| Environmental carcinogens-induced lung cancer and related mechanism is being paid more attention to worldwide. Changes in the epigenome induced by envireonmental exposures may regulate development and progeression of cancer. micro RNAs(mi RNAs) have been recognized as key players in development of cancer. Aflatoxin B1(AFB1) is normally predominant in fungal cultures as well as in food products, it has been classified as potent carcinogen, airborne AFB1 may relate with lung cancer.AFB1 is an indirect carcinogen that is bioactivated by cytochrome P450 2A13. Our previous study showed that treatment of 0.1 n M AFB1 for 50 passages(P50) could induce malignant transformation of immortalized human bronchial epithelial cells(BEAS-2B) stably expressing CYP2A13(P50 B-2A13 cells). However, the role of mi RNAs in this carcinogenic proceeding is still unclear. Therefore, Making sure the effects and mechanisms of mi RNAs on the malignant transformation induced by AFB1 in BEAS-2B cells is helpful to fully understand the roles of mi RNAs in carcinogenesis induced by Environmental carcinogens.In this study, we identified dysregulated mi RNA expression between P50 B-2A13 cells and P50 B-Vector cells by microarray analysis, screening those mi RNAs that are associated with malignant transformation of cells. Then the effect and mechanism of mi RNAs on cell function are detected. The present study will be helpful to strength the understanding of AFB1 in relating to lung cancer, and provide biomarkers and strategy in prediction, therapy and prevention of cancer.Part Ⅰ: Screening of the key mi RNAs related to the malignant transformation of B-2A13 cells induced by AFB1Objective: Screening the key mi RNAs that regulating the malignant transformation induced by AFB1 in B-2A13 cells.Methods: Dysregulated mi RNAs were identified by Affymetrix micrarray analysis in P50 B-2A13 cells. Target genes of mi RNAs were predicted with Target Scan. Key mi RNAs that participate in cell malignant transformation or lung cancer carcinogenesis were screened. Then using quantitative real-time polymerase chain reaction(RT-q PCR) validated the expression of them. And the effect of mi RNAs(mi R-138-1*, mi R- 1271, mi R-708 and mi R-296-3p) on cell proliferation and migration in P50 B-2A13 cells were also evaluated by using cell proliferation experiment and scratch test.Results: 1. There were 36 upregulated and 27 downregulated mi RNAs in P50 B-2A13 cells were identified. 2. The preliminary screening of dysregulated mi RNAs: there were 13 dysregulated mi RNAs were selected, including seven upregualted mi RNAs, mi R-432, mi R-382, mi R-370, mi R-335, mi R-183, mi R-494 and mi R-421; the other six downregulated mi RNAs were mi R-138, mi R-138-1*, mi R-708, mi R-125-1b*, mi R-1271, mi R-296-3p. 3. RT-q PCR validation: There was a high correlation between RT-q PCR and microarray data. Besides mi R-370, mi R-138 and mi R-494, alteration of the others were all in accord with microarray data. 4. Cell proliferation: compared with control group(mi R-Control), mi R-138-1* can dramatical inhibit proliferation of P50 B-2A13 cells. However, mi R-1271, mi R-296-3p and mi R-708 had no affect the proliferation of P50 B-2A13 cells. 5. Cell migration: All the four mi RNAs detected can suppress the migration of P50 B-2A13 cells, the 24 h wound closure(%) of mi R-Control, mi R-138-1*, mi R-1271, mi R-296-3p and mi R-708 were respectively47%, 27%, 30%, 37% and 32%. Part Ⅱ:Effects and regulating mechanisms of mi R-138-1*in AFB1-induced malignant transformation of B-2A13cellsObjective: Exploring the effect and mechanism of mi R-138-1* on the malignant transformation induced by AFB1 in B-2A13 cells.Methods: ectopic mi R-138-1* expression or si RNA-mediated knockdown of PDK1 were maked by transient transfection with mi RNA mimics or si RNA. In vitro studies were carried out to evaluate the role of mi R-138-1* or PDK1 in P50 B-2A13 cells. RT-q PCR, Western blot, Luciferase reporter assay,cell motility, invasion and colony formation assays were performed according to standard procedures.Results: 1. Compared with control cells, ectopic expression of mi R-138-1* can dramatical suppress the proliferation, migration, invasion and colony formation of P50 B-2A13 cells. 2. A reporter assay revealde that mi R-138-1* repressed the luciferase activity of a reporter gene coupled to the 3′-untranslated region of PDK1. Mi R-138-1* overexpression downregulated PDK1 expression at m RNA level and protein level. 3. si RNA-mediated PDK1 knockdown did not affect the expression of mi R-138-1*. 4. The regulation of mi R-138-1* on the PI3K/PDK1/Akt signal pathway: the expressions of p-PDK1, p-Akt, and p-GSK-3β significantly increased in P50 B-2A13 cells in comparison of P0 B-2A13 cell, P50 B-vector cells and BEAS-2B cells. Same as this, the expression of PDK1 decreased in P50 B-2A13 cells transfected with mi R-138-1*. However, the p-PDK1 was almost inhibited by mi R-138-1*, so were its downstream proteins p-Akt and p-GSK-3β. 5. Compared with control cells, the si RNA-mediated knockdown of PDK1 phenocopied the inhibiting effect of mi R-138-1* on the proliferation, migration, invasion and colony formation of P50 B-2A13 cells.Conclusions1. There were 63 dysregulated mi RNAs in malignant transformation P50 B-2A13 cells were identified, including 36 upregulated and 27 downregulated mi RNAs. increasing the expression of mi R-138-1* can dramatical suppress the proliferation, migration, invasion and colony formation of P50 B-2A13 cells.2. Ectopic expression of mi R-138-1* significant suppressed the m RNA level and protein level expression of PDK1, however. PDK1 was one of the target genes of mi R-138-1*.3. PI3K/PDK1/Akt signal pathway was activated in the malignant transformation of P50 B-2A13 cells. mi R-138-1* can regulate the activation of PI3K/Akt/GSK-3β pathway by targeting PDK1.4. si RNA-mediated knockdown fo PDK1 could significant inhibt the proliferation, colony formation, migration, and invasion of P50 B-2A13. mi R-138-1* at least partially targeting PDK1, played an important role in AFB1-induced malignant transformation of B-2A13 cells. |
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