Primairy Study Of MIR-138Targeting RMND5A ND Its Biological Function

As a class of non-coding small RNA, miRNAs are about22nt single-strandnucleotides which inhibit target gene expression through post-transcriptionalregulation, and then participate in various activities of life. In the nucleus,miRNAs are generated into miRNA primary transcripts by RNA polymerase IIor III transcription initially, and cut by a complex microprocessor whichcontains the RNase III Drosha and DGCR8(DiGeorge cirtical region8)constitute, generate about60-70nt precursor miRNAs (pre-miRNAs). Exportin-5recognite the2-nt3’overhangs of pre-miRNAs and export pre-miRNAs intothe cytoplasm depending on Ran-GTP mechanism[1][2]. In the cytoplasm,another RNase III Dicer cut pre-miRNAs into22nt miRNAs duplex——miRNA/of miRNA*. One chain of double-strands that has a lower stability with the5’end of the complementary base usually, generates mature miRNA guidancestrand, then binds with AGO family proteins selectively to form miRISC,interact with the3’UTR of target gene mRNA, resulting in the reduction of thetarget gene mRNA or protein levels by translational repression. While the otherstrand of miRNA*is usually degraded.The expression profiles of miRNAs are very different in different tissues,organs, cell types and growth and development process. They are closely relatedto many physiological and pathological processes, such as growth anddevelopment, cell differentiation, apoptosis, lipid metabolism, hormon secretion,tumor formation and viral infection [3][4][5]. A specific miRNA can target hundreds of target genes, a target gene can beregulated by different miRNAs. Recently, many reports showed that miRNAscan affect multiple functions of cells, and participate in the development ofhuman cancer. Expression level of miRNAs is generally low in tumor cells.miRNA can be a tumor promoter miRNA or a tumor suppressor miRNA incancer cells. miR-138often act as a tumor suppressor miRNA and has manybiological functions in tumor progression and metastasis, cell differentiation,DNA damage and disease correlation. It’s reported that the expression level ofmature miR-138is low but the pre-miR-138-2level is relatively high in HeLacells.Cervical cancer ranks second place of the most common female malignancyin the worldwide; in the tumor-related causes of death in women, cervical cancerranks second. Currently, the detection of cervical cancer miRNA expressionprofiling is used to find specific biomarkers. Further study of specific miRNAand its functional target genes may find the key potential target molecules in theoccurrence and development of cervical cancer.The purpose of our study is to investigate the biological function ofoverexpressed miR-138in HeLa cells. Then the target gene of miR-138will beconfirmed and its biological function will be eluciated in HeLa cells. is theresearch will be divided into the following four parts:Part Ⅰ. Prediction and identification of the RMND5A targeted by miR-138.RMND5A was the highest score target gene of miR-138predicted by thebioinformatic software TargetScan. We verified the two miR-138target seedsites in the RMND5A3’UTR by dual-luciferase reporter gene assay. When HeLacells were transfected with miR-138, the mRNA and protein levels of RMND5Awere decreased significantly. These results suggested that miR-138could target the3’UTR of RMND5A mRNA and inhibit the expression of RMND5A.We further confirmed that the mRNA expression of RMND5A wassignificantly inhibited in tumor cells such as HepG2, H460, H1299and H4100when transfected with miR-138mimics,which suggested that miR-138may beinvolved in a variety of biological processes of tumor cells.Part Ⅱ. miR-138target RMND5A thereby affect the protein level ofExportin-5.As a scaffolding protein, RanBPM can bind Ran[24]. The small GTPase Ranbelongs to the Ras superfamily, and is essential for the RNA and protein throughthe nuclear pore complex. By co-immunoprecipitation assays, we discovered theassociation of RMND5A and Exportin-5surpringly. After knocking downRMND5A in HeLa cells, the protein expression of Exportin-5was suppressed,but the mRNA level was not affected. It is suggested that RMND5A does notaffect the transcriptional level of Exportin-5, and may be interact with Exportin-5to participate in the regulation of biological processes such as precursormiRNA nuclear export. When we used cycloheximide to inhibit proteinsynthesis, we found RMND5A repressed protein degradation of Exportin-5through the interaction with it.Part Ⅲ. miR-138impact nuclear export of precursor miRNA and reduce theoverall level of miRNA by targeting RMND5A.We transfected the Control siRNA, miR-138mimics and RMND5A siRNAinto HeLa cells, then extracted the RNA of cytoplasm and nuclear and detectedthe level of precursor miRNA in the cytoplasm and the nucleus by real-timequantitative PCR, and also the expression levels of mature miRNAs in wholecells. We found that a lot of precursor miRNAs stayed in the nucleus and couldnot be transported to cytoplasm both in the cells tranfacted with miR-138 mimics and RMND5A siRNA. In order to verify the universality of miR-138affect miRNA processing, miRNA microarray was used to identify theexpression of miRNAs. Results showed that there were at least75miRNAswere significantly reduced in HeLa cells transfected with miR-138mimics. It isindicated that miR-138could block the precursor miRNA nuclear exporting andresult in decreasing levels of lots of miRNAs by targeting RMND5A. Byanalyzing the potential target genes of these miRNAs, we found that most oftarget genes may be relevant to cell migration.Part Ⅳ. miR-138inhibit cell migration in HeLa cells.We found that transfection with miR-138mimics or RMND5A siRNA couldinhibit wound healing capacity and migration ability of HeLa cells. Our resultssuggested that miR-138, as a tumor suppressor miRNA, might inhibit themigration of tomor cells.In summary, we found for the first time that over-expression of miR-138could target RMND5A in HeLa cells and affect the precursor miRNA nuclearexport through the interaction with Exportin-5. We also found that over-expression of miR-138could reduce the overall level of miRNAs and inhibit themigration of HeLa cells. A stretch of adenosines is always form poly(A) tail at most mRNA3′-end.Poly(A) tail plays important roles in controlling mRNA stability, translationalefficiency and nuclear export. Here, we showed that short poly(A) transcribedby H1promoter in vivo could accelerate the formation of3’-poly (A) tail ofexogenous GFP mRNA and speed up mRNA export from the nucleus to thecytoplasm in MCF-7cells. Similar results were also observed on exogenous P53gene. Our results indicated that, by using the strategy of transcription of poly(A)in vivo, we could accelerate exogenous gene expression in cells which may bevery useful in gene therapy or in fast exploring a specific gene function.