The Inlfuence Of STAT3Silencing By Shrna On Apoptosis Of ECA109Cell Line

Esophageal cancer is a common malignant gastrointestinal tumors, with highmortality rate. The disease incidence of esophageal cancer is very high in China. Andthe disease incidence of esophageal cancer has a rising trend with living standardsimproved and lifestyle changes. Traditional treatment is surgery, supplemented byradiotherapy and chemotherapy. Although a variety of techniques has improved, theoverall5-year survival rate is still low, especially for advanced esophageal cancerpatients. Therefore, gene technology treatment of esophageal cancer is an emerginghot spot in recent years. It may bring a new way for curing esophageal cancer.In recent years, many studies have reported the pathogenesis of esophageal cancer atthe genetic level. Wild-type p53is a tumor suppressor gene, but the function ofimmune surveillance disappears after p53mutations in tumor cells. The tumorsuppressor gene p16is closely related with the cell cycle regulation. The loss of p16gene happens in esophageal cancer cells frequently.P16after mutation can not inhibitCDK4,that leads to the combination of cyclin D and CDK4.So the loss of p16wouldpromote the proliferation and can canceration of cell.The amplification andover-expression of c-myc has an important relationship with tumor tissuesoccurrence, development and evolution. Related researches have reported theoverexpression of c-myc in Barrett’s esophageal adenocarcinoma and basaloidsquamous cell carcinomas.Signal transducer and activator of transcription (STAT3) is a class of DNA-bindingprotein within cells. It can be activated with the external stimulation. STAT can transferred the signal to the nucleus directly to cause the transcription of targetgene.STAT3signal transduction pathways involved in cell proliferation,differentiation and apoptosis directly. JAK-STAT signal transduction pathway has asignificant impact on cell growth, proliferation and transformation, Sustainedactivation of this pathway can lead to abnormal proliferation and malignanttransformation.STAT3activation and continued high expression leads to thetransformation of fibroblast, which confirms the truth that STAT3is aproto-oncogene. Studies have shown that in a variety of malignancies such asleukemia, breast, lung and head and neck squamous cell carcinoma STAT3isover-expression and activation. Many studies have demonstrated abnormally highexpression of STAT3is related to the mechanism of tumor in a variety of malignanttumors. So blocking STAT3signal transduction pathway in cancer cell may curetumor in theory.With the development of RNA interference technology, the target gene can beremoved or silence.RNA interference technology is currently widely used to exploregene function and the treatment of diseases and tumors hard to cure.Objective:ECA109cells will be inhibited to proliferate and induced to apoptosis withJAK-STAT3signaling pathway interference.Methods:We aimed at STAT3,using plasmid vectors carrying small interfering RNA andliposome transfection methods, transfers vector plasmid pSliencer3.1-sh-stat3carrying small interfering RNA and negative control plasmidpSliencer3.1-sh-scramble into esophageal ECA109cells.expression level of recombinant plasmids and related genes were analyzed by Western blot.Earlymarkers of apoptosis were detected by DNA Ladder apoptosis kit.Early apoptoticchanges in mitochondrial membrane potential were detected by mitochondrialmembrane potential detection kit.Early and late apoptotic of cells were detected byAnnexin-V-FITC apoptosis detection kit.Cell cycle and apoptosis peak were detectedby flow cytometry.The degree of apoptotic cells were measured by TUNEL kit.Results:The experiment confirmed that the recombinant plasmid into esophageal cancer cellswas expressed and played a reserve role.Western blot analysis showed that the expression of STAT3of the group transfectedwith pSilencer3.1-sh-stat3was significantly lower, and the same with cyclinD1andc-myc. Apoptotic protein activated caspase-3decreased significantly while it’s17KDlarge subunit cleaved caspase-3increased.Apoptosis and cell cycle were detected to prove the biological effects of recombinantplasmid.1.DNA fragmentation experiments showed that after transfectionpSliencer3.1-sh-stat3plasmid early apoptosis marker DNA Ladder appeared.2.The results of the mitochondrial membrane potential show that depolarizationof mitochondrial membrane the early stage of apoptosis24hours after transfectionoccurred.3.The percentage of early apoptosis and late apoptosis reached35%, withstatistical significance.4.Hypodiploid apoptotic peaks before normal diploid cells in G0/G1peaks werefound and a significant increase in the number of cells in G0/G1phase, and the G0/G1arrest phenomenon happened.5.TUNEL assay showed apoptotic genomic DNA breakage, the recombinantplasmid to induced apoptosis of tumor cells.Therefore, this study used plasmid vectors carrying small interfering RNA andliposome transfection methods, and transfers the recombinant plasmid and negativecontrol plasmid pSliencer3.1-sh-scramble into esophageal ECA109cells. Expressionlevels of target gene STAT3decreased significantly, and JAK-STAT3signaltransduction pathways were successful interfered. Expression levels of downstreamgenes significantly reduced, and G0/G1arrest happened. So silencing STAT3inECA109cells can inhibit proliferation of tumor cells and induce apoptosis.