Inhibit The Of ISL1Expression Of Gastric Cancer Cells Characteristics Influence Research

Gastric cancer is the most common gastrointestinal malignancyworldwide, with a high incidence and high mortality, low rate of earlydiagnosis, survival rates and low. Many studies have shown that gastricpresence of the gene expression abnormalities, such as P15, P53, FHIT, CD44,etc., but these are far less than for the study of gastric cancer cellcharacteristics and its resistance. ISL1(insulin gene enhancer binding protein1) factor-Islet-1transcriptional regulation of islet cells mature, has animportant role in the proliferation and differentiation. But now a growingnumber of studies have shown to have a close relationship with the digestivetract tumors. ISL1expression in BGC823, RNA interference suppressioncharacteristics of gastric cancer cells to detect what type of change, and toprovide a better theoretical basis for the treatment of gastric cancer.Part Ⅰ Down the ISL1the expression BGC823characteristicsObjective:Building for the human insulin gene enhancer binding protein1(ISL1),small interfering RNA, small interfering RNA transfection into BGC823cellsdown ISL1expression to detect gastric cancer cell characteristics change.Method：Get people ISL1gene coding sequence in full length, from the NCBIdatabase using siRNA design software design interference target and randomnegative control that does not target any gene. Small interfering RNAtransfection of cultured human gastric cancer cell line BGC823, reversetranscriptase chain reaction (RT-PCR) and Western blot (Western-blotting)biochemical techniques, detection ISL1gene mRNA and protein expressionlevelschanges. And cell proliferation was measured by MTT. CCK-8detection sensitivity of cells to chemotherapeutic agents.Result:1Small interfering RNA sequence:ISL1-siRNA sequence:5’-GCUCCAAGGUGUAUCACAUTT-3’(Justice)5’-AUGUGAUACACCUUGGAG-3’(Antisense)Negative control sequence:5’-UUCUCCGAACGUGUCACGUTT-3’(Justice)5’-ACGUGACACGUUCGGAGAATT-3’(Antisense).GAPDH positive control sequence:5’-GUAUDACAACAGCCUCAAGTT-3(Justice)5’-CUUGAGGCUGUUGUCAUACTT-3’(Antisense).2ISL1gene transfection mRNA and protein expression in gastric cancer BGC823cells were measured.2.1ISL1mRNABGC823cells decreased expression.Semi-quantitative RT-PCR analysis, ISL1siRNA cut ISL1mRNA expression so that the expression was significantly decreased. Transfected after ISL1siRNA BGC823cells compared with the blank control group after24h, ISL1mRNA expression levels were (0.658±0.032) and (1.371±0.022), with a statistically significant (P<0.05).2.2ISL1protein expression in gastric cancer BGC823cells decline.ISL1protein detected by Western-Blot found, after ISLlsiRNA,48h after transfection compared with the blank and negative control group, ISL1siRNA group ISL1protein expression levels (0.900±0.133) than the control group (1.710±0.150) and negative control group (1.630±0.126) is reduced, compared with a significant difference (P<0.05).3MTT colorimetric determination of cell proliferationOD value by detecting cells in each group at each time point, the incubation time for the X-axis, Y-axis corresponding OD value obtained cell growth curve, the results show that the siRNA-BGC823cell proliferative capacity in vitro was significantly lower than the other two groups of cells, the difference was statistically significant (P<0.05).4CCK-8assay sensitivity of cells to chemotherapeutic agentsBGC823cells after PTX,5-Fu, VP-16three drugs for48h drug survival were44%;57%;61%;siRNA-BGC823resistance cell survival rates were26%;39%;43%. The results showed that siRNA-BGC823the sensitivity ofcells to chemotherapeutic agents BGC823cells (P <0.05) was significantlyhigher than that.Conclusion;Building people ISL1siRNA successfully transfected BGC823cellssignificantly inhibited ISL1mRNA and protein expression were identified. TheISL1downregulation can significantly inhibit the growth and proliferation ofgastric cancer BGC823speed and enhances their sensitivity tochemotherapeutic drugs. ISL1gene in gastric cancer cell growth andproliferation and resistance has an important impact. part Ⅱ Enrichment BGC823side population cells, observed lowered ofISL1its characteristicsObjective:BGC823side population cells isolated in serum-free suspensionculture method to detect of BGC823cells and side population cells inproliferation and drug sensitivity changes. Small interfering RNA transfectioninto BGC823-SP cells down ISL1expression, to observe the changes in thecharacteristics of gastric cancer stem cells.Method:Serum-free suspension culture were enriched BGC823side populationcells using MTT and CCK-8method to detect changes in proliferation anddrug sensitivity. Small interfering RNA transfection BGC823-SP cells, reversetranscriptase chain reaction (RT-PCR) and Western blot (Western-blotting)biochemical techniques to detect the the ISL1gene mRNA and proteinexpression levels change. And cell proliferation was detected by MTT. CCK-8detection sensitivity of cells to chemotherapeutic agents.Result:1Successful enrichment of side population cells in suspension into a ball 2BGC823and BGC823-SP cells proliferation and chemosensitivitycontrast2.1MTT colorimetric assay BGC823BGC823-SP cells proliferationcontrastOD value by detecting cells in each group at each time point, time totrain for the X-axis, Y-axis corresponds to the OD value derived cell growthcurve, results show that the BGC823-SP cells in vitro proliferative capacitywas significantly stronger than BGC823cells, the differencewas statisticallysignificant (P <0.05).2.2CCK-8assay BGC823BGC823-SP cells chemosensitivityBGC823cells after PTX,5-Fu, VP-16three drugs for48h drug survivalwere44%,57%,61%; BGC823-SP cells resistant survival rates were56%,67%,70%. The results show that BGC823-SP cells to chemotherapeutic drugsensitivity was significantly lower than BGC823cells (P <0.05).3Determination of transfection ISL1gene mRNA and protein expression ingastric cancer BGC823-SP cells circumstances.3.1ISL1mRNA decreased expression in gastric cancer BGC823-SP cells.Semi-quantitative RT-PCR analysis, ISL1siRNA cut ISL1mRNAexpression so that the expression was significantly decreased.Transfected afterISL1siRNA BGC823cells compared with the blank control group after24h,ISL1mRNA expression levels were (0.403±0.001) and (1.428±0.302), witha statistically significant (P <0.05).3.2ISL1protein expression in gastric cancer BGC823-SP cells decreased.ISL1protein detected by Western-Blot found, after ISL1siRNA,48hafter transfection compared with the blank and negative control groupISL1siRNA group ISL1protein expression level (0.592±0.187) than thecontrol group (1.847±0.193) and negative control group (1.700±0.165) isreduced, compared with a significant difference (P <0.05).4MTT colorimetric determination of cell proliferationOD value by detecting cells in each group at each time point, theincubation time for the X-axis, Y-axis corresponding OD value obtained cell growth curve, the results show that the siRNA-BGC823-SP cells in vitroproliferative capacity was significantly weaker than BGC823-SP cells, thedifference was statistically significant (P <0.05).5CCK-8Determination of the sensitivity of cells to chemotherapeuticagentsPTX,5-Fu, VP-16three drugs for48h BGC823-SP cells resistantsurvival rates were56%,67%,70%; siRNA-BGC823-SP cells resistantsurvivalwere20%,31%,33%. The results showed that siRNA-BGC823-SPcells to chemotherapeutic drug sensitivity was significantly higher thanBGC823-SP cells (P <0.05).Conclusion:BGC823cell proliferative capacity in vitro gastric BGC823-SP and itsresistance is much higher than with human ISL1siRNA transfected gastriccancer BGC823-SP cells can significantly inhibit the proliferation rate, andsuppress ISL1mRNA and protein expression, andincrease its sensitivity tochemotherapeutic drugs. Description the ISL1of the growth and proliferationof gastric cancer BGC823and chemosensitivity are likely to achieve throughits regulation of stem cells, the specific mechanism needs further study.