Experimental Study Of Inhibitory Effect Of SiRNA Targeting Against EPAS1/HIF-2α Gene In Human Pancreatic Carcinoma Mediated By RAAV2

Partâ… The significance and correlation of EPAS1 and VEGF expression in pancreatic carcinomaObjective To investigate the expressions of Endothelia PAS domain protein1 (EPAS1),vascular endothelial growth factor(VEGF) and microvessel density(MVD) as well as the relationships among them,and the relationships to the clinicopathologic features of human pancreatic carcinoma.Methods In 60 cases of resected specimens of pancreatic carcinoma and their corresponding normal pancreatic tissues,RT-PCR was used to detect the expression levels of EPAS1 and VEGF mRNAs,western blotting and immunohistochemical SP method were used to examine the expression levels and distributions of EPAS1 and VEGF proteins respectively. MVD regarded as a marker of angiogenesis was explored also.The correlations among them and their relationships to the clinicopathologic characteristics of human pancreatic carcinoma were analyzed.Results Higher expressions of EPAS1,VEGF and MVD were detected in pancreatic carcinoma than in normal pancreatic tissue,for VEGF both at mRNA and protein level(t=6.10,P=0.0003; t=98.41,P=0.0001), for EPAS1 only at protein level (t= 22.51,P=0.0001). Furthermore, significant correlations were observed among them (between EPAS1 and VEGF, r=0.73583,P=0.0041; between VEGF and MVD, r= 0.85783, P=0.0001; between EPAS1 and MVD, r=0.64062, P=0.0003), the positive expressions of EPAS1 and VEGF were closely related with TNM staging and tumor size of pancreatic carcinoma.Conclusions Both EPAS1 and VEGF are overexpressed in pancreatic carcinoma, and the expression of EPAS1 is directly correlated with that of VEGF and the value of MVD significantly. EPAS1 may be involved in the angiogenesis of pancreatic carcinoma by upregulating the expression of VEGF, and play an important role in the carcinogenesis and aggression in pancreatic carcinoma.Partâ…¡Design and screening of the small interfering RNA targeting EPAS1 geneObjective To construct a plasmid expression vector coding for the short hairpin RNA (shRNA) targeting EPAS1 mRNA.Methods Three plasmid expression vectors coding for shRNA targeting EPAS1 gene sequence were constructed.The recombinant plasmids were identified by PCR and sequencing, and then transfected stably into PANC-1 cells respectively. The EPAS1 gene silencing effect was detected by quantitative RT-PCR and western blotting.Results The expected bands were amplified from the plasmids coding for shRNA by PCR,then sequencing confirmed that the EPAS1-shRNA plasmids were successfully constructed. Transfection of PANC-1 cells with shRNA plasmids resulted in an inhibition of EPAS1 mRNA and protein expressions respectively.For EPAS1-shRNA-1, EPAS1- shRNA-2 and EPAS1-shRNA-3,the inhibitory rates of EPAS1 mRNA expression were 67.5%, 46.8% and 47.9% in normoxic condition (P<0.05),as 86.6%, 55.1% and 56.4% in hypoxic condition (P<0.05);And the inhibitory rates of EPAS1 protein expression were 48.8% , 18.9 % and 28.9% respectively in normoxic condition (P<0.05),compared with 73.6%, 40.0% and 37.2% respectively in hypoxic condition (P<0.05).The most potent effect of silencing EPAS1 gene expression was conducted by EPAS1-shRNA-1.Conclusions The plasmid expression vectors coding for shRNA targeting EPAS1 mRNA have been constructed successfully, of which pGCsi-U6/Neo/GFP/EPAS1- shRNA-1 effectively silences EPAS1 gene the most in PANC-1 cells.Partâ…¢Construction and identification of rAAV2-EGFP-U6-EPAS1-siRNAObjective To construct and prepare the recombinant adeno-associated virus serotype 2 (rAAV2) vector for expressing short hairpin RNA(shRNA) targeting EPAS1 mRNA. Methods By inserting the sequence that contained EGFP-U6-EPAS1-siRNA into the EcoR I and Sal I site of vector plasmid pSNAV2.0-lacz-α,we constructed the recombinant vector of plasmid pSNAV2.0-EGFP-U6-EPAS1-siRNA.The recombinant vector of plasmid,BHK cell lines and helper virus HSV1-rc/ΔUL2 were used for the package of rAAV2-EGFP-U6-EPAS1-siRNA vector.The viral purity,identity,titer of rAAV2 viral stock were analyzed by the methods of SDS-PAGE,PCR and dot-blot respectively.Results The results of SDS-PAGE and PCR indicated that the shRNA fragment targeting EPAS1 was successfully packaged into rAAV2,and the viral purity of rAAV2 was above 95%.The titer of rAAV2 was approximately 1×1012v.g. /L.Conclusions The viral vector of rAAV2-EGFP-U6-EPAS1-siRNA is successfully constructed and prepared.Part IV Study of the impact of biological behaviors on human pancreatic carcinoma cells by EPAS1-siRNA in vitroObjective To observe the inhibitory effects of EPAS1-siRNA mediated by rAAV2 on human pancreatic carcinoma cells cultured in vitro.Methods rAAV2-EGFP-U6-EPAS1-siRNA was transfected into human pancreatic carcinoma cell line PANC-1, and the efficiency of transfection was detected. The cells were cultured under normoxic and hypoxic conditions respectively.Then RT-PCR, immunohistochemical SP method and western blotting were used to detect EPAS1, VEGF mRNA and protein expression. While MTT assay was used to determine proliferation rate of PANC-1. Detection of apoptosis was manipulated by the means of Giemsa staining, Hoechst 33342/PI double staining, Annexin V-FITC/PI double staining and DNA Ladder.And secretory VEGF level was measured by ABC-ELISA.Furthermore, CAM assay was used to detect angiogenesis in CAM.At last,a transplanted tumor model of pancreatic carcinoma on CAM was established to check the growth ability of tumor.Results rAAV2-EGFP-U6-EPAS1-siRNA was successfully transfected into PANC-1 cells, and the transfection efficiency detected by FCM was about 40%. Expressions of EPAS1 and VEGF mRNA were significantly inhibited in PANC-1 cells of experimental group,compared with in that of negative control group or blank control group (for EPAS1 mRNA: in normoxic condition, F=44.64, P=0.0002; in hypoxic condition, F=53.33, P=0.0012. for VEGF mRNA: in normoxic condition, F=25.36, P=0.0012; in hypoxic condition,F=56.62, P=0.0001). The same results were found as detecting their protein expressions (for EPAS1 protein: in normoxic state, F=46.12,P=0.0002; in hypoxic state, F=602.11,P=0.0001. for VEGF protein: in normoxic state, F=46.69, P=0.0002; in hypoxic state, F=64.56, P=0.0001).Meanwhile,in experimental group cells, ability of proliferation was inhibited(F=83.85, P=0.0001), apoptotic rate increased (in hypoxic state, F=124.98, P=0.0001), and secretory VEGF level decreased(in hypoxic state,F=9.83, P=0.0128).The count of newly formed blood vessels of experimental group was significanly less than that of control groups(in normoxic condition, F=29.04, P=0.0008; in hypoxic condition, F=88.01, P=0.0001). Lastly, growth of transplanted tumor on CAM was depressed, as tumor of experimental group was smaller than that of control groups (F=64.04, P=0.0001).Conclusions rAAV2-EGFP-U6-EPAS1-siRNA can inhibit expressions of EPAS1 and VEGF gene, cell proliferation, and promote apoptosis in human pancreatic carcinoma cell of PANC-1 in vitro. It can also inhibit angiogenesis and growth of transplanted tumor on CAM.Part V Research for inhibition human pancreatic carcinoma by EPAS1-siRNA in vivoObjective To research inhibition of biological behaviors of human pancreatic carcinoma such as growth,metastasis and invasion by rAAV2-EGFP-U6-EPAS1-siRNA in vivo.Methods Human pancreatic carcinoma transplanted subcutaneously in nude mouse,peritoneal metastasis model of human pancreatic carcinoma and model of surgical orthotopic implantation of human pancreatic carcinoma were established firstly. rAAV2- EGFP-U6-EPAS1-siRNA, rAAV2-EGFP and NS were intratumorally injected with 1×1012 v.g./kg respectively in subcutaneous xenograft model, inhibitory effect of tumor growth was observed , EPAS1 and VEGF protein expressions as well as value of MVD were detected by immunohistochemical SP method, TUNEL staining and transmission electron microscopy were used to determine apoptosis. rAAV2-EGFP-U6-EPAS1-siRNA, rAAV2- EGFP and NS were intraperitoneal injected with 1×10¹² v.g./kg respectively in peritoneal metastasis model of nude mouse, the number of metastases was counted. rAAV2-EGFP- U6-EPAS1-siRNA, rAAV2-EGFP and NS were intraperitoneal injected with 1×10¹²v.g./kg respectively in the model of surgical orthotopic implantation, inhibitory effect of tumor growth and metastasis of tumor were observed.Results Growth rate of subcutaneous xenograft significantly decreased in experimental group , the tumor's size and weight of experimental group were significantly lesser than those of negative control group and blank control group (for volume, F=171.09, P=0.0001; for weight, F=199.50, P=0.0001).For experimental group, expressions of EPAS1 and VEGF proteins were weaker than those of control groups (for EPAS1, H=3.18, P=0.2043; for VEGF, H=6.16, P=0.046), values of MVD and apoptotic index(AI) were significantly lesser than those of control groups (for MVD, F=55.42, P=0.0001; for AI, F=286.57, P=0.0001). And number of peritoneal seeding was less in experimental group than in control groups(F=52.23, P=0.0001). The size and weight of orthotopic implantation in experimental group were significantly less than those in control groups (for volume, F=27.52, P=0.0001; for weight, F=102.41, P=0.0001). While distant metastasis rate was 16.7% in experimental group, compared with 50% in control groups.Conclusions rAAV2-EGFP-U6-EPAS1-siRNA can dramatically inhibit growth and metastasis, promote apoptosis and reduce neovascularization of human pancreatic carcinoma in vivo.