The Study Of The Relationship Between MiR-21 And PDCD4 In HeLa Cervical Carcinoma Cells

Objective:To predict miR-21 targets by bioinformatics and molecular biology techniques, analysis and verify them by functional experiments in Hela cervical carcinoma cells. To investigate the possible mechanism of the miR-21 and its relationship of targets during cervieal cancer development and progression. Our study will contribute to better understanding of the molecular mechanisms involved in cervical carcinogenesis and be helpful to development of new approaches for cancer gene therapy.Methods:1,Bioinformatic analysis was performed for chromosomal localization and conservation analysis of miR-21.Three online softwares for bioinformatics analysis, i.e., picTar, MicroCosm Targets and targetscan , were used for miRNAs target prediction.2,lipid-mediated transfection was used to transfect pre-miR-21,pre-miR negative control and anti-miR-21,anti-miR negative control into hela cervical cancer cells. The varying levels of miR-21 was assessed by real-time quantitative reverse transcription-polymerase chain reaction.3,Potential target genes expression were assessed by western blot for proteins.4,In order to verify the predicted miR-21 target is a real target, we will forecast the 3'UTR sequence of the PDCD4 gene miR-21 binding site in the upstream and downstream part of fragment into pMIR-Luc expression vector, to construct a reconstitute vector, pMIR-Luc-PDCD4-3'UTR. Verification by restriction enzymes digestion and sequencing were performed. The reconstitute vector pMIR-Luc-PDCD4-3'UTR, the control vector for normalization, pMIR-REPORT-β-gal, and pre-miR-21 or anti-miR-21 were co-transfected to HeLa cells (the test group). Seventy-two hours after transfection,β-Galactosidase and luciferase activities were determined. Three control groups were set up, namely, non-miRNA control group, pre-miR-negative control group and anti-miR-negative control group. All transfection data were expressed as luciferase activity normalized byβ-galactosidase activity. By this means, the reliability of miR-21 targets was verified.Results:1,Three online softwares for bioinformatic analysis, i.e., picTar, MicroCosm and targetscan, were used for predicting targets of miR-21. Our results demonstrated that PDCD4 is a potential target of miR-21.2,Western blot results showed that after transfected with pre-miR-21,the expression of PDCD4 protein was significantly down-regulated in Hela cervical carcinoma cells; after transfected with anti-miR-21 , the expression of PDCD4 protein was significantly up-regulated in Hela cervical carcinoma cells.3,Using luciferase reporter vector, pMIR-REPORT, we constructed reconstitute vectors:pMIR-Luc-PDCD4-3'UTR. The results of target verification experiments demonstrated that three control groups had similar Luc/β-gal values.In the test groups, Co-transfected with pre-miR-21,luciferase activity decreased43%; co-transfected with anti-miR-21, luciferase activity ascended 37%, indicating that PDCD4 is a real target for miR-21.Conclusions:1,These results suggest that the tumor suppressor protein Programmed Cell Death 4(PDCD4) is regulated by miR-21 and demonstrated that PDCD4 is a functionally important target for miR-21 in Hela cervical carcinoma cells.2,miR-21 may play an oncogenic role in the cellular processes of cervical cancer and may serve as a target for effective therapies.