The Study On HSF2-Knockout/Activation HCT-116 Cell Using CRISPR/Cas9 System And Mechanism Of HSF2 On The Mucosa Healing In Gut

Objective:We established the HSF2-knockout/activation HCT-116 cell by using CRISPR/Case9 system to disrupt/activate the gene expression of Heat Shock Transcription Factor 2(HSF2)in cell.Study on the effect of HSF2 on the mucosa healing relevant cytokines,protein and signal path in gut.Methods:1.HCT-116 cell was cotransfected by HSF2 CRISPR/Cas9 KO Plasmid(h)and HSF2 HDR Plasmid(h)for disruption the gene expression of HSF2.The transfected cell selected by puromycin to establish the HSF2-knockout monoclonal HCT-116 cell.Then,grouping them into normal control,knockout,negative control;2.HCT-116 cell was transfected by HSF2 CRISPR Activation Plasmid(h)to activate the gene expression of HSF2,grouping them into Normal control,Activation group,Negative control.3.Above groups were measured the expression level of TGF-β1 mRNA by RT-PCR and the expression level of TGF-β1 in cell supernate by ELISA.4.Adding the HSF2 recombination protein into HSF2-knockout cell,then the expression level of Occludin,Smad2 were detected by Western Blot respectively.Results:1.HSF2-knockout HCT-116 cell was confimed by RT-PCR,sequencing and Western Blot,that HSF2-knockout HCT-116 cell was established successfully.2.The HSF2 mRNA and protein expression of HSF2-activation HCT-116 cell is significantly increased,which was detected by RT-PCR and Western Blot.3.MTS assays shows that the proliferation of knockout group was lower than Normal control and Negative control after 72 hours.On the contrary,compared with Normal conrol group and Negative control group,the proliferation of Activation group was significantly increased after 72 hours.4.After stimulated by LPS,the TGF-β1 expression of cell supemate in knockout group was lower than Normal control group and Negative control group(p<0.05);the TGF-β1 expression of cell supernate in Activation group was higher than Normal control group and Negative control group(p<0.05).5.Adding HSF2 recombination protein into the Knockout group,comparing this group with Normal control group,Knockout group and Activation group,the protein levels expression of Occludin and Smad2 had significantly differency(p<0.05).Conclusions:1.The expression of HSF2 would have effect on the proliferation of HCT-116 cell.Compared with the Normal control group and Negative control group,the proliferation of Knockout group was significantly decrease.Inversely,the Activationa group was significantly increased.2.The protein expression of Occludin in HSF2-knockout group was lower than Normal control.Comparing with Normal control group,the Occludin expression of the Activation group and adding HSF2 recombination protein group was markedly increase,which suggests the HSF2 has positive correlation with Occludin.3.Compared with Normal control group,the TGF-β1 and Smad2 expression of HSF2-knockout group was descended significantly.However,the TGF-β1 and Smad2 expression of HSF2-activation group was increased markedly.These studies suggest that HSF2 could improve the expression of TGF-β1 that activates the expression of Smad2 to promote the intestinal mucosa healing.