Interference And Overexpression Of Heat Shock Transcription Factor 2 And Its Influences On The Apoptosis And Migration Of The Human Colonic Epithelial Cell

Objective:To study the role of HSF2 by method of interference and overexpression on the human colonic epithelial cell lines-HT-29 cell in regulating sodium butyrate (SB) induced apoptosis and healing ability of cell migration assays.Methods:?Induced apoptosis of HT-29 cells with different time and dose of sodium butyrate,cytotoxicity was estimated by the MTT assay; the cell cycle and apoptosis were observed by Flow cytometry.According to the experimental results to choose the optima time and concentration of sodium butyrate.?HT-29 cells were transfected with HSF2 siRNA and Overexpression (Ubi-MCS-3FLAG-SV40-EGFP) using lentivirus. The expression of Overexpression and siRNA of HSF2 were detected by Leica DMIRB and western blot.? Cell migrating ability was measured with wound healing assay and Transwell assay after transfection.? After the HT-29 were transfected, we induced apoptosis of HT-29 cells with the optima time and concentration of sodium butyrate, the cell proliferation was studied by MTT assay,the cell cycle and apoptosis were observed by Flow cytometry.Results:?Compared with the NC group,2.5?5.0?10mmol/L sodium butyrate can cause growth inhibition after 48h,with significant differences between the groups (P< 0.01), the effect were time-dose -dependent.The cell apoptosis assay showed thatrthe apoptosis rate of NC group (0.548±0.113%), and the experimental group treated with sodium butyrate(1.25mmol/L,2.5mmol/L,5.0mmol/L,10mmol/L)after 48h, the apoptosis rate(51.588±5.11%,77.732± 2.746,90.115±1.438,94.247±1.243)showed significant difference to the NC group (P< 0.01). When the sodium butyrate concentration> 2.5mmol/L, the apoptosis rate increased significantly (P<0.01).Cell cycle analysis:when treated with 1.25mmol/L sodium butyrate for 48h, the popolation of The GO/G1 phase cell did not show significant difference with the negative control group (NC), (P=1> 0.05);when the concentration is higher than 2.5mmol/L (5.0?10mmol/L),sodium butyrate could induce G1/G0 phase arrest of HT-29 cell (P< 0.01).?After lentiviral transfection,a large number of HT-29 cells with green fluorescence was observed by Leica DMIRB (X100)(transfection efficiency> 80%). Western-blot of results showed that lentivirus transfection siRNA can effectively inhibit HT-29 cell expression of HSF2,and lentivirus transfection(Ubi-MCS-3FLAG-SV40-EGFP)can Overexpression HT-29cell expression of HSF2.?The MTT assay shows:the proliferation of overexpression group was higher than Blank vector group(P<0.05), the siRNA group significantly inhibit the proliferation of HT-29 cell compared with the Negative siRNA group (P<0.05).?Cell migration assays:compared with the control group,overexpression group of HSF2 showed higher migration ability(P< 0.05), while compared with NC group, migration ability of the siRNA group was lower(P<0.05).?There are no significant differences of the cell cycle distribution between NC and lentivirus transfected groups(HSF2 siRNA/Overexpression)(P>0.05).?Cell apoptosis:There are significant differences of cell apoptosis rate between Blank vector B group and overexpression group, cell apoptosis rate of the overexpression group decreased significantly (P<0.05); while compared with negative control group, cell apoptosis rate of the siRNA group increased significantly(P<0.05).Conclusions:?Sodium butyrate can inhibit proliferation and induce apoptosis of HT-29 cells,the effect were time-dose-dependent.?lentivirus transfection siRNA can effectively inhibit HT-29 cell expression of HSF2,and (Ubi-MCS-3FLAG-SV40-EGFP)can Overexpression HT-29 cell expression of HSF2.?When silenced HSF2 significantly decreased the HT-29 cell proliferation compared with the Negative siRNA group, but when overexpression HT-29 cell expression of HSF2,We got the opposite result. HSF2 may be the protective factor of the intestinal epithelial cells;?No significant differences among the cell cycle distribution of overexpression group?siRNA group and NC group, which suggesting that HSF2 may through the cell cycle regulation to achieve the effects on cell protection;?The migration rate of overexpression group of HT-29 cell was significantly higher than the NC group, and the result of siRNA group was on the contrary.These indicating that HSF2 may have the function of repair the intestinal epithelial cell during apoptosis;?These cell apoptosis studies indicated that HSF2 might be a protective factor for epithelial cell in apoptosis.