Effects Of STAT3Gene Overexpression By On Proliferation And Apoptosis Of Gastric Cancer Cell Line

STAT3, signal transducer and activator of transcription3, signal transducer and activator of transcription factor family, is also an acute phase response factor and nuclear transcription factor. STAT3exist in the cytoplasm, the cells receiving the exogenous signal transduction is activated, and be able to cross the nuclear membrane is bonded to the corresponding DNA sequence on the information carried by the outer source signal by changing the expression of the corresponding gene demonstrated out.The incidence of gastric cancer by multiple genes, multiple factors involved in a multi-step process. Although in recent years, incidence of gastric cancer is high, but the specific molecular mechanisms of gastric cancer development is not yet clear. Has been detected, STAT3in a variety of malignant tumors in the activated form, such as leukemia, head and neck squamous cell carcinoma, malignant glioma, breast cancer, colorectal cancer, lung cancer, malignant melanoma, cervical cancer. Therefore, this study suggests that, STAT3has played an important role in the occurrence and development of gastric cancer.Tumor tissue cells abnormal transformation, proliferation and differentiation barriers and formed. STAT3can promote the proliferation of tumor cells. STAT3activation due to phosphorylation of tyrosine residues705points,727silk acid residues phosphorylated STAT3protein binding capacity of the target gene can improve. Using siRNA interference STAT3mRNA expression can reduced due to the increase of IL-6caused by HIF1-alpha protein. STAT3can also be through the inhibition of apoptosis of tumor cells so as to promote the proliferation of tumor cells. In prostate cancer, STAT3expression reduce down BCL-2and PCNA increase in caspase-3expression. Gastric cancer tissues by immunohistochemistry experimental results show that the expression of STAT3releated to expression of survivin. In breast cancer, STAT3binding to the P53promoter to inhibit the transcription of P53.Objective:This study, through the use of Eukaryotic expression plasmid and transfected into gastric cancer cell lines were cultured in vitro, observed in the case of elevated expression of the target gene STAT3, the impact of the situation on the growth of gastric cancer cell lines to explored the role of STAT3in the development of gastric cancer.Method:1. Vector was constructed by PCR amplification of human STAT3gene sequence, with the vector pcDNA3connected host strain DH5a was transformed to screening of the positive clones of ampicillin-resistant, plasmid was extracted and identified.2. Transfection using cationic liposomes transfected gastric cancer cell lines MGC803(P53gene wild type), were divided into three groups, the blank control group (liposomal transfection), empty vector group (transfected with pcDNA3) and The recombinant plasmids group (transfected with pcDNA3-STAT3).Gastric cancer cell protein and mRNA expression levels48hours after transfection, total protein was extracted and total RNA, the the WESTERN BLOTTING and RT-PCR method for detection of STAT3, P-STAT3(Y705), P-STAT3(S727)HIF-1α, BCL-2, SURVIVIN, P53, CASPASE-3, PCNA expression at the protein and mRNA levels in gastric cancer cell lines MGC803.The4. Gastric cancer cell proliferation levels transfection24h,48h after72hours by CCK-8assay proliferation of gastric cancer cell lines MGC803.Results:1. Successfully constructed recombinant plasmid pcDNA3-STAT3agarose gel electrophoresis results recombinant plasmid pcDNA3-STAT3molecular weight of about7.6KB, I double digested fragments and PCR products HIND Ⅲ and XHO molecular weight of about2.3KB.Verified by sequencing the inserted sequence of recombinant plasmid and the design of the oligonucleotide fragments to match completely.2. Target gene STAT3in gastric cancer cells at the protein and mRNA levels surpassed expression the WESTERN BLOTTING and RT-PCR results in gastric cancer cell lines MGC803transfected with the recombinant plasmid group, compared with the blank control group transfected with empty vector group target gene STAT3protein and mRNA expression levels were increased (P<0.05).3. Gastric cancer cell proliferation level of CCK-8experimental results show that, compared with the blank control group and transfected with empty vector group, transfected with the recombinant plasmid promote the proliferation of gastric cancer cell lines MGC803. The the WESTERN BLOTTING and RT-PCR results showed that, compared with the blank control group transfected with empty vector group in gastric cancer cell lines MGC8O3transfection the recombinant plasmids Group P-STAT3(Y705), P-STAT3(S727) of HIF-la, BCL-2, SURVIVIN, PCNA expression average increase, P53, CASPASE-3expression were reduced.Conclusion:The eukaryotic expression vector pcDNA3-STAT3in gastric cancer cell lines MGC803can effectively improve the STAT3target gene expression by upregulating P-STAT3(Y705), P-STAT3(S727), HIF-la BCL-2gene expression SURVIVIN, PCNA significantly promote the proliferation of gastric cancer cells down the P53CASPASE-3gene expression thereby inhibiting apoptosis of gastric cancer cells, for further study of the STAT3gene regulation in vitro culture of gastric cancer cell mechanism to provide an experimental basis.