RNAi Specifically Inhibits The Expression Of Heparanase Gene In Esophageal Carcinoma Cells

Esophageal carcinoma is a kind of the most common malignancies in China, especially in Henan province. Esophageal carcinoma is characterized by high incidence, nonspecific appearance, easily metastasis and high death rate.The treatments include surgery,chemotherapy, radiotherapy and so on, but the therapeutic effect is unsatisfactory. The formation of tumor is a course of many steps, and activity of oncogene is one of the reasons that cause tumors. More investigators have considerd that RNAi technology is used to inhibit expression of oncogege in order to cure tumor.RNA interference (RNAi) is the process of sequence-specific posttranscriptional gene silencing (PTGS) triggered by double stranded RNA (dsRNA). The dsRNA introduced or generated in cells is subjected to digestion with a dsRNA-specific endonuclease, Dicer, into 21-25 nucleotide (nt) RNA duplexes, and the resultant duplexes, referred to as short interfering RNAs (siRNAs) duplexes, function as essential sequence specific mediators of RNAi in the RNA-induced silencing complexes (RISC). These reactions result in the cleavage of the target mRNA. The exploratory development of RNAi is manly practiced by synthesized in vitro siRNA and prodused in vivo siRNA. SiRNA can obviously inhibit expression of target gene; moreover, specificness of depressive effect is powerful. At present, experimental studies in the domain of anti-virus, anti-tumor and so on are frequently carried out by means of RNAi technology.Heparanase (HPA) is one kind of cysteine proteases in cytolysosome. It can directly degrade extracellular matrix(ECM) or indirectly degrade ECM by activating plasminogen activator(PA) and matrix metalloproteinase so on; thus participate invasion and matastasis process of tumor. In addition, HPA can promote hyperplasy of tumor vessel, strengthen motor ability of tumor cell; moreover its level of transcription and expression have positive correlation with tumorous prognosis. It has been discovered that expression and activity of HPA is double even 3-9 times higher than near normal tissue in many malignant tumor tissue such as gastric cancer, lung cancer, colon carcinoma, breast cancer, prostatic carcinoma, glioma, bladder carcinoma, ovarian cancer, melanoma, thyroid cancer by using in situ hybridization(ISH), western blot, immunohistochemistry, et al. Some scholars think that HPA is likely to become a new target of tumorous gene therapy and a index of auxiliary diagnosis.In this study, RNAi technology was used to inhibit heparanase gene in esophageal carcinoma EC9706 cells. After the treatment, The expression of HPA protein and mRNA in esophageal carcinoma deteted by immunohistochemistry and in situ hybridization. Our study would provide the laboratory evidence for the future gene therapy of esophageal carcinoma using RNAi.Materials and MethodsThe culture of the esophageal carcinoma EC9706 cells: The EC9706 cells were adherent-cultured with culture fluid of 10% fetal bovine serum under condition of 37℃and 5%CO₂.Synthesis and identification of siRNA: The DNA templates were synthesized firstly, which included T7 RNA Polymerase promoter sequence at 5' end that could bind to the T7 RNA Polymerase to transcript the target siRNA in vitro. The synthesized two siRNA were 21 nt; were specific and unspecific respectively. 4% gel electrophoresis was used to detect the length and concentration of the synthesized siRNAs.Transfection: Lipofectamine^RM2000 siRNA Transfection Reagent was used to Transfect the siRNAs into EC9706 cells.HPA siRNA inhibited expression in vitro: 10μmol/L, 15μmol/μl,20μmol/L of HPA siRNA which synthesized in vitro were transfected into EC9706 cells, then cultured 24h,48h,72h.The non-sense sequence group and non-transfected group were used to control.The detection after transfection: In situ hybridration and immunohisto-chemistry , and nude mice model with transplanted human esophageal cancer cells were used to detect the expression level of the target gene.Statistical analysis: The SPSS 11.0 statistical software package was used for all analyses. The date were expressed by meanlstandard deviation [x|-±s] and analyzed using the ANVOA. The level of significant difference is a =0.05.Results1. SiRNAs were synthesized successfully in vitro and their lengths were 21bp.2. Immunohistochemistry results: The expression quantity of HPA proteinum was lower in experimental group compared with control group after transfecting for 24h, 48h, 72h, especially at 72h. It was not significantly different among different transfecting time(P>0.05) . The inhibition effect was in dosage-dependent manner at every transfecting time and there was significant difference in three kind of dosage(P <0.05). Inhibition effect didn't been observed in control group.3. In situ hybridration result: The expression quantity of HPA mRNA was lower in experimental group compared control group after transfecting for 24h, 48h, 72h, especially at 72h. It was not significantly different among different transfecting time(P>0.05) . The inhibition effect was in dosage-dependent manner at every transfecting time and there was significant difference in three kind of dosage(P< 0.05). Inhibition effect didn't been observed in control group. 4. After being transfected by siRNA in vitro for 24h, 48h, 72h,the expreesion of HPA protein and mRNA were all suppressed evidently, there was a correlation between the expreesion HPA protein and the expreesion HPA mRNA (P< 0.05).5. Nude mice model Result:â‘ Transfected firstly, then transplanted group: The expression of HPA protein and mRNA in transplanted tumor were inhibited by siRNA. There was significant difference between the non-sense sequence group and non-transfected group(P<0.05);â‘¡firstly transplanted, then transfected: The expressionof HPA protein and mRNA in transplanted tumor were inhibited by siRNA. There was significant difference between the non-sense sequence group and non-transfected group (P<0.05); The inhibitory effects of siRNA in the first group was stronger than that in the second group, but there was no significant difference between them (P>0.05).6. There was a correlation between the expreesion HPA protein and the expreesion HPA mRNA in transfected firstly group or firstly transplanted, then transfected group(P<0.05,r=6.87 or r=7.04)Conclusions1. dsRNA can be synthesized successfully in vitro.2. Both the expression level of HPA protein and mRNA in EC9706 cells were inhibited by different concentrations HPA siRNA, it indicated HPA could inhibit metastasis of esophageal carcinoma or other tumors through inhibiting the expression of HPA.3. wether in transfected firstly group or firstly transplanted, then transfected group,the expression level of HPA protein and mRNA were inhibited by HPA siRNA in transplanted tumors carried by nude mice, it indicated it might be possible that the siRNA is used to silent target gene.4. The expression level of HPA gene after being inhibited by siRNA in firstly transplanted, then transfected group was stronger than that in transfected firstly group,and the later is near to clinic, it provided a basic theory for implying RNAi technique to therapy malignant tumors .