Therapeutic Efficacy Of Bifunctional SiRNA Combining HER2 Silencing With RIG-I Activation In Gastric Cancer

Gastric cancer is not only one of the most common cancers worldwide,but also a common cause of cancer-related death.Currently,chemotherapy and surgical resection are still the main treatment for patients with gastric cancer,however,these two treatment methods are not very effective.Because of poor outcome and high recurrence rate of gastric cancer,targeted drugs are required us to actively look for.The studies have reported that the variants of immune response-related genes,metabolic enzyme genes,DNA repair genes,tumor suppressor genes and gastric mucosal protection genes might have a strong correlation with the risk of gastric cancer.HER2,a proto-oncogene,is located on chromosome 17q21.A large number of studies have shown that with the overexpressed of proto-oncogene,the expression of HER2 receptor on the cell membrane is increased,and the cell carcinoma is promoted,leading to the occurrence of cancer.The association between HER2 overexpression and the poor prognosis and recurrence rates in patients with gastric cancer was also identified.Through fluorescence in situ hybridization and immunohistochemistry,researchers found 7%~35% of gastric cancer patients were HER2-positive patients.Preclinical data show that HER2 specific monoclonal antibodies have significant antitumor effects in in vivo and in vitro experiments,but the HER2 positive patients has low response.At present RNAi has been used in very disease research.Further studies have shown that some siRNAs have an immune-activating effect that recognizes innate immune receptors in the body.This finding opens a new perspective for the treatment of cancer.In this study,we designed a specific HER2 siRNA trough NCBI database,and selected the most efficient HER2 siRNA.Then HER2 3p-siRNA was synthesized by biochemical method.The aim of this present study was to investigate the effect of HER2 3p-siRNA on the expression of HER2 and immune activation of gastric cancer cells,moreover the effect of HER2 3p-siRNA on the proliferation and apoptosis of gastric cancer cells and its molecular mechanism were further researched.This study was divided in three parts.In the first part,the main purpose of the experiment was to synthesize the HER2 3psiRNA by biological methods,and to confirm the identification of the HER2 3p-siRNA.The HER2 gene-specific siRNA was designed based on the NCBI database.We dectected the mRNA and protein level of HER2 after siRNA interfernce,and the siRNA2 with the best interference effciency was selected as the template to synthesize HER2 3p-siRNA.The result of aggallate gel electrophoresis showed that the synthesized HER2 3p-siRNA had the same sequence length 23 bp with siRNA2.Additionally,luciferase activity assay indicated that HER2 3p-siRNA significantly activated the promoter activity of IFN-β.In the second part,the main purpose of the experiment was to explored the effect of HER2 3p-siRNA on the expression of HER2 and immune activation.Furthermore we detected the change of HER2 3p-siRNA on apoptosis and proliferation of gastric cancer cells,and the mechanism also be researched.Our results showed that HER2 3p-siRNA increased the expression of p IRF-3,IP-10,and MHC-I through nonsepcific activation of RIG-I,and decreased the expression of HER2.Moreover,we found that the two functions of HER2 3p-siRNA not only induced cell apoptosis but also inhibited the proliferation of gastric cancer cells.Further studies showed that HER2 3p-siRNA increased the expression of pro-apoptotic protein Noxa and cytochrome c,at the same time the activation of Caspase 3 and Caspase 9 were triggered.In addition,the combination of Cisplatin and HER2 3p-siRNA decreased the proliferation and incresaed the apoptosis of gastric cancer cells,but the effect of HER2 3p-siRNA on the proliferation and apoptosis of gastric immortalized cell was not obvious.The third part experiment aimed to research the effect of HER2 3p-siRNA on tumor apoptosis and proliferation or growth both in vitro and in vivo.In vitro,we synthetized the HER2 3p-siRNA of mouse and investigated the bi-function of HER2 3p-siRNA on mouse gastric cancer cell line MFC.In vivo,we established the mouse tumor model by subcutaneous injection of MFC cells and intravenously injected the HER2 3p-siRNA by tail.Then,tumor growth and the mechanism were studies.The results of bioassay showed that HER2 3p-siRNA inhibited the expression of HER2 in MFC cells and promoted the expression of RIG-I,p IRF-3,IFN-β,MHC-I and IP-10.Moreover,HER2 3p-siRNA induced the apoptosis of MFC and decreased the cell survival.The results of vitro experiment indicated that the synthetic HER2 3p-siRNA had two aspect function and this bi-function impact the apoptosis and proliferation of gastric cancer cells.The experiment of animal model showed that HER2 3p-siRNA increased the concentration of IFN-β and IFN-γ in serum of mice,and the toxicity of HER2 3p-siRNA was weak in mice liver,spleen,kidney and lung.Additionally,tumor growth was been found significantly inhibit by HER2 3p-siRNA.Futhermore,we detected the mechanism and found that HER2 3psiRNA significantly triggered the expression of IP-10,IFN-β and IFN-γ in tumor tissue,and enhanced the tissue infiltration of CD8+T cell.Finally,the combination of HER2 3psiRNA and Cisplatin significantly inhibit the tumor growth,and these provide a new approach for the combination therapy.In this study,HER2 3p-siRNA was synthesized and the effect of HER2 3p-siRNA on apoptosis and proliferation of gastric cancer cells was investigated by in vivo and in vitro experiments.The results of this study lay the experimental basis and provide new ideas to finding new approach for HER2-positive gastric cancer patients.