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Hsa-miR-125a Facilitates Cell Apoptosis Via Wild Type p53 and Bcl-w

Author: ChangJiHong
Tutor: QiuXueShan
School: China Medical University
Course: Pathology and Pathophysiology
Keywords: microRNA miR-125a-3p/5p Lung cancer Apoptosis MDM2 p53 Bcl-w
CLC: R734.2
Type: Master's thesis
Year: 2010
Downloads: 152
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Abstract

Introduction MicroRNA (miRNA) are a class of about 21-24 nucleotides in length non-coding small molecule RNA. miRNA completely complementary or partially complementary way to identify the target gene 3'-UTR direct degradation of the target mRNA or inhibiting their translation inhibition of target gene expression and regulation of apoptosis, proliferation and differentiation. Therefore, miRNA plays an important role in the occurrence and development of tumor. miR-125a is one of the members of the miRNA family, there are two forms of the precursor and mature, their mature body having a biological activity. Found two mature form of miR-125a-3p and of miR-125a-5p. Our group early experiments found miR-125a-3p/5p compared with HBE showed low expression in lung cancer cells transfected the justice miR-125a-3p/5p make lung cancer A549 cells miR-125a-3p/5p content promote apoptosis of A549 cells, while expression of wild-type p53 also will increase, while the antisense miR-125a-3p/5p transfection contrast, but its regulatory mechanism is unclear. Therefore, we believe that miR-125a-3p/5p may promote apoptosis of A549 cells through regulation of wild-type p53. Found, can inhibit the p53 MDM2 oncogene may be a common target genes of miR-125a-3p/5p; predict target genes of miR-125a-3p/5p possible role of Bcl-w gene using a variety of forecasting software may miR-125a-3p/5p common target genes, Bcl-2 gene may only miR-125a-5p target genes. p53 present study more in-depth pro-apoptotic factors; Bcl-w, Bcl-2 is a member of the Bcl-2 family, both of which are inhibitors of apoptosis. Therefore, we speculate miR-125a-3p/5p by MDM2/p53, Bcl-w, Bcl-2 pathway regulation of apoptosis in lung cancer cells. This experiment will be increased by transfection of sense or antisense miR-125a-3p/5p or down in the intracellular level study of lung cancer cells express MDM2/p53 Bcl-w, Bcl-2 impact, further reveal the regulation withered The death of the molecular mechanisms, and provide a new approach for the treatment of lung cancer. Materials and methods, materials and reagents human lung adenocarcinoma cell line A549, H1299 (p53-negative). Mature sequence of miR-125a: justice of miR-125a-3p: 5'-ACA GGU GAG GUU CUU GGG the AGC C-3 '; antisense miR-125a-3p: 5'-GGC UCC the CAA GAA CUG U-CCU CAC-3' ; out-of-order miR-1: 5'-GGU CGG the UGC UCG AUG CAG GUA A-3 '; justice of miR-125a-5p: 5'-UCC CUG AGA CCC UUU AAC CUG UG-3'; antisense miR-125a- 5p: 5'-CAC AGG UUA AAG GGU CUC AGG GA-3 '; disorder miR-2: 5'-GGA CGG CGA the UCA GAU AAG AGUs UC-3'; each bases were 2'-0-A group modification in order to maintain the stability of the RNA sequence, no fluorescent labeling. 2, experimental methods 2.1 Cell culture: 10% fetal bovine serum DMEM (Gibco, USA) and RPMI-1640 (Gibco, USA) culture, 37 ° C, 5% of CO2. 2.2 Real time PCR was: Total cellular RNA was extracted according to TaqMan (?) MicroRNA Reverse Transcription Kit (P / N: 4366596, Applied Biosystems) kits and TaqMan (?) MicroRNA Assays HAS-MIR-125A (P / N: 4380904, Applied Biosystems) according to the instructions operation. All reactions were set three wells. 2.3 transiently transfected: cultured cells to Asian fusion state, transfection the justice or antisense miR-125a-3p/5p and out-of-order group miR-1/2 solution (20 microM) LipofectamineTM mixture is added to the well plate. Incubated for 4-6 hours will be 37 ℃, 5% CO2, the medium was changed and cultured for 24-72 hours as needed. 2.4 RT-PCR: extraction of total cellular RNA by RT-PCR (TaKaRa) kit method reaction. PCR product to agarose gel electrophoresis to detect and semi-quantitative analysis, using gel image analysis system. 2.5 Western Blot: Coomassie blue method for protein quantitation, ECL color. Western Blot experimental results by the gel imaging system acquisition, gray value determination, β-actin as an internal control, repeated three times. 2.6 Flow cytometry (Annetin V-FITC double staining method): each cell after transfection into four groups: blank control group, plus Annetin V group, with FITC-group processing Annetin V FITC group. BD FACSCaliburTMFlow Cytometer (BD Biosciences) to detect apoptosis. Statistical analysis SPSS13.0 statistical analysis software for data analysis using t test cell experimental results, expressed as mean ± SD, P lt; 0.05 was considered statistically significant. Results 1 transfected with sense or antisense miR-125a-3p/5p on the A549 cells miR-125a-3p/5p expression levels and apoptosis in A549 cells were transfected with the justice or antisense miR-125a-3p / 5p, as a control group with out-of-order and idling transfected group 24h after transfection, Real-time PCR method detect miR-125a-3p/5p expression levels using flow cytometry cell apoptosis. Results showed: transfection of justice miR-125a-3p/5p cells in miR-125a-3p/5p group and idling transfected group than out-of-order expression levels and apoptosis rates were significantly higher (P lt; 0.01); turn the antisense miR-125a-3p/5p cells in miR-125a-3p/5p expression levels and apoptosis rates were significantly lower (P lt; 0.01); disorder group and the group and idling transfected group than out-of-order Idling group compared with the untreated group, the miR-125a-3p/5p expression level and apoptosis was no significant change (P gt; 0.05). 2, the sense or antisense miR-125a-3p/5p A549 cells express MDM2, wild-type p53, Bcl-w, and the impact of Bcl-2 in A549 cells were transfected with sense or antisense miR-125a-3p/5p 24h, MDM2, wild-type p53, Bcl-w and Bcl-2 mRNA and protein expression was detected by RT-PCR and Western Blot method. The results showed: the the justice miR-125a-3p/5p cells p53 mRNA and protein expression increased Bcl-w mRNA and protein expression, MDM2 mRNA did not change while lower protein expression of Bcl-2 mRNA and protein did not change; turn of p53 mRNA and protein expression in the the antisense miR-125a-3p/5p cells decreased Bcl-w mRNA and protein expression increased, MDM2 mRNA did not change the elevated protein expression of Bcl-2 mRNA and protein did not change. These results suggest that: miR-125a-3p/5p possible regulation of p53 expression by inhibiting MDM2 expression and thus promote apoptosis; promote apoptosis while inhibiting the expression of Bcl-w. 3, blocking p53 or Bcl-w, miR-125a-3p/5p A549 cell apoptosis in order to clear miR-125a-3p/5p whether apoptosis pathway regulation by p53 and / or Bcl-w, turn dyed justice miR-125a-3p/5p simultaneously with monoclonal antibody closed wild-type of p53 24h; cells transfected antisense miR-125a-3p/5p simultaneously with monoclonal antibody blocking Bcl-w 24h and analyzed by flow cytometry apoptosis. The results showed: the justice, miR-125a-3p/5p induced apoptosis by blocking p53 are subject to varying degrees of inhibition (P lt; 0.05), which blocked p53 inhibition of apoptosis of justice miR-125a-5p significant effects than miR-125a-3p (P lt; 0.05); inhibitor of apoptosis induced by antisense transfection miR-125a-3p/5p varying degrees of recovery (P lt; 0.05) by blocking Bcl-w blocking Bcl-w (P lt; 0.05) than the significant role of antisense miR-125a-5p antisense miR-125a-3p role. In order to further clarify the wild-type p53 and Bcl-w A549 cell apoptosis induced miR-125a-3p/5p, we chose the p53-negative lung cancer cell lines H1299, transfected with sense and antisense miR-125a- 3p/5p 24h, apoptosis was detected by flow cytometry. The results show: the sense of miR-125a-3p justice of miR-125a-5p caused apoptosis was significantly higher (P lt; 0.01); antisense miR-125a-3p miR-125a-5p than antisense inhibition of apoptosis significantly lower (P lt; 0.05). The above results show that: miR-125a-5p may mainly promote apoptosis by regulating MDM2/p53 pathway, miR-125a-3p may be mainly through the regulation of Bcl-w pathways to promote apoptosis. Conclusion 1 miR-125a-3p/5p by inhibiting MDM2, raised wild-type p53 expression; while inhibiting the expression of Bcl-w, and promote apoptosis of lung cancer. 2, miR-125a-5p mainly through regulation of MDM2 / wild-type p53 pathway to promote apoptosis in lung cancer cells, miR-125a-3p may promote apoptosis in lung cancer cells by inhibiting Bcl-w.

Hsa-miR-125a Facilitates Cell Apoptosis Via Wild Type p53 and Bcl-w

Abstract

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