Excessive apoptosis of podocytes caused by dysregulation of microRNA-182-5p and CD2AP confers to an increased risk of diabetic nephropathy

The functions of miR-182-5p in the pathogenesis of diabetic nephropathy (DN) remain largely unclear. Here, we studied the roles and relationship between miR-182-5p and CD2AP in the development of DN. We used real-time polymerase chain reaction (PCR) to compare miR-182-5p expression between DN and control groups, while computational analysis and luciferase assays were used to confirm CD2AP as a miR-182-5p target. Western blot and real-time PCR were then used to measure the messenger RNA (mRNA) and protein expression of CD2AP in the presence of miR-182-5p. The results showed that miR-182-5p was highly expressed in cells isolated from people with DN. In addition, the luciferase activity of cells transfected with wild-type/mutant CD2AP confirmed CD2AP as a direct target of miR-182-5p. The expression levels of CD2AP mRNA and protein were much lower in the DN group compared with that in the normal group. In addition, the expression levels of CD2AP mRNA and protein were evidently increased by a miR-182-5p inhibitor, but notably downregulated by miR-182-5p mimics or CD2AP small interfering RNA (siRNA). Furthermore, miR-182-5p and CD2Ap siRNA significantly reduced the survival rate and viability of transfected cells, while the miR-182-5p inhibitor exhibited an opposite effect. These findings indicated the presence of a negative regulatory relationship between miR-182-5p and CD2AP in podocytes cells and suggested that the overexpression of miR-182-5p contributes to the pathogenesis of DN.     

Thioredoxin-interacting protein deficiency alleviates phenotypic alterations of podocytes via inhibition of mTOR activation in diabetic nephropathy

Thioredoxin-interacting protein (TXNIP) is induced by high glucose (HG), whereupon it acts to inhibit thioredoxin, thereby promoting oxidative stress. We have found that TXNIP knockdown in human renal tubular cells helped prevent the epithelial-to-mesenchymal transition (EMT). Here, we studied the potential effect of TXNIP on podocyte phenotypic alterations in diabetic nephropathy (DN) in vivo and in vitro. In conditionally immortalized mouse podocytes under HG conditions, knocking down TXNIP disrupted EMT, reactive oxygen species (ROS) production, and mammalian target of rapamycin (mTOR) pathway activation. Further, Raptor short hairpin RNA (shRNA), Rictor shRNA, and mTOR specific inhibitor KU-0063794 were used to assess if the mTOR signal pathway is involved in HG-induced EMT in podocytes. We found that Raptor shRNA, Rictor shRNA, and KU-0063794 could all restrain HG-induced EMT and ROS production in podocytes. In addition, antioxidant Tempol or N-acetylcysteine presented a prohibitive effect on HG-induced EMT in podocytes. Streptozotocin was utilized to render equally diabetic in wild-type (WT) control and TXNIP ^−/− (TKO) mice. Diabetes did not increase levels of 24-hr urinary protein, serum creatinine, blood urea nitrogen, and triglyceride in TXNIP ^−/− mice. Podocyte phenotypic alterations and podocyte loss were detected in WT but not in TKO diabetic mice. Oxidative stress was also suppressed in diabetic TKO mice relative to WT controls. Also, TXNIP deficiency suppresses the activation of mTOR in glomeruli of streptozotocin-induced diabetic mice. Moreover, TXNIP expression, mTOR activation, Nox1, and Nox4 could be detected in renal biopsy tissues of patients with DN. This suggests that decreased TXNIP could ameliorate phenotypic alterations of podocytes via inhibition of mTOR in DN, highlighting TXNIP as a promising therapeutic target.     

Deregulation of long noncoding RNA (TUG1) contributes to excessive podocytes apoptosis by activating endoplasmic reticulum stress in the development of diabetic nephropathy

The objective of this study was to investigate the molecular mechanism of how TUG1 interferes with the expression of C/EBP homologous protein (CHOP), peroxisome-proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α), which contributes to the development of diabetic nephropathy. Real-time polymerase chain reaction and western blot analysis were performed to explore the regulatory relationship among TUG1, CHOP, PGC-1α, and caspase-3. Terminal deoxynucleotidyl transferase dUTP nick-end labeling was performed to confirm TUG1 involved in diabetic nephropathy (DN) through influencing podocytes apoptosis. TUG1 was highly expressed in a cell following treatment with high glucose, and PGC-1α and cleaved caspase-3 levels were much lower, while CHOP level was much higher in high glucose group (HG), furthermore, CHOP inhibited PGC-1α expression. TUG1 negatively regulated CHOP expression, and positively regulated PGC-1α expression. Meanwhile, total caspase-3 level in cell treated with or without HG transfected with CHOP small interfering ribonucleic acid (siRNA), TUG1, and TUG1 siRNA showed no evident difference with their corresponding control, while CHOP siRNA and TUG1 evidently decreased, and TUG1 siRNA remarkably increased cleaved caspase-3 level in HG or normal glucose groups in comparison with corresponding control. TUG1 and PGC-1α levels were much lower, while CHOP level was much higher in participants diagnosed with DN. A higher level of CHOP protein and lower level of PGC-1α were observed in subjects diagnosed with DN. Finally, podocytes apoptosis in the DN group was significantly promoted compared with that in nondiabetic renal disease group. Our current study has suggested for the first time that the long noncoding RNA (lncRNA) TUG1 influenced podocytes apoptosis via mediating endoplasmic reticulum stress (ERS)–CHOP–PGC-1α signaling pathway in HG-induced DN.     

Cyclin G2 regulates canonical Wnt signalling via interaction with Dapper1 to attenuate tubulointerstitial fibrosis in diabetic nephropathy

Cyclin G2 (CCNG2) is an atypical cyclin that inhibits cell cycle progression and is often dysregulated in human cancers. Cyclin G2 in the occurrence and development of diabetic nephropathy (DN), one of the most severe diabetic complications, has not been fully identified. In this study, we investigated the function and regulatory mechanism of cyclin G2 in DN. In vivo studies revealed that a deficiency of cyclin G2 significantly increased albuminuria and promoted tubulointerstitial fibrosis in established DN. Cyclin G2 regulated the expression of fibrosis-related proteins via the canonical Wnt signalling pathway in renal tubular epithelial cells. Moreover, the binding of cyclin G2 to Dapper1 (Dpr1/DACT1), a protein involved in Wnt signalling, decreased the phosphorylation of Dpr1 at Ser762 by casein kinase 1 (CK1) and suppressed the Wnt signalling pathway. These findings reveal that cyclin G2 can protect against renal injury and fibrosis associated with DN and, thus, is a new target for the prevention and treatment of diabetic complications.     

Co-inheritance of specific genotypes of HSPG and ApoE gene increases risk of type 2 diabetic nephropathy

The objective of this paper is to investigate co-inheritance of specific HSPG and ApoE genotypes in the development of Chinese type 2 diabetic nephropathy. PCR-RFLP was used to detect HSPG and ApoE genotypes in 385 Chinese subjects including 298 patients with type 2 diabetes mellitus (T2DM) and 87 non-diabetic controls (Non-DM). The T2DM group was subdivided into patients with (TDN; n = 218) and without diabetic nephropathy (Non-DN; n = 80). The latter group was further subdivided into groups of patients with microalbuminuria nephropathy (DN-1; n = 129) and severe diabetic nephropathy (DN-2; n = 89). We then compared the relative frequencies of various HSPG and ApoE genotypes and alleles among the groups, searching for predictive trends. The T allele of the HSPG gene occurred more frequently in the DN-2 group than in the Non-DN or DN-1 or groups, their (Fisher's exact p was 1.05 × 10^–3 and 6.58 × 10^–6; odds ratios were 2.09 (95% CI 1.32–3.30) and 2.48 (95% CI 1.64–3.74), respectively. The E2 allele of the ApoE gene occurred more frequently in the T2DM than in the Non-DM group, the Fisher's exact p was 0.0087; odds ratio was 3.45 (95% CI 1.30–9.81). Genotype analysis showed that the TT or TG of HSPG gene were paired with the E2/2 or E2/3 of ApoE gene significantly more frequently in the TDN group than in the Non-DN group, with an odds ratio of 3.03 (95% CI 1.03–8.90). There was no significant differences in other combinations of genotypes in HSPG and ApoE genes between TDN and Non-DN group. These results suggest that the HSPG T allele is a risk factor for the development of severe diabetic nephropathy in type 2 diabetic patients, and that the ApoE E2 allele is a risk factor for the occurrence of type 2 diabetes mellitus in Chinese general population. In addition, we find that co-inheritance of T/E2 confers a higher risk of type 2 diabetes mellitus progression to diabetic nephropathy in Chinese.     

Promising renoprotective effect of gold nanoparticles and dapagliflozin in diabetic nephropathy via targeting miR-192 and miR-21

Diabetic nephropathy (DN) is a worldwide issue that eventually leads to end-stage renal failure, with limited therapeutic options. Prior research has revealed that gold nanoparticles (AuNPs) have a substantial antidiabetic impact. In addition, sodium-glucose cotransporter2 (SGLT2) inhibitors, including dapagliflozin (DAPA), had renoprotective impact on DN. Therefore, this research attempted to determine the potential AuNPs and DAPA impacts in ameliorating experimentally DN induction and the underlying mechanisms focusing on miR-192 and miR-21, correlating them with autophagy, apoptosis, fibrosis, and oxidative stress. Diabetes induction was through a single intraperitoneal streptozotocin (55 mg/kg) injection, and rats with diabetes received AuNPs (2.5 mg/kg/day) as well as DAPA (2 mg/kg/day) for 7 weeks as a treatment. AuNPs and DAPA treatment for 7 weeks substantially alleviated DN. AuNPs and DAPA significantly increased catalase (CAT) activity as well as serum total antioxidant capacity (TAC), along with a substantial decline in malondialdehyde (MDA). AuNPs and DAPA treatment alleviated renal fibrosis as they decreased transforming growth factorß1(TGF-ß1) as well as matrix metalloproteinase-2 (MMP-2) renal expression, decreased apoptosis through alleviating the proapoptotic gene (caspase-3) renal expression and increased the antiapoptotic gene (Bcl-2) renal expression, and increased autophagy as they increased LC-3 as well as Beclin-1 renal expression. Autophagy activation, inhibition of apoptosis, and renal fibrosis could be due to their inhibitory impact on miR-192 and miR-21 renal expression. AuNPs and DAPA have a protective effect on DN in rats by targeting miR-192 and miR-21 and their downstream pathways, including fibrosis, apoptosis, autophagy, and oxidative stress.     

肠道菌群对2型糖尿病肾病患者肾功能损害及白蛋白尿短期进展的影响

研究2型糖尿病肾病（DN）患者肠道菌群与肾功能损害及白蛋白尿短期进展的关系。

选择2018年2月至2021年2月收治的2型DN患者作为DN组并分为微量白蛋白尿患者（30～300 mg/24 h）和大量白蛋白尿患者（＞300 mg/24 h）,另取同期收治的尿白蛋白水平正常（＜30 mg/24 h）的2型糖尿病患者作为DM组,健康体检者作为对照组,检测各组对象肠道菌群多样性指标Ace指数、Simpson指数、Chao1指数、Shannon指数及肠道菌群门水平的组成,根据2型DN组患者前后2次入院时尿白蛋白水平的变划分为白蛋白尿短期进展和未进展,分析2型DN患者肠道菌群特征与病情及白蛋白尿短期进展的关系。

DN组患者的肠道菌群Ace指数、Simpson指数及厚壁菌门占比均低于DM组和对照组,拟杆菌门占比高于DM组和对照组（均P＜0.05）,Chao1指数、Shannon指数以及放线菌门、变形菌门占比与DM组、对照组比较差异均无统计学意义（均P＞0.05）;DN组中大量白蛋白尿患者的Ace指数、Simpson指数及厚壁菌门占比低于微量白蛋白尿患者,拟杆菌门占比高于微量白蛋白尿患者（均P＜0.05）;DN组中白蛋白尿短期进展患者的Ace指数、Simpson指数及厚壁菌门占比低于未进展患者,拟杆菌门占比高于未进展患者（均P＜0.05）;DN组患者的Ace指数、Simpson指数以及厚壁菌门占比、拟杆菌门占比均对白蛋白尿短期进展具有预测价值。

2型DN患者肠道菌群的多样性降低、厚壁菌门减少及拟杆菌门增多,肠道菌群的变化与病情加重和白蛋白尿短期进展有关。

     

Long noncoding RNA ATB promotes ovarian cancer tumorigenesis by mediating histone H3 lysine 27 trimethylation through binding to EZH2

Ovarian cancer (OC) remains one of the most lethal gynecological malignancies. The unfavourable prognosis is mainly due to the lack of early-stage diagnosis, drug resistance and recurrence. Therefore, it needs to investigate the mechanism of OC tumorigenesis and identify effective biomarkers for the clinical diagnosis. It is reported that long noncoding RNAs (lncRNAs) play important roles during the tumorigenesis of OC. Therefore, the present study aimed to study the role and clinical significance of LncRNAs ATB (lnc-ATB) in the development and progression of OC. In our research, lnc-ATB expression in OC tissues was elevated compared with adjacent normal tissues and high expression of lnc-ATB was associated with poor outcomes of OC patients. The silencing of lnc-ATB blocked cell proliferation, invasion and migration in SKOV3 and A2780 cells. RNA immunoprecipitation and RNA pull-down results showed that lnc-ATB positively regulated the expression of EZH2 via directly interacting with EZH2. Besides, the overexpression of EZH2 partly rescued lnc-ATB silencing-inducing inhibition of cell proliferation, invasion and migration. Chromatin immunoprecipitation assay results demonstrated that the silencing of lnc-ATB reduced the occupancy of caudal-related homeobox protein 1, Forkhead box C1, Large tumour suppressor kinase 2, cadherin-1 and disabled homolog 2 interacting protein promoters on EZH2 and H3K27me3. These data revealed the oncogenic of lnc-ATB and provided a novel biomarker for OC diagnosis. Furthermore, these findings indicated the mechanism of lnc-ATB functioning in the progression of OC, which provided a new target for OC therapy.     

TFPI2 suppresses the interaction of TGF-β2 pathway regulators to promote endothelial–mesenchymal transition in diabetic nephropathy

Endothelial–mesenchymal transition (EndMT) is an important source of myofibroblasts, but also contributes to the progression of diabetic nephropathy (DN). By several differential gene expression analyses from the Gene Expression Omnibus (GEO) database, the tissue factor pathway inhibitor 2 (TFPI2) gene, known as a tumor suppressor, was shown to be dysregulated in DN; however, the potential role and regulatory mechanism of TFPI2 in DN are unclear. Here, we found abnormal upregulation of TFPI2 in the renal cortex of diabetic mice, accompanied by impaired renal function. We also injected a single dose of adeno-associated virus (AAV)2 carrying shRNA targeting TFPI2 intravenously into these mice and found that knockdown of TFPI2 improved renal function and reduced renal fibrosis and cell apoptosis in experimental DN. Furthermore, hyperglycemia-induced EndMT was inhibited in the absence of TFPI2, as evidenced by increased expression of endothelial markers (VE-cadherin and CD31) and decreased expression of mesenchymal markers (α-SMA, desmin, and FSP-1). To further explore the mechanism in vitro, human renal glomerular endothelial cells (hRGECs) were incubated in the presence of high glucose or transforming growth factor beta (TGF-β)2. TFPI2 deficiency inhibited high glucose-induced cell apoptosis and TGF-β2-induced EndMT in hRGECs, while overexpression of TFPI2 had the opposite effects. Importantly, TGF-β2 is a crucial driver of EndMT, and we found that TFPI2 promoted TGF-β2/Smad signaling activation by interferring the interaction of TGF-β pathway regulators (SMURF2 with SMAD7). Our results show that TFPI2 regulates EndMT and the TGF-β2 signaling pathway and is a potential promoter of DN pathogenesis.     

Retracted: Effects of microRNA-370 on mesangial cell proliferation and extracellular matrix accumulation by binding to canopy 1 in a rat model of diabetic nephropathy

As one major diabetic complication, diabetic nephropathy (DN) has been reported to be associated with various kinds of microRNA (miRNA). Thus, we conducted this study to explore the potential of miR-370 in a rat model of DN through investigation of mesangial cell proliferation and extracellular matrix (ECM). A total of 40 healthy adult male Sprague–Dawley rats were enrolled and assigned into normal (n = 10) and DN ( n = 30, DN rat model) groups. Dual-luciferase reporter assay was performed for the targeting relationship between miR-370 and canopy 1 (CNPY1). Mesangial cells were collected and transfected with prepared mimic, inhibitor or small interfering RNA (siRNA) for analyzing the effect of miR-370 on DN mice with the help of expression and cell biological processes detection. CNPY1 was confirmed as a target gene of miR-370. DN mice had increased expression of miR-370, fibronectin, type I collagen (Col I), type IV collagen (Col IV), and plasminogen activator inhibitor-1 (PAI-1) but reduced CNPY1 expression. Cells transfected with miR-370 mimic and siRNA–CNPY1 had increased expression of fibronectin, Col I, Col IV, and PAI-1 but decreased CNPY1 expression. The miR-370 mimic and siRNA–CNPY1 groups showed increased cell proliferation, as well as elevated ECM accumulation and declined cell apoptosis rate as compared with the blank and negative control groups, with reverse trends observed in the miR-370 inhibitor group. Our study concludes that overexpression of miR-370 promotes mesangial cell proliferation and ECM accumulation by suppressing CNPY1 in a rat model of DN.     

Long noncoding RNA MEG3 silencing protects against hypoxia-induced pheochromocytoma-12 cell injury through inhibition of TIMP2 promoter methylation

Hypoxia is a common pathological process caused by insufficient oxygen. Long noncoding RNAs (lncRNAs) have been proven to participate in this pathology. Hypoxia is reported to significantly reduce the secretion of tissue inhibitor of metalloproteinase 2 (TIMP2) and TIMP2 induces pheochromocytoma-12 (PC12) cell cycle arrest. Thus, our study aimed to explore the mechanism by which lncRNA maternally expressed gene 3 (MEG3) was implicated in hypoxia-induced PC12 cell injury through TIMP2 promoter methylation. To elucidate the potential biological significance of MEG3 and the regulatory mechanism between MEG3 and TIMP2, a hypoxia-induced PC12 cell injury model was generated. The hypoxia-exposed cells were subjected to a series of overexpression plasmids and short hairpin RNAs, followed by the measurement of levels of MEG3, TIMP2, lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), reactive oxygen species (ROS), Bcl-2-associated X protein, B-cell lymphoma-2, and caspase-3, as well as the changes in MMP, cell proliferation, apoptosis, and cell cycle progression. On the basis of the findings, MEG3 was upregulated in hypoxia-injured PC12 cells. MEG3 recruited methylation proteins DNMT3a, DNMT3b, and MBD1 and accelerated TIMP2 promoter methylation, which in turn inhibited its expression. Moreover, PC12 cells following MEG3 silencing and TIMP2 overexpression exhibited significantly decreased levels of LDH, MDA, and ROS along with cell apoptosis, yet increased SOD and MMP levels, as well as cell cycle entry to the S phase and cell proliferation. In conclusion, MEG3 silencing suppresses hypoxia-induced PC12 cell injury by inhibiting TIMP2 promoter methylation. This study may provide novel therapeutic targets for hypoxia-induced injury.     

Long noncoding RNA FOXD2-AS1 promotes glioma malignancy and tumorigenesis via targeting miR-185-5p/CCND2 axis

Glioma is the most aggressive malignant tumor in the adult central nervous system. Abnormal long noncoding RNA (lncRNA) FOXD2-AS1 expression was associated with tumor development. However, the possible role of FOXD2-AS1 in the progression of glioma is not known. In the present study, we used in vitro and in vivo assays to investigate the effect of abnormal expression of FOXD2-AS1 on glioma progression and to explore the mechanisms. FOXD2-AS1 was upregulated in glioma tissue, cells, and sphere subpopulation. Upregulation of FOXD2-AS1 was correlated with poor prognosis of glioma. Downregulation of FOXD2-AS1 decreased cell proliferation, migration, invasion, stemness, and epithelial-mesenchymal transition (EMT) in glioma cells and inhibited tumor growth in transplanted tumor. We also revealed that FOXD2-AS1 was mainly located in cytoplasm and microRNA (miR)-185-5p both targeted FOXD2-AS1 and CCND2 messenger RNA (mRNA) 3′-untranslated region (3′-UTR). miR-185-5p was downregulated in glioma tissue, cells, and sphere subpopulation. Downregulation of miR-185-5p was closely correlated with poor prognosis of glioma patients. In addition, miR-185-5p mimics decreased cell proliferation, migration, invasion, stemness, and EMT in glioma cells. CCND2 was upregulated in glioma tissue, cells, and sphere subpopulation. Upregulation of CCND2 was closely correlated with poor prognosis of glioma patients. CCND2 knockdown decreased cell proliferation, migration, invasion, and EMT in glioma cells. In glioma tissues, CCND2 expression was negatively associated with miR-185-5p, but positively correlated with FOXD2-AS1. FOXD2-AS1 knockdown and miR-185-5p mimics decreased CCND2 expression. Inhibition of miR-185-5p suppressed FOXD2-AS1 knockdown-induced decrease of CCND2 expression. Overexpression of CCND2 suppressed FOXD2-AS1 knockdown-induced inhibition of glioma malignancy. Taken together, our findings highlight the FOXD2-AS1/miR-185-5p/CCND2 axis in the glioma development.