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Background: To uncover the diagnostic potential of peripheral blood microRNA-200b (miRNA-200b) in renal interstitial injury in diabetic nephropathy (DN) patients. Methods: A total of 50 diabetes subjects, 50 mild DN subjects, 50 moderate-severe DN subjects and 50 healthy subjects were included. Peripheral blood level of miRNA-200b in every subject was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Serum levels of renal function indicators were determined by enzyme-linked immunosorbent assay (ELISA). Meanwhile, relative levels of fibrosis damage indicators were examined by chemiluminescent immunoassay. Diagnostic potentials of miRNA200b in diabetes, mild DN and moderate-severe DN were assessed by depicting receiver operating characteristic (ROC) curves. Results: Peripheral blood level of miRNA-200b was higher in DN subjects than diabetes subjects without vascular complications, especially moderate-severe DN patients. Peripheral blood level of miRNA-200b in DN subjects was negatively correlated to relative levels of serum creatinine, urinary nitrogen, cystatin, TGF-b, CIV and PCIII. ROC curves demonstrated diagnostic potentials of miRNA-200b in mild and moderate-severe DN. Conclusions: Peripheral blood level of miRNA-200b is closely linked to the degree of renal interstitial injury in DN patients. MiRNA-200b may be a vital indicator in predicting the development of DN.

Aims/Introduction: Long non‐coding RNAs (lncRNAs) exert essential functions in the pathogenesis of diabetic nephropathy (DN). LncRNA T‐cell factor 7 (TCF7) and semaphorin‐3A (SEMA3A) have been found to be involved in the progression of diabetic nephropathy. However, whether the effect of TCF7 on the pathogenesis of diabetic nephropathy is mediated by SEMA3A remains unclear. Materials and Methods: TCF7, miR‐16‐5p, and SEMA3A were quantified by a qRT‐PCR or immunoblotting method. A CCK‐8 assay gauged the cell viability. Measurement of cell apoptosis was done using flow cytometry. RNA immunoprecipitation (RIP), dual‐luciferase reporter, and RNA pull‐down assays were utilized to assay the targeted interactions among the variables. Results: The TCF7 and SEMA3A levels were elevated in serum from patients with diabetic nephropathy. TCF7 silencing or SEMA3A depletion ameliorated high glucose (HG)‐induced podocyte injury. Moreover, TCF7 silencing protected against HG‐induced podocyte injury by down‐regulating SEMA3A. TCF7 targeted miR‐16‐5p, and miR‐16‐5p targeted SEMA3A. Furthermore, TCF7 affected the expression of SEMA3A by competing specifically for shared miR‐16‐5p. Conclusions: These findings suggested that TCF7 silencing attenuated high glucose‐induced podocyte damage partially through the miR‐16‐5p/SEMA3A regulation cascade. [ABSTRACT FROM AUTHOR]

Loss or absence of hearing is common at both extremes of human lifespan, in the forms of congenital deafness and age-related hearing loss. While these are often studied separately, there is increasing evidence that their genetic basis is at least partially overlapping. In particular, both common and rare variants in genes associated with monogenic forms of hearing loss also contribute to the more polygenic basis of age-related hearing loss. Here, we directly test this model in the Penn Medicine BioBank–a healthcare system cohort of around 40,000 individuals with linked genetic and electronic health record data. We show that increased burden of predicted deleterious variants in Mendelian hearing loss genes is associated with increased risk and severity of adult-onset hearing loss. As a specific example, we identify one gene–TCOF1, responsible for a syndromic form of congenital hearing loss–in which deleterious variants are also associated with adult-onset hearing loss. We also identify four additional novel candidate genes (COL5A1, HMMR, RAPGEF3, and NNT) in which rare variant burden may be associated with hearing loss. Our results confirm that rare variants in Mendelian hearing loss genes contribute to polygenic risk of hearing loss, and emphasize the utility of healthcare system cohorts to study common complex traits and diseases. Author summary: Age-related hearing loss is relatively common and has a genetic component to risk, though little is known about specific genes that are involved. Here, we study participants in the Penn Medicine BioBank to identify factors that increase risk of hearing loss. We show that loss-of-function variants in genes that are known to cause Mendelian forms of hearing loss also increase risk of age-related hearing loss. Because many such genes have been identified, they can be used to interpret the results of genome-wide association studies and to investigate the biological basis of age-related hearing loss. Our results also emphasize that many reported Mendelian hearing loss variants are incompletely penetrant and may act cumulatively. Finally, our study shows how hospital-recruited biobank cohorts can aid in the study of common conditions like hearing loss, even when those conditions are not the primary reason for contact with the health system. [ABSTRACT FROM AUTHOR]

Glomerular mesangial cell (MC) hypertrophy is regarded as one of the earliest pathological characteristics of diabetic nephropathy (DN), which plays a critical role in the pathogenesis of glomerulosclerosis. This study investigated the role of microRNAs (miRNAs) in MC hypertrophy due to exposure to high glucose. With a microarray, we screened the differential profiles of miRNAs in the renal cortex of DN mice, as verified by reverse transcription PCR with subsequent analysis of bioinformatics. We found miR-196a was downregulated remarkably in DN mice and increased the hypertrophy-related gene of p27kip1 in high-enrichment gene ontologies. Furthermore, transfection of the miR-196a mimic greatly inhibited the expression of p27kip1 with recovery of MC hypertrophic morphology. With flow cytometry, we also found that overexpression of miR-196a significantly reduced the percentage of G1 phase arrest in the cell cycle. Cotransfection of the miR-196a mimic with a wild type of 3′ UTR of the p27kip1 vector reduced the activity of the luciferase reporter significantly in contrast to the miR-196a mimic with a mutant of the counterpart in HEK293 cell lines, suggesting that miR-196a directly targets p27kip1. Finally, knockdown of p27kip1 with specific small interfering RNA in MCs substantially reversed MC hypertrophy induced by transfection of the miR-196a inhibitor. This study revealed that miR-196a acts as an important molecular regulator in high glucose-induced MC hypertrophy by targeting p27kip1. [ABSTRACT FROM AUTHOR]

Aims/introduction: Long non-coding RNAs (lncRNAs) exert essential functions in the pathogenesis of diabetic nephropathy (DN). LncRNA T-cell factor 7 (TCF7) and semaphorin-3A (SEMA3A) have been found to be involved in the progression of diabetic nephropathy. However, whether the effect of TCF7 on the pathogenesis of diabetic nephropathy is mediated by SEMA3A remains unclear., Materials and Methods: TCF7, miR-16-5p, and SEMA3A were quantified by a qRT-PCR or immunoblotting method. A CCK-8 assay gauged the cell viability. Measurement of cell apoptosis was done using flow cytometry. RNA immunoprecipitation (RIP), dual-luciferase reporter, and RNA pull-down assays were utilized to assay the targeted interactions among the variables., Results: The TCF7 and SEMA3A levels were elevated in serum from patients with diabetic nephropathy. TCF7 silencing or SEMA3A depletion ameliorated high glucose (HG)-induced podocyte injury. Moreover, TCF7 silencing protected against HG-induced podocyte injury by down-regulating SEMA3A. TCF7 targeted miR-16-5p, and miR-16-5p targeted SEMA3A. Furthermore, TCF7 affected the expression of SEMA3A by competing specifically for shared miR-16-5p., Conclusions: These findings suggested that TCF7 silencing attenuated high glucose-induced podocyte damage partially through the miR-16-5p/SEMA3A regulation cascade., (© 2022 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

In an effort to generate a desired expression construct for making heart-specific expression transgenic zebrafish, a Tol2 plasmid, which can drive EGFP reporter gene specifically expressed in the heart, was modified using subcloning technology. An IRES fragment bearing multiple cloning site (MCS) was amplified directly from pIRES2-EGFP plasmid and was inserted between the CMLC2 promoter and EGFP fragment of the pDestTol2CG vector. This recombinant expression plasmid pTol2-CMLC2-IRES-EGFP can drive any interested gene specifically expressed in the zebrafish heart along with EGFP reporter gene. To test the effectiveness of this new expression plasmid, we constructed pTol2-CMLC2-RED-IRES-EGFP plasmid by inserting another reporter gene DsRed-Monome into MCS downstream of the CMLC2 promoter and injected this transgenic recombinant plasmid into one-cell stage embryos of zebrafish. Under fluorescence microscope, both the red fluorescence and the green fluorescence produced by pTol2-CMLC2-RED-IRES-EGFP were detected specifically in the heart tissue in the same expression pattern. This novel expression construct pTol2-CMLC2-IRES-EGFP will become an important tool for our research on identifying heart development candidate genes' function using zebrafish as a model.