Study Of ShRNA Silencing IKCa1 Gene In HeLa Cell Line Of Human Cervical Cancer

Abstract:Objective:The eukaryotic expression vectors special for knocking down IKCal gene were constructed to research the specificity and efficiency of IKCal gene and protein silence in HeLa cells of cervical cancer and the changes of HeLa cells biological characteristics. Methods:To construct the plasmid-mediated shRNA. The first, two oligonucleotides IKCal cDNA and one negative control oligonucleotide were designed referring to that of GenBank,then double-stranded RNA was derived through annealing, finally cloned into pGenesil-1.1 vector directed orientation. The pGenesil-1.1 vector has been signed by EGFP. The recombinant plasmid was transformed into the competence germ DH5a. The germ was cultured by using LB medium to extract the plasmid which was incisived and sequenced. Transfection was done with the help of lipofectamine 2000. The expression of IKCal mRNA was detected with FQ-PCR (fluorescence quantitative PCR) and the expression of IKCal protein was detected with western blotting. Cell proliferation was detected with WST-1 assay. Cell cycle was detected with flow cytometry. Results:Identification by incision enzyme and sequencing. Report showed the recombinant plasmid expressing shRNA was successful. FQ-PCR showed that the expression of IKCal mRNA was lower in the HeLa cell transfected with pGenesil-IK1 and pGenesil-IK2 than that of transfected with pGensil-HK, and the difference was statistically significant (P<0.05).Western Blotting showed that the expression of IKCal protein in pGenesil-IK1 group was lower than pGenesil-HK group and HeLa group(P<0.05),the pGenesil-IK2 group was not decreased(P>0.05). After transfected with pGenesil-IK1, the proliferation of HeLa cell was inhibited.Compared with pGenesil-HK group and HeLa group,the difference was statistically significant(P<0.05).FCM analysis indicated that the pGensil-IKl group G0/G1 phase cell ratio was 55.0±1.8% and S phase cell ratio was 29.2±0.9%,compared with control groups, the difference was statistically significant(P<0.05).The PI(proliferation index) of pGensil-IKl group was 44.97±1.75%, while the control group (HeLa) and the plasmid control group (pGenesil-HK) was 50.2±0.1%,49.3±0.44%, compared with control groups, the difference was significant (P<0.05).Conclusion:The expression of IKCal gene and protein in HeLa cell can be inhibitted by IKCal shRNA eukarytic vectors. Silencing expression of IKCal gene in HeLa cell inhibited the proliferation and growth of HeLa cell by block the cell cycle at G0/G1 phase.