| Objective:To investigate the association of Rafl expression with clinical characteristic and prognosis in non-small cell lung cancer (NSCLC). To clarify the mechanism of miRNA-643 affected radiation sensitivity and invasion metastasis in NSCLCcell lines by regulating Rafl.Methods:The expression of Rafl in paraffin embedded tissues of 110 NSCLC were detected by immunohistochemistry; and analyzed the effects of the Rafl expression on clinical pathological parameters and therapeutic effect of radiotherapy. Cox proportional hazards regression models were performed to estimate the risk prognostic factors. We detected Rafl expression by real-time quantitative PCR (RT-PCR) and western blot in NSCLC cell lines, interference of Rafl was executed in A549 and H1299 cell lines with Rafl high-expression and low-expression respectively by siRNA technology. The cellular growth activity was measured by CCK-8 and cell colony assay.The cellular radio-sensitivity, migration and invasion ability were detected by cell scratch test and transwellchamber.We utilized three microRNA (miRNA) target prediction programs, and information from the existing literature to find the miRNA-643 which targets Raf1; The expression of miRNA-643 in lung cancer cells A549 and H1299 was detected by real-time PCR.The banding relationship of miRNA-643 with Rafl 3’UTR was observed via luciferase reporter assaytest. The add-back rescue assay was conducted to validate the effect of miRNA-643 on cellular radio-sensitivity, migration and invasion ability in A549 and H1299 cell lines via regulating the target gene Rafl, and then we detected the altered expression of AKT/NF-κB signaling pathway-associated proteins and pro-apoptotic proteins.Results:1. Among the 110 NSCLC patients,49 (44.5%) cases of Rafl expression were positive and high expression was 21(19.1%) cases, the low was 28 cases (25.5%); The expression of Rafl are correlated with lymph node metastasis and T stage(P<0.05);The time to progression and 3-years overall survival in patients with Rafl-positive were significantly less than those with Rafl-negtive (p<0.05).2. The expression of Rafl is higher in HI299 and is lower in A549 and H827. The proliferation, migration and invasion ability were significantly weakened with the radio-sensitivity increased after silencing Rafl gene expression in HI299. However, the opposite results were observed after over expressing Rafl genein A549.3. We predicted that miRNA-643 may regulate the expression of Rafl by online software TargetScan, miRanda and TarBase, and then we confirmed that Rafl is a specific target gene of miRNA-643 via luciferase reporter assay test. Over expressing miRNA-643 inhibited the expression of Rafl mRNA and protein.4. Further study demonstrated that add-back rescue over expressing Rafl could reverse the inhibition effect of cellularradio-resistance, proliferation, migration and invasion ability induced by miRNA-643 stably expressing in HI299 cell line; In contrast, blocking Rafl gene could down-regulate the cellular radio-resistance, proliferation, migration and invasion ability induced by silencing miRNA-643 in A549.5. The expression and activity of AKT, p65 and Vimentin were up-regulated along with down-regulation of E-cadherin, on account of Rafl gene overexpression; while silencing Rafl gene expression, the result of the expression and activity of the protein above changed on the contrary, along with down-regulation of caspase-3 and caspase-9.Conclusion:Rafl is associated with early time to progression and poor prognosis in NSCLC patients after treatment with radiotherapy, and increase malignant progression and radiation resistance. miRNA-643 plays an important role in lung cancer cell proliferation invasion metastasis and incresease radiosensitivity via down-regulated. High expression of miR-643,the biological behavior of lung cancer cells may be decreased and the radiosensitivity increased, it serves as antioncogene in NSCLC, provide a new exploration direction for molecular targeted therapy and radiotherapy in NSCLC. |
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