The Effects of miR-4492 on Nos1ap/CAPON and its Intragenic miRNAs in PC-3 Cells

摘要

MicroRNAs (miRNAs/miRs) are short noncoding RNAs of approximately15-22 nucleotides in length, depending on the organism. They are expressed plants, animals, bacteria, and some viruses and imperfectly binds the 3' untranslated region (3'-UTR) of mRNAs to inhibit translation and cause mRNA degradation. They regulate several cellular processes, and they have been implicated in a plethora of diseases and non-diseases. Their mechanisms of actions are still being vastly studied that they can be developed for therapeutic purposes. The overall goal of this study is to clone hsa-miR-4492 into an expression DNA vector and analyze its effects on Nitric Oxide Synthase 1 Adaptor Protein (NOS1AP), which is also known as CAPON. NOS1AP/CAPON have been linked to countless human diseases, such as cancer, cardiovascular disease, diabetes, dystrophy, schizophrenia, post-traumatic stress disorder (PTSD), and autism. Currently, there are three NOS1AP/CAPON isoforms (one CAPON-S and two CAPON-L). NOS1AP/CAPON has been found to play a role in cell cycle regulation, cell proliferation, and cell migration. The aims for this study are: (1) to demonstrate that miR-4492 will target the 3'-UTR of NOS1AP/CAPON mRNA as predicted at the miRDB to reduce its protein expression in PC-3 cells; and (2) to demonstrate that miR-4492 will either equally or unequally target the 3' untranslated region (3'-UTR) of the two isoforms of NOS1AP/CAPON-L and NOS1AP/CAPON-S, since their 3'-UTR nucleotide sequence is similar, but the length of their mRNAs is different; and (3) to demonstrate that reduced expression of NOS1AP/CAPON mRNA by expression of miR-4492 will also cause reduced expression of two intragenic miRNAs, miR-4654 and miR-556, localize within introns 2 and 5 of the NOS1AP/CAPON gene. Three working hypotheses are developed: (1) expression of hsa-miR-4492 will inhibit the translation of NOS1AP/CAPON mRNA and reduce its RNA; (2) inhibition of NOS1AP/CAPON mRNA by expression has-miR-4492 will either equally or unequally reduce the expression of NOS1AP/CAPON mRNA isoforms; and (3) degradation of NOS1AP/CAPON RNA by expression of hsa-miR-4492 will diminish the expression of the two intragenic miRs, miR-4654 and miR-556, which are localized in the introns of the NOS1AP/CAPON gene, and they are likely expressed and localized within the pre-mRNA of NOS1AP/CAPON. The purpose of this study is to examine the expression effects of hsa-miR-4492 on NOS1AP/CAPON and its intragenic miRs in PC-3 cells. This study validates that expression of hsa-miR-4492 targets mRNA of NOS1AP/CAPON and downregulate protein expression of the short (S) isoform (NOS1AP/CAPON-S), but not the long (L) isoform (NOS1AP/CAPON-L). Furthermore, this study also shows that the RNA expression of the intragenic miRs localized within introns two and five of the NOS1AP/CAPON gene are altered by the expression of hsa-miR-4492, inferring that both target mRNA of a gene and its intragenic miR can be regulated by miRNAs provided they are present within a single pre-mRNA transcript.