Identifiction And Function Of Kidney-specific MicroRNA

MicroRNAs (miRNAs) are 19-22nt non-coding RNAs that widely exist in eukaryotes. miRNAs are highly conserved among various species and can suppress gene expression at posttranscriptional level. It has been widely shown that miRNAs play critical roles in regulating a variety of cellular developmental and physiological processes, including development, hematopoiesis, organogenesis, viral defense, fat metabolism, cell proliferation and apoptosis. Profile studies have already shown that many miRNAs are specifically expressed in certain organs, cell types and developmental stages and these organ-specific miRNAs are essential to maintain normal physiological function of such organ. However, so far no kidney-specific miRNAs have been identified and their role in modulating renal function remains unknown. Here, we performed an unbiased assessment of genome-wide miRNA expression profile using Solexa sequencing and selected kidney-specific miRNAs. Then we further studied the possible role of these miRNAs in kidney, which may provide us more clues about the roles of miRNAs in the kidney.Part Ⅰ:Identification and function prediction of kidney-specific microRNAsObjective:Solexa sequencing was used to screen for kidney-specific miRNAs. The target genes of the identified miRNAs were analysed by bioinformatics to predict the biological functions of the miRNAs.Methods:To screen for the miRNAs specifically expressed in kidney,11 tissues or organs from 8 weeks C57/BL6 male mice were collected. Total RNA was extracted, followed by small RNA isolation. Solexa sequencing was performed to profile the small RNA samples. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to validate the results from the sequencing. Then, in order to identify the distribution of miR-196a and miR-196b in the kidney, in situ hybridization was performed using normal kidney tissue sections from mice and human. To further clarify the different expression of miRNA196a/b between glomerular and tubular, we performed qRT-PCR assay using isolated mouse or human glomerular and tubulointerstitial fractions. Bioinformatic analysis was performed to predict the target genes of the kidney specific miRNAs, then for function predition the predicted target genes were subject to GO (gene ontology) and KEGG pathway analyses.Results:(1) Solexa sequencing results showed that miR-196a and miR-196b were the kidney specific miRNAs in normal mice. The distribution of miR-196a and miR-196b in 11 tissues or organs of mice were validated by qRT-PCR, and the results were consistent with those from sequencing; (2) In situ hybridization clearly showed that miRNA196a/b were widely distributed in human and mouse kidneys and miR-196a and miR-196b were strongly expressed in both glomerular and tubulointerstitial cells; the qRT-PCR assay confirmed that miR-196a and miR-196b were heavily expressed in both glomerular and tubulointerstitial fractions and the amount of miR-196a/b in glomeruli was higher than in tubulointerstitial; (3) The number of overlapping predicted target genes by TargetScan6.2 and PicTar was 74. GO analysis showed that the genes participated in the regulation of platelet-derived growth factor binding, transcriptional activity and extracellular matrix structural constituent and the genes were also associated with several biological processes, including metabolic process and developmental process(p<0.01). The pathway analysis showed that the genes significantly enriched in GnRH signaling, ECM-receptor interaction, and glioma and prostate cancer (p <0.05).Conclusion:miR-196a/b specifically and extensively expressed in the kidney, and their functions are predicted to be closely related with cell metabolism, growth and development. These results provide us clues to further study the role of miR-196a/b in the physiology and pathology of kidney.Part Ⅱ:The role of kidney-specific miRNA196a/b in renal fibrosisObjective:To study the change of miR-196a/b expression in unilateral ureteral obstruction (UUO) mice, and explore its impact and moleular mechanisms in renal fibrosis which can help us reveal new targets to intervent fibrosis.Methods:In order to explore the role of miR-196a/b in renal fibrosis, first of all, we randomly divided 15 C57/BL6 male mice(8 weeks) into three groups:sham group, UUO 3d group and UUO 7d group. The mice in each group were sacrificed at the corresponding time points. Masson staining was used to evaluate the degree of renal fibrosis among the groups and qRT-PCR was used to detect the expression of miR-196a/b in each group. Subsequently, hydrodynamic injection (HDS) method was performed to overexpress miR-196a/b in renal tissue of UUO. Then qRT-PCR was utilized to detect the levels of miR-196a/b and Masson staining was used to assess the renal fibrosis after overexpressing miR-196a/b. To further investigate the possible molecular mechanism of miR-196a/b involved in renal fibrosis, by target prediction, we found renal fibrosis-related protein such as TGFβ receptor 2(TGFβR2), collagen al chain (COLA1) and collagen a2 chain (COLA2) mRNA 3’UTR were the target of miR-196a/b, and they were verified by luciferase reporter assay. In order to clarify the role of miR-196a/b in TGFβ/Smad signaling pathway, we suppressed the expression of miR-196a/b in human proximal tubular epithelial cells (HK2) and the protein level of TGFβR2, COLI and Smad were detected. In order to further validate the role of miR-196a/b in renal fibrosis, in vivo, the renal fibrosis-related protein levels were detected by immunohisto chemical and Western.Results:(1) miR-196a/b expression levels were gradually decreased along with the worse of UUO renal fibrosis. (2) miR-196a/b has a protective role in UUO mice:Masson staining showed that overexpressed miR-196a/b can ameliorate the renal fibrosis of UUO mice; (3) luciferase reporter assays demonstrate that miR-196a/b can binding with the 3’UTR of TGFβR2, COLIA1 and COLIA2 mRNA, thereby miR-196a/b can inhibite the expression of TGFβR2 and COLI; (4) after inhibiting the expression of miR-196a/b in HK2, the protein level of TGFβR2、COLI can be up-regulated and the phosphorylation level of Smad2 and Smad3 can also be increased; (5) in vivo, Western and immunohistochemistry results showed that the elevated protein level of TGFβR2, α-SMA and COLI in miR-196a/b overexpressed group were significantly lower than the UUO group.Conclution:Our results firstly provided that the kidney-specific miR-196a/b play an inhibitory role in the progress of renal fibrosis by downregulating TGFβR2 and COLI, and maintaining renal miR-196a/b level via delivery of miR-196a/b-expressing vector may be a potential therapeutic strategy for renal fibrosis in the future.