The Research About Intronic Mirnas Feedback Regulating The Expression And Function Of Their Host Genes

In recent years the research about miRNAs has been a hotspot of non-encoding RNAs. MiRNAs are endogenous, 22-nucleotide (nt) RNAs that are expressed at specific stages of tissue development or cell differentiation. MiRNAs are involved in the regulation of gene expression during development, cell proliferation, apoptosis, glucose metabolism, stress resistance, and cancer development at the post - transcriptional level. Most cellular miRNAs are transcribed by RNA polymerase II as long, capped, polyadenylated primary miRNA (pri-miRNA) transcripts bearing a stem-loop hairpin structure of ~80-nts. Most mature miRNAs were generated by processing of pri-miRNAs in two sequential cleavage steps mediated by two RNase III enzymes, Drosha and Dicer. The miRNA-target mRNA recognition is mainly due to the perfect complementarity between a short 7~8 nt stretch at 5` end of the miRNA molecule (miRNA seed) and the corresponding stretch on the 3` UTR of the target gene (seed match). This results in a reduced translation or a decreased level of the mRNA .Following the intensive research, the function of more and more miRNAs have been identidied. Meanwhile a sort of miRNAs which be located in the protein-encoding genes(host genes), called intronic miRNAs, have been drawn attention gradually because most of these intronic miRNAs are co-expressed with their host gene, suggesting that they and their host genes have some potential relationship. Moreover some intronic miRNAs have been reported to feedback regulate their host genes in transcriptional and/or functional level. This paper is intended to find out whether this feedback regulation is prevalent.In the first part, we found that miRNA-301 highly expresses in A549.When miRNA-301 was blocked by special inhibitor, the transcription of its host gene, Ska2, decreased. Meanwhile it led to increase of mitotic index and decrease of colony formation in soft agar, suggesting that miRNA-301 affect the function of Ska2.More research showed that miRNA-301 up-regulated REK pathway through directly targeting MEOX2, which enhanced the activity of CREB, a downstream molecular of ERK pathway. By bioinformatic analysis and ChIP assay, CREB was confirmed as a transcriptional activator of Ska2. The increase of CREB activity led to the increase mRNA level of Ska2. Meanwhile miRNA-301 up-regulated Ska1, a partner of Ska2, suggesting miRNA-301 feedback regulated its host gene in function level. As the increase of CREB activity may up-regualating its downstream oncogenes, such as c-fos and egr1, it is suggested that miRNA-301 feedback regulating its host gene may be involved in the lung tumorigenesis.In the second part, to verify the prevalence of this kind of feedback regulation, all miRNAs information were obtained from Sanger database and all the conserved intronic miRNAs (about 65) were picked up. Ten intronic miRNAs (miR-26a/93/107/140-5p/191/153-1/196b/208a/218-1/338-5p) ,most highly expressed in the common tumor cells , were picked up from the conserved miNRAs by referring to Ambion's miRNA expression profile. Then inhibitors of these intronic miRNAs were synthesized. These inhibitors were transfected to five common tumor cell lines, the expressions of their host genes were analyzed by qPCR. We found that most of intronic miRNA feedback regulated their host genes in different cells. These results provide a better understanding for intronic miRNAs.