The Role Of MiRNA In Development Of Diseases And Prediction Of Drug Efficacy

MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression in plants and animals. Through miRNA guides the binding of the RNA-induced silencing complex to complementary sequences in the 3’-untranslated region or the open reading frame of target mRNA molecules, miRNAs either degrade mRNA or block the gene translation. MiRNAs play a key role in the cellular processes of differentiation, proliferation, maturation, and apoptosis. Recent studies have demonstrated that miRNAs are stably expressed in animal serum/plasma and that their unique expression patterns serve as "fingerprints" of various diseases. Further studies suggest that cells can selectively secret miRNAs via MVs into target cells, in which they silence their target genes and affect recipient cell function. In this thesis, we study the miRNA function in development and treatment of diseases.First we researched the role of platelet-secreted miRNA-223 affecting vascular endothelial cell apoptosis. First, platelets expressed abundant miRNAs, and miR-223 had the highest level of expression. Platelet miR-223 and other miRNAs can be upregulated by the stimulation with thrombopoietin (TPO) or thrombin. Unlike leukocytes, platelets contained high levels of pre-miRNAs, and upregulation of mature platelet miRNAs by TPO was correlated with decreased pre-miRNAs. Second, under stimulation with TPO, platelets released a large amount of MVs, which also contain higher levels of miR-223. Elevation of miR-223 inside circulating platelet MVs (P-MVs) was also observed in plasma samples from patients with enteritis, hepatitis, nephritis, or atherosclerosis. Third, incubation of P-MVs with HUVECs, which had significantly lower levels of miR-223 than platelets, showed that P-MVs effectively delivered miR-223 into HUVECs. Finally, in HUVECs, exogenous platelet miR-223 decreased the level of insulin-like growth factor 1 receptor and thus promoted HUVEC apoptosis induced by advanced glycation end products.Second, we studied the circulating HCMV-miR-US4-1 as an indicator for predicting the efficacy of IFNa treatment in chronic hepatitis B patients. Identification of an easy accessible biomarker that can predict the outcome of IFNa treatment for individual CHB patients would be greatly helpful to CHB patients. Recent reports show that miRNAs encoded by human cytomegalovirus (HCMV) were readily detected in human serum and can interfere with lymphocyte responses required by IFNa therapeutic effect. We thus postulate that differential expression profile of serum HCMV miRNAs in CHB patients may serve as indicator to predict the efficacy of IFNa treatment for CHB patients. Blood was drawn from 56 individual CHB patients prior to IFNa treatment. By quantifying 13 HCMV miRNAs in serum samples, we found that the levels of HCMV-miR-US4-1 and HCMV-miR-UL-148D were significantly higher in IFNa-responsive group than in IFNa-non-responsive group. In a prospective study of 96 new CHB patients, serum level of HCMV-miR-US4-1 alone classified those who were and were not responsive to IFN-a treatment with correct rate of 84.00% and 72%, respectively. In conclusion, our results demonstrate that serum HCMV-miR-US4-1 can serve as a novel biomarker for predicting the outcome of IFNa treatment in CHB patients.Third, we report that miR-200b/c can promote colorectal cancer cell proliferation via targeting the reversion-inducing cysteinerich protein with Kazal motifs (RECK). MicroRNA-200b and microRNA-200c (miR-200b/c) are 2 of the most frequently upregulated oncomiRs in colorectal cancer cells. Firstly, bioinformatics analysis predicted RECK as a conserved target of miR-200b/c. By overexpressing or knocking down miR-200b/c in colorectal cancer cells, we experimentally validated that miR-200b/c are direct regulators of RECK. Secondly, an inverse correlation between the levels of miR-200b/c and RECK protein was found in human colorectal cancer tissues and cell lines. Thirdly, we demonstrated that repression of RECK by miR-200b/c consequently triggered SKP2 (S-phase kinase-associated protein 2) elevation and p27Kipl degradation in colorectal cancer cells, which eventually promotes cancer cell proliferation. Finally, promoting tumor cell growth by miR-200b/c-targeting RECK was also observed in the xenograft mouse model. Taken together, our results provided a potential new target for colorectal therapy in the future.Finally, we research the mechanism of miR-221 promoting breast cancer progression.In this study we reveal a novel mechanism that E-cadherin is post-transcriptionally regulated by Slug-promoted miR-221, which serves as an additional blocker for E-cadherin expression in metastatic tumor cells. Profiling the predicted E-cadherin-targeting miRNAs in breast cancer tissues and cells showed that miR-221 was abundantly expressed in breast tumor and metastatic MDA-MB-231 cells and its level was significantly higher in breast tumor or MDA-MB-231 cells than in distal non-tumor tissue and low-metastatic MCF-7 cells, respectively. MiR-221, which level inversely correlated with E-cadherin level in breast cancer cells, targeted E-cadherin mRNA open reading frame (ORF) and suppressed E-cadherin protein expression. Furthermore, both in vitro and in vivo results showed that Slug-promoted miR-221 enhanced the breast tumor progression through reducing E-cadherin protein expression.In summary, we investigated the mechanism of miRNA involved in development and treatment of different diseases, and our results may has significant clinical value.