The Effects Of Advanced Glycation End Products On MirNA-92b And Its Role In The Pathogenesis Of Diabetic Nephropathy

In recent years, some studies have shown that microRNAs (miRNAs) play animportant role in the pathogenesis of diabetic nephropathy. This kind of small RNAmolecules influence mRNA gene expression regulation in the form of incompletecomplementary to its target mRNA3’noncoding region (UTR) blocking the specificbinding of the translation of mRNA, but, the specific mechanism involved deeply isstill unclear. Advanced glycation end of products (AGEs) which is one of theimportant pathogenesis of DN, however, its effect on the expression of miRNAs hasnot reported. In this study, firstly, miRNAs chip technology was introduced to detectkidney miRNAs expression changes of rat which given AGEs. Then, Real Time PCRwas applied to verify the expression of miRNAs changed obviously.Ultimately, themiRNA target genes were determined, and the mechanism of the miRNA and itstarget genes were elucidated.Our aim is to explore the effects of AGEs on miRNA andits role in the pathogenesis of diabetic nephropathy in rats.Experiment I The construction of AGEs renal injury animal modelsNormal rats blood serum was separated and collected, and AGEs and pure serum(RSA) were prepared respectively. Rats were divided randomly into3groups. Normalrats (Control group) do not exert any treatment; RSA group animal were given pureserum100mg/kg by tail intravenous one time daily; AGEs group were given AGEsserum100mg/kg by tail intravenous one time daily. Kidney damage was tested after6w. Results show that24h urine protein excretion rate and kidney organizationstructure has no obvious change in RSA group comparing with the control group.However, in AGEs group rats,24h urine albumin excretion rate trends to be higherthan that in RSA group and Control group animal, and histopathological examinationshowed glomerular mesangial cells appeared mild hyperplasia, glomerularextracellular matrix had increased and congregate locally agglomeration. Theseresults indicated animal in AGEs group had appeared pathological changes similar todiabetic nephropathy. Experiment II miRNA microarray screen miRNA expression profiling of renaltissue and Real time-PCR verificationThis part, firstly, three rats was selected in AGEs group and the RSA groupsaccording to24h urine albumin excretion rate to detect miRNA expression profilingusing miRNA microarray. And then, select miRNAs distinguishing obviously and inputmiRBase database to obtain the corresponding miRNAs sequence information and therelevant comments, input TargetScan databases in turns to find corresponding targetgenes of miRNAs (target genes include conservative genes and nonconservativegenes). Several miRNAs changed obviously was selected according to the cell factorclosely related to the pathogenesis of diabetic nephropathy. Finally, Real time-PCRverificated these selected miRNAs.According to the results of miRNA microarray, nine miRNAs changed obviouslywere observed (p <0.05) between AGEs and RSA group, they are respectivelymiRNA-7d-3p, the miRNA-92b-3p, the miRNA-181b-5p, the miRNA-196c-5p,miRNA-7a-1-3p, miR-186-5p, the miRNA-192-5p, the miRNA-196b-5p and themiRNA-345-5p, of which the miRNA-92b-3p changed the most obviously (p <0.01).There are still other20changed miRNA (p <0.10) between the two groups, they arerespectively the miRNA-7c-1-3p,miRNA-129-5p, miRNA-7d-3p, miRNA-375-3p,miRNA-92b-3p, miRNA-7b-3p, miRNA-196b-5p, miRNA-181b-5p, miRNA-187-3p,miRNA-196c-5p, miRNA-1306-3p, miRNA-411-3p, miRNA-126a-3p,miRNA-301a-3p, miRNA-339-5p, miRNA-186-5p, miRNA-192-5p, miRNA-582-5p,miRNA-200a-5p, miRNA-196a-5p, miRNA-345-5p, miRNA-140-5p, miRNA-872-3p,miRNA-7a-1-3p,miRNA-19b-3p, miRNA-466c-3p, miRNA-3584-5p, of which themiRNA-196b-5p, miRNA-7d-3p, the miRNA-126a3p are high abundance miRNA(>500).Besides these30miRNAs which was statistically significant, there are stillseveral miRNA (miRNA-21-5p, miRNA-30b-3p, miRNA-30b-5p, miRNA-140-3p)which has a close relationship with diabetic nephropathy, showed a changed trend (p>0.10).In a word, the miRNAs profiles of rat kidney was changed after given AGEs,furthermore, several miRNA for example miRNA-21-5p, miRNA-192-5p which has a close relationship with diabetic nephropathy, showed also a changed trend.Experiment III Prediction and validation of target genesThis part, firstly, the miRNA target genes prediction software (Targetscan andPicTar, miRanda) was used to predict that smad7was the target genes ofmiRNA-92b-3p. Then the metheds such as Immunohistochemistry, Western Blot wereused to further validate smad7gene expression in different group rat.Immunohistochemical results showed that Smad7expression of renal glomerular cellnucleus and cytoplasm in AGEs group decreased significantly comparing with that inRSA group and control group. Western results showed that Smad7express grey valuein AGEs group decreased significantly comparing with that in RSA group and controlgroup. The results suggest Smad7is probably the target gene of miRNA-92b-3p.Conclusion:1. Expression profiles of the miRNAs in rat kidney are changed after givenAGEs.While more than30miRNAs have differences between the twogroups.2. The rat kidney miR-92b-3p expression increased significantly and Smad7protein expression decreased after given AGEs.3. Smad7is probably one of the target genes of miR-92b-3p.AGEs may involved in the pathogenesis of diabetic nephropathy throughincreasing the expression of miR-92b-3p then inhibiting the expression ofsmad7,followed causing extracellular matrix accumulation.Innovative:1.For the first time to explore the role of AGEs in the pathogenesis of diabeticnephropathy from the view point of miRNAs.2.For the first time to proposed smad7is probably one of the target genes of miR-92b-3p.