CircRNAs in exosomes from high glucose-treated glomerular endothelial cells activate mesangial cells.

Understanding the relationships between glomerular endothelial cells (GECs) and glomerular mesangial cells (GMCs) is important to identify the molecular mechanisms underlying diabetic nephropathy (DN). Exosomes carried with mRNA, microRNA, and protein play important roles in cell-to-cell communication. In this study, we showed that high glucose (HG)-treated GECs secreted a higher number of exosomes enriched in circRNAs compared with normal glucose (NG)-treated GECs. Differentially expressed circRNAs (DECs) were obtained by high-throughput sequencing. Of these DECs, the expressions of 217 DECs and 484 DECs in HG-treated GEC exosomes were significantly downregulated and upregulated, respectively, compared with NG-treated GEC exosomes. The functions of the DEC target genes were involved in the PI3K/AKT and MAPK pathways. Five DECs were randomly selected for identification by quantitative real-time PCR (qRT-PCR). Two DECs (circRNF169 and circSTRN3) were further selected for functional validation. Moreover, we demonstrated that exosomes released by HG-treated GECs promoted α-smooth muscle actin (α-SMA) expression. It also inhibited proliferation and promoted epithelial-mesenchymal transition (EMT) in GMCs. In addition, cell functional studies indicated that the knockdown and over-expression of two DECs (circRNF169 and circSTRN3) effectively inhibited or promoted cell proliferation and promoted or inhibited EMT, respectively. Thus, the results of this study provide new insights into the pathogenesis of DN that involves the intercellular transfer of circRNAs from GECs to GMCs via exosomes.

Long noncoding RNA ENSRNOG00000037522 is involved in the podocyte epithelial­mesenchymal transition in diabetic rats.

Diabetic nephropathy (DN) is one of the most common complications associated with type I and II diabetes mellitus. Long noncoding RNAs (lncRNAs) have been implicated in various physiological and pathological processes, and recent evidence has demonstrated that they are involved in the process of the epithelial­mesenchymal transition (EMT). In the present study, the potential functions of lncRNA ENSRNOG00000037522 during the EMT process in DN were investigated. The results identified that the level of the lncRNA ENSRNOG00000037522 was significantly increased in kidney tissues collected from rats with streptozocin (STZ)­induced DN accompanied by impairment of the glomerular podocytes. It was further demonstrated that the silencing of lncRNA ENSRNOG00000037522 by small interfering RNA transfection partially restored the podocyte function. In addition, knockdown of lncRNA ENSRNOG00000037522 repaired the damage to the podocytes via regulating vimentin, podocalyxin­like 1 and nephrin expression. In conclusion, the current results demonstrated that lncRNA ENSRNOG00000037522 serves a pivotal role in the podocyte EMT in DN.

High glucose induces podocyte epithelial­to­mesenchymal transition by demethylation­mediated enhancement of MMP9 expression.

Abnormal expression of matrix metalloproteinase 9 (MMP9) is correlated with podocyte epithelial-to---mesenchymal transition (EMT) in diabetic nephropathy (DN). However, the mechanisms underlying this process are not well defined. Site­specific demethylation may sustain high expression levels of target genes. In the present study, in order to investigate the association between DNA demethylation of MMP9 promoter and podocyte EMT in DN, human podocytes were cultured in high­glucose (HG) medium and a rat model of DN was established by intraperitoneal injection of streptozotocin (STZ) to determine whether site­specific demethylation of the MMP9 promoter was involved in regulating podocyte EMT in DN. The MTT assay was used to assess the effects of HG culture on the growth of podocytes, and the demethylation status of the MMP9 promoter was assessed by bisulfite sequencing polymerase chain reaction. mRNA and protein expression levels of MMP9, α­smooth muscle actin (α­SMA), podocalyxin and fibronectin­1 in podocytes were assessed by reverse transcription­quantitative PCR (RT­qPCR) and western blot analyses. The results demonstrated that HG treatment up regulated the expression of MMP9, α­SMA and fibronectin­1, but down regulated the expression of podocalyxin in podocytes. The MMP9 promoter region was revealed to contain a variety of demethylated CpG sites, and HG treatment reduced the rate of MMP9 promotermethylation, which, in turn, enhanced its promoter activity. In summary, these data suggested that demethylation of the MMP9 promoter may serve an important role in podocyte EMT in DN. The demethylation status of the MMP9 promoter maybe used as an important prognostic marker of DN in clinic.

[Effect of Metformin on Proliferation of Multiple Myeloma Cells].

OBJECTIVE: To investigate the effects of metformin on proliferation and apoptosis in multiple myeloma cell line RPMI8226 and U266, and to clarify the molecular mechanism of proliferation inhibition and apoptosis induced by metformin. METHODS: RPMI8226, U266 cells were treated with 0, 5, 10, 20, 40, 80 mmol/L of metformin for 24, 48 and 72 hours, then the inhibition rate was detected by CCK-8; RPMI8226 cells were treated with 0, 10, 20, 40 mmol/L of metformin for 48 hours, the apoptosis rates were detected by flow cytometry with Annexin-V-FITC/PI double staining; RPMI8226 cells were treated with 0, 5, 10, 20 mmol/L of metformin for 48 hours, the expressions of Caspase-3, PARP, STAT3, p-STAT3, BCL-2, Cyclin D1 and P21 were detected by Western blot. RESULTS: The inhibition rate increased in RPMI8226 and U266 cells treated with metformin in the dose- (r=0.982, r=0.967, P<0.05) and time-dependent (r=0.956, r=0.962, P<0.05) manner; the apoptosis rate increased(r=0.976, P<0.05) in RPMI8226 cells treated with metformin; it also was found that procaspase-3 was degraded and PARP was cleaved when treated with metformin. Proliferation inhibition and apoptosis of RPMI8226 cells were related with inhibition of STAT3 phosphorylation, down-regulation of BCL-2 and Cyclin D1, and up-regulation of P21. CONCLUSION: Metformin can inhibit the proliferation and induce apoptosis of RPMI8226 and U266 cell lines, which may be related to down-regulation of STAT3 signal transduction pathway.

Ghrelin improves vascular autophagy in rats with vascular calcification.

BACKGROUNDS: This study aimed to investigate whether ghrelin ameliorated vascular calcification (VC) through improving autophagy. METHODS: VC model was induced by nicotine plus vitamin D3 in rats and ß-glycerophosphate in vascular smooth muscle cell (VSMC). Calcium deposition was detected by von Kossa staining or alizarin red S staining. ALP activity was also detected. Western blot was used to assess the protein expression. RESULTS: Ghrelin treatment attenuated the elevation of calcium deposition and ALP activity in VC model both in vivo and in vitro. Interesting, the protein levels of autophagy markers, LC3 and beclin1 were significantly upregulated by ghrelin in VC model. An autophagy inhibitor, 3-methyladenine blocks the ameliorative effect of ghrelin on VC. Furthermore, protein expressions of phosphate-AMPK were increased by ghrelin treatment both in calcified aorta and VSMC. The effect of ghrelin on autophagy induction and VC attenuation was prevented by AMPK inhibitor, compound C. CONCLUSIONS: Our results suggested that ghrelin improved autophagy through AMPK activation, which was resulted in VC amelioration. These data maybe throw light on prevention and therapy of VC.

Wnt3a regulates proliferation, apoptosis and function of pancreatic NIT-1 beta cells via activation of IRS2/PI3K signaling.

Wnt-signaling pathway is implicated in pancreatic development and functional regulation of mature beta-cells. Wnt3a/Wnt pathway activation expands islet cell mass in vitro by increasing proliferation and decreasing apoptosis of beta-cells, thereby enhancing its function. However, the signaling pathways that mediate these effects remain unknown. By using a clonal beta-cell line (NIT-1), we examined the role of IRS2/PI3K in the mediation of Wnt3a-stimulated beta-cell growth. Real-time PCR and Western blot were employed to investigate the activity of Wnt/ß-catenin and IRS2/PI3K signaling. Proliferation of NIT-1 cells was assessed by BrdU incorporation, and apoptosis was quantitatively determined by TUNEL and flow cytometry (FCM). Dkk1, an inhibitor of Wnt signaling, and wortmannin, an inhibitor of PI3K, were also used. Results showed that Wnt3a rapidly activated Wnt/ß-catenin signaling, promoted IRS2 expression and Akt phosphorylation in NIT-1 cells. These effects were completely abrogated by Dkk1 or partially eliminated by wortmannin. Wnt3a also promoted NIT-1 cell proliferation, inhibited cytokine-induced beta-cell apoptosis, and increased insulin secretion. Both of these effects were also eliminated by Dkk1 or wortmannin. Our results demonstrated that Wnt3a regulates proliferation, apoptosis and enhances function of pancreatic NIT-1 beta cells via activation of Wnt/ß-catenin signaling, involving crosstalk with IRS2/PI3K signaling, with the effect of Wnt signaling on beta-cells also being IRS2/PI3K/AKT dependent.