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miR-106a and miR-17-5p in the trans- BPDE induced malignant transformation in the role of

Author: WuYan
Tutor: JiangYiGuo
School: Guangzhou Medical College
Course: Health Toxicology
Keywords: Trans- BPDE Malignant transformation microRNA miR-106a miR-17-5p Human bronchial epithelial cells Mimetic Inhibitors
CLC: R114
Type: Master's thesis
Year: 2010
Downloads: 63
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Abstract

Background and Purpose benzo [a] pyrene (B [a] P) is the first to be found widely distributed in the environment, environmental chemical carcinogens, which produce the final carcinogen metabolic activation by anti-BPDE after carcinogenic . MiRNA is a class of large number of small non-coding RNA molecules that can target gene through the 3 'untranslated region of pair-bonding causing translational repression or mRNA degradation of oncogenes or tumor suppressor genes play a role. In this study, the use of anti-BPDE-induced malignant transformation of human bronchial epithelial cells (16HBE-T) study of miRNA in the possible mechanism of chemical carcinogenesis. QRT-PCR method using the measured body and the miR-106a mature miR-17-5p expression of mature form, and further use of specific miRNA inhibitors and mimetics malignant transformed cells upward or downward in the miR-106a or miR-17 -5p activity, in order to determine the level of its expression in malignant biological characteristics of transformed cells. The results showed that compared with control cells (16HBE-N), miR-106a and miR-17-5p in malignant transformation of cells in the presence overexpression. Transfection Inhibitors silence 16HBE-T cells, miR-106a or miR-17-5p will inhibit cell proliferation and induce cell cycle arrest and apoptosis, inhibition of non-independent growth. In contrast, transfection mimetic 16HBE-T cells increased in the miR-106a or miR-17-5p levels of cell characteristics exhibit the exact opposite. In addition, the miR-106a also conducted experiments in nude mice and found that the injection of inhibitors of miR-106a transfected cells was significantly slowed tumor growth group, and injected transfected miR-106a mimetic group of cells in the tumor significantly faster growth rate. These results suggest that miR-106a and miR-17-5p in chemical carcinogen-induced transformation may play the role of oncogenes. Therefore, the knock-crime becomes cells miR-106a and miR-17-5p may be a potential therapeutic approach. Method 1. Detection of miRNA expression using TaqMan MicroRNA assays kit identification 16HBE-N and 16HBE-T cells miRNA expression levels. (2) transient transfection miRNA mimetics and inhibitors Lipofectamine 2000 according to instructions for transient transfection. Mimetic transfected group set up the test group (miR-106a mimic and miR-17-5p mimic) and negative control group (mimic NC), inhibitors transfected group set up the test group (anti-miR-106a and anti-miR-17 -5p) and negative control group (inhibitor NC), in addition, also set up a transfection reagent Lipofectamine 2000 negative control group (Lipo-2000 NC). Evaluated after transfection 5 hours the transfection efficiency, and 72 hours after the detection of miRNA expression levels changed circumstances. 3 transfected cell cycle 72 hours after transfection experiment, the PI and RNase A coloring, flow cytometric analysis of cell cycle. 4 Cell proliferation assay in 96-well plates inoculated with the same number of 16HBE-T cells, cultured for 24 hours, and then transfected packet. Fifth day after transfection with CCK-8 microplate detection reagents and cell proliferation. 5 of apoptosis 72 hours after transfection experiments using Annexin V / PI kit and flow cytometry transfected and non-transfected cells apoptosis. 6 soft agar experiment 500 cells were resuspended in 1ml 0.3% low melting point agarose, and then spread to the semisolid included 1ml 0.6% low melting point agarose in 12-well plates. With low melting point agarose medium containing 10% serum configuration. Place in incubator for 3 weeks, the calculation of the number of clones. 7 mice after 24 hours transfection experiment, collect transfected or transfected cells with PBS, resuspend the cell to a cell density of 1 × 107 个 / ml, taking 0.2 ml injected subcutaneously into 5-week-old BALB / c nude inside ham. Tumorigenicity tumor volume was measured every five days after the growth curve, keeping 42 days, mice were killed, remove the tumor weighed, fixed in 4% formaldehyde for pathological identification. 8 Statistical analysis of data in each experimental group were expressed as mean ± standard deviation, using SPSS17.0 statistical analysis software. Meet the normal distribution and homogeneity of variance with the differences between the two groups compared miRNA expression, the use of two independent samples t-test; three groups were compared using analysis of variance above, pairwise comparisons between groups homogeneity of variance while using LSD-t test. Does not meet the normal distribution using the non-parametric tests. Significance level of a = 0.05. Results 1. 16HBE-T cells, miR-106a and miR-17-5p overexpression 16HBE-T cells mature miR-106a body and miR-17-5p expression levels of mature body is 16HBE-N of 2.9 ± 0.2-fold and 2.3 ± 0.1-fold (P lt; 0.05). 2. 16HBE-T cells transfected with miRNA expression changes 16HBE-T compared to transfected miR-106a inhibitors or mimetics after miR-106a expression levels were 0.3 ± 0.1 and 1.9 ± 0.3-fold (P lt; 0.05 ), transfected with miR-17-5p inhibitors or mimetics after miR-17-5p expression levels were 0.4 ± 0.1 and 1.7 ± 0.2-fold (P lt; 0.05). 3 transfected cell cycle distribution changes and inhibitor NC group, anti-miR-106a group and anti-miR-17-5p group were increased proportion of cells in G0/G1 phase occurs, S phase cells decreased (P lt; 0.05 ). Compared with mimic NC transfected miR-106a mimic reduced proportion of cells in G0/G1 phase, S phase cells increased (P lt; 0.05). 4 after transfection changes in cell proliferation compared with the control group, transfected or miR-106a mimetic inhibitors of cell proliferation was reduced by 0.37 ± 0.01 and 0.45 ± 0.07-fold elevated (P lt; 0.05) transfected with miR-17-5p inhibitors or mimetics of cell proliferation was reduced by 0.29 ± 0.03-fold and increased by 0.34 ± 0.02-fold (P lt; 0.05). 5 after transfection rate of change inhibitor of apoptosis inhibitor test group than in the control group had a higher apoptosis rate (P lt; 0.05), while the negative control group compared with untransfected group, apoptosis rate differentials . Meanwhile, the simulant mimics the experimental group and the control group compared to the apoptosis rate dropped significantly (P lt; 0.05). 6 transfected cell colony formation rate of change inhibitor test group than the control group and the untreated group inhibitor cells showed fewer number of clones (P lt; 0.05). The simulant mimics the experimental group and the control group compared to the number of clones increased (P lt; 0.05). 7 nude mice in 6 treatment groups, anti-miR-106a group, the slowest rate of tumor growth (P lt; 0.05), miR-106a mimic the fastest growth of the tumor group (P lt; 0.05), and 16HBE-T group, Lipo-2000 NC group, inhibitor NC group, mimic NC group, no significant difference in tumor growth. Harvested tumor inoculation 42 days and found, anti-miR-106a group mean tumor weight (35.7 ± 9.6 mg) was significantly lower than the inhibitor NC group, the mean tumor weight (110.8 ± 15.3 mg) (P lt; 0.05). miR-106a mimic group mean tumor weight (340.4 ± 23.1 mg) was significantly higher than that mimic NC group, the mean tumor weight (127.6 ± 21.9 mg) (P lt; 0.05). Identified by pathology found this six kinds of cells injected mice were grown squamous cell carcinoma. Conclusion 1.anti-BPDE-induced malignant transformation of human bronchial epithelial cells 16HBE-T exists miR-106a and miR-17-5p mature body upregulated. 2.miR-106a and miR-17-5p expression levels of mature body changes can affect 16HBE-T cell biology. 3.miR-106a and miR-17-5p in the environmental chemical carcinogen-induced malignant cells plays an important role, are two small molecule cancer genes, can inhibit apoptosis, increased cell malignancy. The project innovation: The first explores the miR-106a and miR-17-5p toxicology, especially in the field of chemical carcinogenesis function mechanism for cancer research provides a new basis.

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