The Function Analysis Of MiR-506 In Lung Caner

microRNAs(miRNAs)are about 22 nucleotide length RNAs expressed in eucaryotic organism.miRNAs as important regulatory molecules involved in all biological processes including cell differentiation,cell proliferation,cell apoptosis and so on.It was reported that many kinds of external stimuli and cell intracellular signaling regulators can affect the expression and function of miRNAs.Importantly,miRNAs and protein regulators consist of a sophisticated regulation network in organism to orchestrate developmental and cellular processes.It was demonstrated that abnormal changes of miRNAs often causes and contributes to a variety of diseases including cancers.Lung cancer morbidity and mortality became the highest cancer in China,the diagnostics and therapy of lung cancer has been a major challenge worldwide.During lung cancer genesis and development processes,many signaling pathways,including oncogenic and tumor suppressive pathways,were found altered,hinting that the expression and function of miRNAs were altered accordingly.So it is worthy to identify and illustrate the abnormal expressing miRNAs in order to understand the signaling pathway regulation and mechanism of development of lung cancer,and provide important theoretical references for the clinical diagnosis,treatment and prognosis of lung cancer.Our laboratory previously has studied miRNA expression profiling,and found a number of aberrant expression of miRNAs in lung cancer patients.This thesis research selected a dozen miRNAs based on the miRNA expression profiling,and found miR-506 that can markedly inhibit the proliferation and induce apoptosis of lung cancer cells by EdU and Caspase-3/7 detection assays.We identified that p65 and cyclin-dependent protein kinase 6(CDK6)are two potential targets of miR-506 by bioinformatics and biochemical experiments.The further experimental data showed that miR-506 directly regulates p65 expression resulting in the Caspase-8 activation,which triggers apoptosis in the lung cancer cells.Besides this,in lung cancer cells miR-506 induced cell cycle arrest in G1 Phase by directly suppressed CDK6 gene,resulted in inhibition of cancer cell proliferation.Interestingly,we found that the expression of miR-506 is regulated by p53 transcriptional factor in lung cancer cells.The expression of miR-506 was upregulated after p53 activation by Adriamycin treatment.But after silencing p53 gene by specific siRNA,the expression of miR-506 failed to elevate after Adriamycin treatment.However,the re-expression of exogenous wild type p53 can restore the miR-506 expression in the p53-null H1299 cells.Chromatin immunoprecipitation(ChIP)assay using p53-specific antibodies revealed that miR-506 uptream region is bound by p53 in A549 cells subjected to Adriamycin treatment.These findings suggest that miR-506 transcription may be induced directly by p53.Further experiments showed that downregulation of p65 by miR-506 causes a p53-dependent feed-forward activation of miR-506.We also found that the p65 downstream antioxidant reductase SOD2 expression was decreased by treated with miR-506,leading to rise cell ROS(Reactive oxygen species)level,and the accumulation of ROS activated p53 expression.In addition,the exogenous hydrogen peroxide treatment of A549 cells found that the ROS can increase the expression of miR-506 through the activation of p53,thus inhibiting the p65 protein levels leads to the generation of apoptosis,which can be inhibited by Antagomir-506 treatment.We analyzed the miR-506 expression in cancer tissue compared with the normal counterpart in 40 lung cancer patients.The results showed that miR-506 was significantly up-regulated in 33/40 lung cancer tissues,and the mRNA expression of p65 and CDK6 had a reverse relationship with miR-506 expression.We further verified that tumor volume in animals that were administered cells pre-treated with miR-506 mimetics was significantly smaller than tumor volume in animals that were administered cells treated with an identically formulated No-target control and Lipo2000.In summary,we show in this thesis that miRNA-506 regulates the cross talk between p53 and NF-kB in lung cancer cells,providing a potential novel target for diagnosis and therapeutic treatments.