Construction Of MGMT Interference Plasmid Vector And Its Effect On Oxaliplatin In Anti-hepatoma Cells

Objective:To construct a MGMT interference plasmid vector and study its anti hepatoma effects on oxaliplatin.Methods:1?MGMT interference plasmid vectors were constructed by means of RNA interference.The plasmids were identified by sequencing and identification of bacteria examination by PCR.The interference vectors pENTR-U6-shRNA2-GFP and pENTR-U6-shRNA2-GFP were constructed.2?The cells were divided into 4 groups consist of blank control group?KB group?with smmc-7721 cells without transfection,shRNA1 group with smmc-7721 cells transfected with plasmid pENTR-U6-shRNA1-GFP,shRNA2 group with smmc-7721 cells transfected with plasmid pENTR-U6-shRNA2-GFP and negative control group?NC group?with smmc-7721 cells transfected with blank plasmid.The transfection efficiency was observed by fluorescence microscope.The expression levels of MGMT mRNA before and after transfection into smmc-7721 cells were detected by Realtime-PCR to verify the interference effects of MGMT shRNAs.3?The 4 group of smmc-7721 cells were treated with oxaliplatin at concentration of2?g/mL,4?g/m L,8?g/m L,16?g/mL,32?g/mL,64?g/m L,128?g/mL and 256?g/m L to compare the IC₅₀ by MTT to study the effects that MGMT interference plasmid vectors made on oxaliplatin.4?Analyze the data by SPSS software.Results:1?By DNA sequencing,the target sequence of MGMT interference plasmid vector was consistent with the design sequence,which means successful construction of the plasmid vector named as pENTR-U6-shRNA-GFP.2?Transfect the smmc-7721 cells with the plasmids.By fluorescence microscope,green fluorescence was found in the shRNA1 group that smmc-7721 cells transfected with pENTR-U6-shRNA1-GFP,the shRNA2 group that smmc-7721 cells transfected with pENTR-U6-shRNA2-GFP and negative control group that smmc-7721 cells transfected with blank plasmid,indicating that the interference plasmid were successfully transfected into hepatoma cells.The interference efficiency of shRNA1 and shRNA2 measured by Realtime PCR were 56%and 63%,respectively.3?The IC₅₀ of experimental groups smmc-7721 cells that the IC₅₀ of shRNA1 group and shRNA2 group were 2.31?g/m L and 1.98?g/m L,respectively,were significantly lower than IC₅₀ of control group smmc-7721 cells?blank control group 5.00?g/m L,negative control group 6.17?g/m L??P₁<0.05,P₂<0.05,P₃<0.05,P₄<0.05?.Conclusion:We successfully constructed MGMT interference plasmid vector and transfected the plasmids into smmc-7721 cells,which effectively and significantly decreased the expression level of MGMT mRNA,as well as reducing the IC₅₀ of smmc-7721 cells to oxaliplatin after transfection with shRNAs,laying the foundation for further study to explore the molecular mechanism for MGMT interference vector promoting the cytotoxicity effect of oxaliplatin to hepatoma cells,and to reveal the relationship between MGMT and hepatocellular carcinoma.