The Cancer Target Of Small Interference RNA Expression Vector Containing HTERT/U6 Combining Promoter

Background and objective In recent yeas, the RNA interfernce technology has becomewidespread in research of gene target therapy. However, the small interference RNA (siRNA) has not the ability of targeting malignant tumor cells, so that the indeterminateness of the ffect is caused. It has been reported that there is high expression of the telomerase in various malignant tumor cells, and no or low expression in human normal cells expect of generative cell and hematopoietic cell. The human telomerase reverse transcriptase is a key control factor of telomerase, and it plays an important role in the tumorgenesis and angiogenesis. In present study, the small interference RNA expression vectors containing hTERT/U6 combining promoter targeting to enhanced green fluorescence protein (EGFP) gene were constructed, and the effects of those vectors n EGFP in various cells lines with different expression of human telomerase reverse transcriptase (hTERT) were evaluated, so that to provide the foundation of targeted gene therapy of tumors. Methods1. Hepatocellular carcinoma cell SMMC-7721 of stable expressing enhanced green gluorescence protein (EGFP-7721) and human normal fibrocyte cell HELF of stable expressing fluorescence protein (EGFP-HELF) were cultured to provide transfecting effect test.2. Two genes of small interference RNA (siRNA) expression vector (siRNA-EGFP-PTZU6+1) targeting EGFP gene were constructed, and identified by the enzyme digestion and DNA sequencing. The recombinant expression plasmid siRNA-EGFP-PTZU6+1 was transfected into hepatocarcinoma cell lines (SMMC-7721) of stable expressing fluorescence protein, and screened the inhibition effects by fluorescence microscope, RT-PCR and Western-blot.3. The Core sequence of the promoter region of hTERT gene was amplified by PCR, and cloned into vector PTZU6+1-EGFP-siRNA to construct the Positive and opposite insert plasmid EGFP-siRNA-PTZU6+1-U6/hTERT separately. The recombinant plasmids were identified by DNA sequencing.4. Real-time SYBR Green PCR and Western-blot were applied to investigate the different effects of plasmid siRNA-EGFP-hTERT/U6-PTZU6+1 on the EGFP gene expressions in EGFP-772 and EGFP-HELF cell, to test the effects and target of the recombinant plasmids. Results1. The expression vectors siRNA-EGFP-PTZU6+1 were successfully constructed proved by the enzyme digestion and DNA sequencing. The difference of EGFP gene between recombinant plasmid siRNA-EGFP-PTZU6+1 transfected cells and PTZU6+1 transfected cells was statistically significant in results of RT-PCR and western-blot (all P<0.01), but there is no significant difference of EGFP expression between the two cell lines.2. The results of DNA sequencing showed that plasmid siRNA-EGFP-hTERT/U6-PTZU6+1 was successfully constructed. The results of RT-PCR showed that the EGFP gene was only significantly inhibited by the opposite inserted recombinant plasmid in hepatocarcinoma cell (P<0.01).3. Real-time SYBR Green PCR and Western-blot showed that there was no significant difference in the EGFP gene expression between the recombinant plasmid siRNA-EGFP-hTERT/U6-PTZU6+1 (neither opposite nor positive inserted) and the empty vector transfected EGFP-HELF cells. On the contrary, only the opposite inserted siRNA-EGFP-hTERT/U6-PTZU6+1 recombinant plasmid significantly inhibited the EGFP gene expression in the EGFP-7721 cells (P<0.01). As compared with the empty plasmid, the plasmid siRNA-EGFP-PTZU6+1 significantly inhibited the EGFP gene expression in both EGFP-7721 and EGFP-HELF cells groups (P<0.01).Conclusion1. The plasmid siRNA-EGFP-hTERT/U6-PTZU6+1 was successfully constructed.2. The opposite inserted siRNA-EGFP-hTERT/U6-PTZU6+1 recombinant plasmid can induce gene silencing.3. The opposite inserted recombinant plasmid siRNA-EGFP-hTERT/U6-PTZU6+1 has the ability to target the malignant tumor cells, which could provides the foundation for tumor gene-targeted therapy.