Construction Of Bach1 Clone And Its Effect On The Drug Sensitivity In Hepatocellular Carcinoma Cells

BackgroundTranscription factor BTB and CNC homolog 1(Bachl) which belongs to the CNC transcription factor family, has a characteristic inhibitory action on Heme oxygenase 1(HO-1). HO-1 is a key cytoprotective enzyme. Some studies discovery that the expression of HO-1 can influence the growth of tumor and the sensitivity of tumor cells to the chemotherapy. After treat the tumor tissues with radiotherapy, chemotherapy and so on, the expression of HO-1 should be increased. In view of the protection of HO-1 on tumor cells in the cancer therapy, we try to enhance the expression of Bach1 to inhibit the expression of HO-1, and observe whether the sensitivity of tumor cells to vincristine (VCR) changes, and what the changes will happen.Objectsconstructing a eukaryotic expression vector pBI-EGFP-Bachl, transfecting it into Hepatoma cells SMMC-7721 to reduce the expression of HO-1 protein, and detecting whether the drug sensitivity of the transfected Hepatoma cells to the VCR have changed by MTT. Methodsl.We amplified gene Bachl from the plasmid pQE30-bachl by using PCR, plasmid pBI-EGFP and the Bachl gene amplification from PCR were digested respectively with NheI and MluI, and linked by using T4 ligase to form the recombinant plasmid;2.When detecting Bachl gene was successfully inserted by PCR, we co-transfected the Tet-Off plasmids and recombinant plasmids into human Hepatoma cells SMMC-7721. After 48 hours, we detected its expression in cells by using RT-PCR;3. Detecting whether the drug sensitivity of the transfected Hepatoma cells to the VCR have changed by MTT, and get the value of the IC50;4. Using RT-PCR method to detect the impact of the expression of Bach 1 and HO-1 in group VCR+Bachl and group VCR;5. We use DNA Ladder to show the bands of apoptosis of VCR+Bachl group and VCR group in low concentration of vincristine;6. The cell morphology of group VCR+Bachl and group VCR was observed after adding the vincristineResults1. Using PCR and digesting with Nhel and MluI, the result shows that Bachl gene has been embedded successfully into the answering vector pBI-EGFP;2. When the reorganized vector pBI-EGFP-Bachl and the Tet-Off transfect in cancer cell 48 hours later, RT-PCR results prove that the expressing of Bachl gene has increased and the expression of HO-1 gene has reduced;3. Under the same dose of drug, the apoptosis of the transfected cells is higher than non-transfected cells. When the drug concentration is 0.15,0.25μg/ml, the p value is less that 0.05 and when the drug concentration is 0.20μg/ml, the p value is less that 0.01. MTT assay the IC50 of the group VCR to vincristine is 0.15μg/ml and the group VCR+Bachl is 0.21μg/ml. Compared to the VCR+Bachl group, the IC50 of the VCR+Bachl group is lower 28.6%;4. Detecting the expression of Bachl and HO-1 after dosing in the different group, the expression of Bachl in the VCR+Bachl group is increased, and the expression of HO-1 in the VCR group is increased and is higher than the control group;5. Extracting DNA of each group after adding 15μg/ml of VCR 48h, electrophoresis showed that there is apoptotic band in VCR+Bachl group, and under the same dose of drug, the VCR group did not have the apoptotic band;6. After adding the low dose VCR, the morphology of SMMC-7721 cells in the VCR group did not change much, in contrast the VCR+Bachl group cells became smaller and round. We can observe a large number of death cell, the cell membrane is integrity but have vesicles.Conclusions1.We successfully constructed a eukaryotic expression vector pBI-EGFP-Bachl which can express Bachl and inhibit the expression of HO-1 in human hepatocellular carcinoma SMMC-7721 cells;2. We tested the reorganized plasmid pBI-EGFP-Bach can enhance the SMMC-7721 cells' susceptibility of vincristine by MTT susceptibility testing in this study, the IC50 reduced from 0.21μg/ml to 0.15μg/ml, reduced 28.6%;3. Confirmation may know after DNA the Ladder, under the low concentration medicine function the recombinant plasmid transfected hepatoma cells SMMC-7721 happen apoptosis, but under the same concentration of SMMC-7721 cells did not produce significant apoptosis band. The morphological observation shows the same results.