular carcinoma HepG2cells.There are two parts in the experiment:Part one:Objective:To investigate the role of TRPM3in hepatocarcino-genesis, we examined the expression of TRPM3in human HCC samples and analyzed the association of TRPM3expression with its clinical significance. Methods:Tissue samples and clinical data were obtained from56patients with HCC who underwent hepatectomy between2005and2009at the Department of Surgery, Xiangya2nd Hospital, Central South University. The expression of TRPM3were assessed by immunohistochemistry, real time quantitative PCR and western blot in the study. Furthermore, we analyzed the correlations between expression of TRPM3and clinicopathological parameters including age, sex, tumor cell differentiation, tumor size and number, portal vein tumor embolus, capsular invasion and capsular formation.Results:1. Western blot:The higher expression of TRPM3was detected in HCC tissues compared with non-tumor tissues.2. Real time PCR: TRPM3mRNA expression quantity in tumor tissues was increased1.5-fold with respect to non-tumor tissues.3. Immunohistochemistry:High expression of TRPM3was observed in32(64.3%) non-tumor tissues samples of56HCC, while32(57.1%) cases were overexpressed in the corresponding tumor tissues.4. Clinicopathological evaluation indicated a significant correlation between TRPM3expression and clinicopathological parameters of histopathological differentiation and capsular invasion (P<0.05).Conclusions:Our results indicate that TRPM3may play roles in hepatocarcinogenesis and can be potentially used as a prognostic indicator of HCC.Part two:Objective:To study the effect of TRPM3siRNA on the proliferation of human hepatocellular carcinoma HepG2cells.Methods:We designed TRPM3siRNA sequence. The proliferation of siRNA-transfected HepG2cells was detected by MTT assay.Result:TRPM3siRNA significantly inhibited the proliferation of HepG2cells.Conclusion:TRPM3siRNA may play roles in the proliferation of human hepatocellular carcinoma cells.