Experimental Research On Pancreatic Carcinoma Cells Transfected By Synthetic MicroRNA Targeting To STAT3

Pancreatic carcinoma is one of the highest malignant tumors with covert origination in digestive tract of human being. Mostly, clinic symptoms appear late and the patients are almost in the advanced stage when diagnosis is definite. The rate of resection with operation is rather low while recurrence and metastasis generate earlier. In addition, the pancreatic carcinoma is insensitivity to chemotherapeutical drugs and radiotherapy, which accounts for the poor prognosis. With the progression of gene therapy research, the RNA interference technology has been applied extensively and pulls the research of tumor gene therapy into a rapid stage of development.SiRNA is used as an effective molecule to apply to gene therapy now, and the achievement is remarkable. But the deficiency of siRNA is also well known, because siRNA is not a kind of endogenous mechanism of action in the cell of advanced creature, and exists the indefiniteness phenomenon of target gene mRNA. Whereas, another small RNA—microRNA (miRNA) has more advantage in RNAi study than siRNA theoretically, because miRNA can not only induce RISC to leave the target gene mRNA though the classic RNA pathway, but also suppress translation of target gene and direct rapid deadenylation of target mRNAs to lead to target gene mRNA decay. The diversification of mechanism makes miRNA possess a certain fault tolerance that assures the effective of RNAi to target without complete complementary between miRNA to target mRNA.Signal transduction and activator of transcription 3(STAT3) is an important factor of signal transduction pathway,which is constantly activated in lots of human tumor cell. It links closely with the occurrence, development and prognosis of human malignant tumor, so it has been one of important target in gene therapy to tumor. The purpose of our study is to construct plasmids which express precursor of miRNA (pre-miRNA) targeting to STAT3mRNA by simulating natural miRNA, and to survey regulation effective to pancreatic carcinoma cell in vitro and vivo. The experiment was separated into three parts:Part one: Constructing and screening of miRNA effective expression plasmid targeting STAT3 gene.Objective: To construct the plasmid which regulation target genes, and screen effective plasmid, and to confirm efficient positions in which miRNA direct against STAT3 gene, and to observe regulative effect of candidate plasmids.Method: We constructed four miRNA plasmid targeting different positions which miRNA might direct against STAT3 gene, and transfected them into Panc-1 cells respectively, and study the effect of their regulation, RT-PCR was used for detecting expression level of STAT3 mRNA and Western blot was used for detected the protein expression level of STAT3 at 48h after transfection. MTT was used for detected the hyperplasia condition in different plasmid transfected groups after 24, 48 and 72h. Flow cytometry was used for detecting the cell cycle status after 48h and cell apoptosis after 24h, 48h and 72h in different plasmid transfected groups.Results: The sequencing results of four expression plasmid miRNA-STAT3-1, 2, 3, and 4 were correct in structure. After 48h in different plasmid transfected groups, the levels of STAT3 mRNA were down-regulated to 78.5%, 57.1%, 53.1% and 54.7% respectively; the levels of STAT3 protein were down-regulated to 89.1%, 76.1%, 67.4% and 53.3% respectively. Among them, the effective of miRNA-STAT3-1 was the highest, and the gene loci from 1242 to 1262 were more effective to regulate STAT3 gene than other gene loci. The inhibition rate of miRNA-STAT3-1 group cells was 31.6%, 55.3% and 62.2% after 24h, 48h and 72h respectively. The translation from G1 phase to S phase in the Panc-1 cell was inhibited. Flow cytometry analysis showed that the apoptosis rate in miRNA-STAT3-1 group was significant higher than that in the control groups.Part two: The inhibition experiment of synthesis miRNA plasmid targeting STAT3mRNA in vitro after stable expressionObjective: To construct Panc-1 cell lines transfected miRNA effective plasmid and expressing miRNA-STAT3-1 stably, and survey the inhibitory effect of Panc-1 cell by regulating expression of STAT3 gene.Method: MiRNA-STAT3-1 and miRNA-negative (miRNA-neg)-plasmids were used to transferred to Panc-1 cell line respectively, and generate stable cell lines that constitutively express miRNA by antibiotics selection. The effects of stable transfection of the miRNA plasmids are surveyed by RT-PCR and Western blot respectively.Results: The two Panc-1 cell lines expressed stably miRNA-STAT3-1 or miRNA-neg, in stable cells expressing miRNA-STAT3-1, the levels of mRNA of STAT3, Cyclin-D1 and Bcl-2 were down-regulated to 53%, 57% and 34% respectively. The levels of protein of STAT3, Cyclin-D1 and Bcl-2 were down-regulated to 39%, 30% and 17% respectively.Part three: The study of the inhibitory effect of transfection after miRNA-STAT3-1 plasmid in the model of human pancreatic carcinoma in nude mice.Objective: A human pancreatic carcinoma model in nude mice was established to study the inhibitory effect of miRNA target STAT3 gene in vivo.Method: Model of human pancreatic carcinoma in nude mice was established successfully. The volume of tumor was determined every five days. The nude mice were killed after 40 days. The STAT3 protein was detected by western blot and immunohistochemisty method.Results: The appearance of tumor in nude rice was delay, and the growth of tumor was inhibited obviously after transfection of miRNA-STAT3-1 plasmid. The level of protein of STAT3 was down-regulated to 84% or so.Conclusion:1) The recombination plasmid vectors which target STAT3 gene was constructed successfully. The plasmid vectors which miRNA targeting STAT3 gene down-regulate the expression of STAT3 gene efficiently and detent the Panc-1 at G0/G1 stage, and induce cell apoptosis. The miRNA targeting STAT3 gene loci from 1242 to 1262 down-regulate STAT3 gene more effectively than the others.2) In vitro, miRNA plasmid of STAT3 down-regulated expression of STAT3, Cyclin-D1 and Bcl-2 at mRNA and protein level effectively in pancreatic carcinoma cell line Panc-1.3) In vivo, the animal model of Panc-1 tumor in nude mice was established successfully. The recombination plasmid targeting STAT3 gene inhibited the growth of Panc-1 cell in vitro and transplanted tumor in vivo efficiently.