The Role And Molecular Mechanism Of MicroRNAs In Breast Cancer And Neuronal Dysplasia

MicroRNAs (miRNAs) are highly conserved non-coding RNAs that functions by gene regulation at post-transcriptional level. They interact with the specific segment of 3'-untranslated region (3'-UTR) contained in mRNA of the target genes by incomplete complementary to inhibit gene expression. Almost one-third of the protein-coding genes are susceptible to miRNAs regulation, while every miRNA has hundreds of target genes.miRNAs play pivotal roles in regulation of the complex gene network. Therefore, they have important functions in biological processes, such as cellular growth, differentiation,apoptosis, and occurrence of human illness. The research of miRNAs in expression pattern and mechanisms during the process of disease can provide new markers for the diagnosis and treatment.1. The roles and molecular mechanisms of miR-101 and miR-185 in the occurrence of breast carcinogenesisRecent years, increasing evidence indicates that the abnormal expression of miRNAs closely associate with the occurrence and development of tumors. miRNAs have abnormal expression profiles in many kinds of cancers, such as lung cancer, breast cancer, esophageal cancer, prostatic cancer, pancreatic cancer, gastric cancer, colon cancer and et al. Our lab once used human breast cancer tissues and adjacent normal breast tissues as research subjects to screen the differential expression of miRNAs during the occurrence of breast carcinogenesis by the method of screening the candidate genes.We found that miR-101 and miR-185 were differentially expressed. Then we studied the specific roles they play in the progress of breast cancer.Our study found that miR-101 was significantly down-regulated in triple negative,ER/PR and HER2 positive, ER/PR negative and HER2 positive, ER/PR positive and HER2 negative breast cancer tissues compared with that in adjacent normal breast tissues.Up-regulation of miR-101 inhibited cell proliferation, migration and invasion, and promoted cell apoptosis in breast cells. Down-regulation of miR-101 displayed opposite effects on cell growth and metastasis. Further investigation revealed a significant inverse correlation between the expression of miR-101 and Stathminl (Stmnl), and miR-101 could bind to the 3'-untranslated region (UTR) of Stmnl to inhibit Stmnl translation. The inhibition of cell growth and metastasis induced by up-regulation of miR-101 was partially restored by overexpresson of Stmn1. Knockdown of Stmnl attenuates the down-regulation of miR-101-mediated enhancement of cell growth and metastasis. More importantly, in vivo analysis found that Stmn1 mRNA and protein level in different subtypes of human breast cancer tissues, contrary to the down-regulation of miR-101,were significantly elevated.MiR-185 was significantly decreased in triple negative, ER/PR and HER2 positive,ER/PR negative and HER2 positive human breast cancer tissues compared with that in adjacent normal breast tissues, while mildly down-regulated in ER/PR positive and HER2 negative breast cancer tissues. We researched the functions of miR-185 in breast cancer by overexpression of miR-185 in relatively low-expressed breast cancer cells MDA-MB-231 and MDA-MB-453 , and knockdown of miR-185 in relatively high-expressed breast cancer cells MCF7 and T47D. The results showed that up-regulation of miR-185 inhibited cell proliferation, migration and invasion, and promoted cell apoptosis in breast cancer cells, vice versa. Meanwhile, we verified that vascular endothelial growth factor a (Vegfa) is one of the target genes of miR-185. The inhibition of cell growth and metastasis induced by up-regulation of miR-185 was partially restored by overexpresson of Vegfa. Knockdown of Vegfa attenuates the down-regulation of miR-185-mediated enhancement of cell growth and metastasis.Additionally, Vegfa mRNA and protein level in triple negative, ER/PR and HER2 positive,ER/PR negative and HER2 positive human breast cancer tissues, were significantly elevated.In summary, miR-101 and miR-185 are down-regulated in different subtypes of breast cancer tissues, and have similar regulation to breast cancer cells, which closely associates with the enhancement of the ability of cellular proliferation and invasion by their respectively target genes. All of these results may provide valuable clues for the clinic diagnosis of breast cancer in the future.2. The functions and regulation mechanisms of miR-206 in abnormal development of nerve cellsMiR-206 is one of the members in miRNAs family. miR-206 plays important roles in skeletal muscle. However, whether miR-206 functions in other kinds of cells is still unclear. In our previous study, we found that differential expression of miR-206 in SA-treated chick embryos with neural tube defects (NTDs) by microRNA microarray analysis. Therefore, we speculate that miR-206 may play important roles in the development of nervel cells. We verified the differential expression of miR-206 in SA-treated chick embryos by Northern blot analysis. Ectopic expression of miR-206 inhibited cell proliferation, and promoted cell apoptosis in human glioma cell U343 and neuroblastoma cell SK-N-SH cell by using MTT, Edu Apollo assay and Flow cytometry analysis, vice versa. Bioinformatics predicts that Orthodenticle homeobox 2 (Otx2) may be one of the targets of miR-206. So we cotransfected 3' -UTR of Otx2 with miR-206 mimics or miR-206 inhibitor into cells. The dural luciferase activity analysis showed that the luciferase activity was attenuated by overexpression of miR-206, while enhanced by knockdown of miR-206. These results indicated that miR-206 may interact with the 3'-UTR of Otx2. Overexpression of miR-206 down-regulated the endogeneous Otx2 expression, whereas knockdown of miR-206 obviously up-regulated the expression of Otx2 by western blot. The results further verified the relationship between miR-206 and Otx2. Meanwhile, OTX2 was also significantly down-regulated in SA-treated chick embryos with NTDs by whole-mount immunofluoresence and western blot. All of these results indicate that miR-206 may modulate the development of nerve cells by targeting Otx2.