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The Study of the Relationship between miR-21 and PDCD4 in HeLa Cervical Carcinoma Cells

Author: DaiGuiHong
Tutor: LiGuoLi
School: Yangzhou University
Course: Pathology and Pathophysiology
Keywords: microRNA-21 Cervical Cancer Hela Target genes PDCD4
CLC: R737.33
Type: Master's thesis
Year: 2010
Downloads: 210
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Abstract


Objective: bioinformatics methods and molecular biology techniques in the prediction and validation of miR-21 target genes, and to investigate the interaction of miR-21 target genes and their relationship with the incidence of cervical cancer, cervical cancer Hela cells for further study of the molecular mechanisms of cervical cancer and cervical cancer gene therapy method to provide new ideas and experimental evidence. Method: 1, bioinformatics methods chromosomal localization of miR-21, a conservative analysis, the use of network sharing software MicroCosm Targets, TargetScan and PicTar analysis of the possible role of the target gene. Liposomal transfection methods, the United States of Ambion synthetic pre-miR-21, pre-miR negative control and anti-miR-21, anti-of miR negative control instantaneous transfected into HeLa cells, real-time fluorescence Quantitative PCR (qRT-PCR) was used to detect the relative expression level of miR-21. Protein immunoblotting (Western blot): total protein was extracted by electrophoresis, transferred to a membrane of PDCD4 antibody incubation, exposed piece developing software PDCD4 protein relative content analysis. 4, luciferase reporter experiments: PDCD4 molecules 3'UTR sequence of miR-21 binding sites on the downstream part of the fragment into pMIR-REPORT-luciferace luciferase reporter gene vector, the recombinant vector (pMIR-Luc-PDCD4 ), and restriction analysis and sequencing. Standardized control vector (pMIR-REPORT-β-gal) to the recombinant carrier (pMIR-Luc-of PDCD4) were measured pre-miR-21 or anti-miR-21 were co-transfected Hela cells (experimental group), 72 hours after Hela cells, luciferase (Luc) and β-galactosidase activity of the enzyme, and to calculate the ratio between the two (the Luc / β-gal). Set up three of the control group (no miRNA blank control group, pre-miR-negative control group and anti-miR-negative control group) were compared among groups Luc / β-gal ratio, to verify the authenticity of the role of miR-21 targets . Results: 1, the use of online software PicTar, role MicroCosm Targets and TargetScan predicted miR-21 target genes. The results found the PDCD4 possible role of miR-21 target genes. Western blot results found that pre-miR-21 after transfection, cells PDCD4 protein expression levels lowered (P lt; 0.01); transfected with anti-miR-21, PDCD4 protein expression levels in the cells increased (P lt; 0.05). 3 pMIR-REPORT luciferase reporter gene vector successfully constructed by restriction endonuclease and sequencing the recombinant vector pMIR-Luc-of PDCD4-3 'UTR. luciferase pMIR-REPORT experimental results show that the three control groups (the no miRNA blank control group, pre-miR-negative control group, and Anti-miR-negative control group), the Luc / β-gal ratio close. Experimental group, pre-miR-21 transfection group, luciferase activity decreased by approximately 43% (P lt; 0.001); anti-miR-21 co-transfection group, luciferase activity was increased approximately 37% (P lt ; 0.001), indicating that PDCD4 is the real role of miR-21 target. Conclusion: 1 in Hela cells, the expression of miR-21 regulation of tumor suppressor gene PDCD4 protein, PDCD4 is an important target genes of miR-21. 2, miR-21 in cervical cancer process may play the role of a proto-oncogene, and may be an effective target for cervical cancer gene therapy.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Female genital tumors > Uterine tumors
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