MiR-130b increases fibrosis of HMC cells by regulating the TGF-β1 pathway in diabetic nephropathy

Basement membrane thickening, glomerular hypertrophy, and deposition of multiple extracellular matrix characterize the pathological basis of diabetic nephropathy (DN), a condition which ultimately leads to glomerular and renal interstitial fibrosis. Here, we identified a novel microRNA, miR-130b, and investigated its role and therapeutic efficacy in alleviating DN. Introduction of miR-130b dramatically increased cell growth and fibrosis in DN cells. We found that transforming growth factor (TGF)-β1 was a functional target of miR-130b in human glomerular mesangial cells (HMCs) and overexpression of miR-130b increased expressions of the downstream signaling molecules of TGF-β1, t-Smad2/3, p-Smad2/3, and SMAD4. An ectopic application of miR-130b increased messenger RNA and protein expressions of collagen type I (colI), colIV, and fibronectin, whose expression levels were correlated with the expression of miR-130b. Taken together, the findings of this study reveal that miR-130b in HMC cells plays an important role in fibrosis regulation and may thus be involved with the pathogenesis of DN. Therefore, miR-130b may serve as a novel therapeutic target for the prevention and the treatment of DN.     

LncRNA-PCAT-1 promotes non–small cell lung cancer progression by regulating miR-149-5p/LRIG2 axis

Long noncoding RNAs (lncRNAs) are key players in the development and progression of human cancers. The lncRNA PCAT-1 has been shown to be upregulated in human non–small cell lung cancer (NSCLC); however, its role and molecular mechanisms in NSCLC cell progression remain unclear. Here, we found that the higher expression of PCAT-1 led to a significantly poorer survival time, and multivariate analysis revealed that PCAT-1 was an independent risk factor of prognosis in NSCLC. Furthermore, we also found that the knockdown of PCAT-1 remarkably suppressed cell growth by inducing cell cycle arrest and apoptosis promotion in NSCLC cells. Moreover, the bioinformatics analysis and luciferase reporter assay revealed that PCAT-1 directly bound to the miR-149-5p, which has been reported to act as a tumor suppressor in diverse cancers. In addition, our results confirmed that the tumor-promoting effects of PCAT-1 in NSCLC cells are at least partly through negative modulation of miR-149-5p. Finally, mechanistic investigations showed that PCAT-1 upregulated the expression of miR-149-5p target gene leucine-rich repeats and immunoglobulin (Ig)-like domains 2 (LRIG2) through competitively “spongeing” miR-149-5p. Therefore, we concluded that PCAT-1 may promote the development of NSCLC through the miR-149-5p/LRIG2 axis.     

Mechanically stimulated osteocytes reduce the bone-metastatic potential of breast cancer cells in vitro by signaling through endothelial cells

Bone metastases occur in 65% to 75% of patients with advanced breast cancer and significantly worsen their survival and quality of life. We previously showed that conditioned medium (CM) from osteocytes stimulated with oscillatory fluid flow, mimicking bone mechanical loading during routine physical activities, reduced the transendothelial migration of breast cancer cells. Endothelial cells are situated at an ideal location to mediate signals between osteocytes in the bone matrix and metastasizing cancer cells in the blood vessels. In this study, we investigated the specific effects of flow-stimulated osteocytes on the interaction between endothelial cells and breast cancer cells in vitro. We observed that CM from flow-stimulated osteocytes reduced endothelial permeability by 15% and breast cancer cell adhesion onto endothelial monolayers by 18%. The difference in adhesion was abolished with anti-intercellular adhesion molecule 1 (ICAM-1) neutralizing antibodies. Furthermore, CM from endothelial cells conditioned in CM from flow-stimulated osteocytes significantly altered the gene expression in bone-metastatic breast cancer cells, as shown by RNA sequencing. Specifically, breast cancer cell expression of matrix metallopeptidase 9 (MMP-9) was downregulated by 62%, and frizzled-4 (FZD4) by 61%, when the osteocytes were stimulated with flow. The invasion of these breast cancer cells across Matrigel was also reduced by 47%, and this difference was abolished by MMP-9 inhibitors. In conclusion, we demonstrated that flow-stimulated osteocytes downregulate the bone-metastatic potential of breast cancer cells by signaling through endothelial cells. This provides insights into the capability of bone mechanical regulation in preventing bone metastases; and may assist in prescribing exercise or bone-loading regimens to patients with breast cancers.     

Fibroblast growth factor 21 protects against pathological cardiac remodeling by modulating galectin-3 expression

Background/Aims: Fibroblast growth factor 21 (FGF21) plays a protective role in ischemia/reperfusion induced cardiac injury. However, the exact molecular mechanism of FGF21 action remains unclear. This study was designed the protective effect of FGF21 on the heart and its mechanism. Method: Adenovirus vector expressing FGF21 or control β-galactosidase was injected into the myocardium of mice. Myocardial injury was observed by tissue staining and immunohistochemical staining. The expression level of caspases-3 and galectin-3 in myocardial cells were observed by immunoblotting. Then, hypoxia-induced cell model was established. Small interfering RNA (SiRNA) and plasmid were transfected into H9c2 using Lipofectamine 2000 reagent (Invitrogen). The expression levels of galectin-3, ECM and cystatin-3 in cells were observed by immunoblotting, and the relationship between fibroblast growth factor 21 and galectin-3 was analyzed. Result: Cell test in vitro showed that FGF21 could inhibit apoptosis and decrease the expression of ECM (ColIaI, fibronectin, and alpha-SMA) under hypoxia. Western blot data showed that hypoxia-induced cell damage increased galectin-3 levels, while FGF21 decreased galactose lectin-3 levels. In addition, inhibition of galactose agglutinin-3 expression by siRNA enhanced the cardioprotective effect of FGF21, while overexpression of galectin-3 reduced the cardioprotective effect of fibroblast growth factor 21. Conclusion: FGF21 may be a novel therapy for hypoxia-induced cardiac injury by regulating the expression of galectin-3.     

Microvesicles-mediated communication between endothelial cells modulates,endothelial survival,and angiogenic function via transferring of miR-125a-5p

Endothelial cells (ECs) released microvesicles (EMVs) could modulate the functions of target cells by transferring their microRNAs (miRs). We have reported that miR-125a-5p protected EC function. In this study, we determined whether EMVs provided beneficial effects on ECs by transferring miR-125a-5p. Human brain microvessel ECs were transfected with miR-125a-5p mimic or miR-125a-5p short hairpin RNA to obtain miR-125a-5p overexpressing ECs and miR-125a-5p knockdown ECs, and their derived EMVs. For the functional study, ECs or hypoxia/reoxygenation injured ECs were coincubated with various EMVs. The survival and angiogenic function of ECs were measured. Western blot and quantitative real time polymerase chain reaction (qRT-PCR) were used for measuring the levels of phosphoinositide 3-kinase (PI3K), phosphorylation-Akt (p-Akt)/Akt, p-endothelial nitric oxide synthase (p-eNOS), cleaved caspase-3, and miR-125a-5p. PI3K inhibitor was used for pathway analysis. EMVs promoted the proliferation, migration, and tube formation ability of ECs, and alleviated the apoptotic rate of ECs. These effects were associated by an increase in p-Akt/Akt and p-eNOS, and a decrease in cleaved caspase-3 could be abolished by LY294002. Overexpression or downregulation of miR-125a-5p in EMVs promoted or inhibited those effects of EMVs. EMVs could enhance the survival and angiogenic function of ECs via delivering miR-125a-5p to modulate the expression of PI3K/Akt/eNOS pathway and caspase-3.     

Antitumor activity of SR splicing-factor 5 knockdown by downregulating pyruvate kinase M2 in non–small cell lung cancer cells

SR splicing-factors (SRSFs) play a vital role in carcinogenesis. SRSF5 was demonstrated to be upregulated in lung cancer and identified as a novel prognostic indicator for small-cell lung cancer. However, the role of SRSF5 in the pathogenesis of non–small cell lung cancer (NSCLC) and the molecular mechanism involved are still undefined. The expression of SRSF5 in NSCLC cells was detected by quantitative real-time polymerase chain reaction and Western blot analysis. The proliferation of cells was evaluated by cell counting kit-8 and BrdU assays. Apoptosis was assessed by flow cytometry and Western blot analysis of apoptosis-associated proteins including B-cell lymphoma 2 (Bcl-2), Bax, and cytochrome C (Cyt C). Glycolysis was detected by determining glucose consumption, lactate production, and pyruvate kinase M2 (PKM2) expression. We found that SRSF5 messenger RNA and protein levels were elevated in NSCLC cells. SRSF5 knockdown inhibited the proliferation and Ki67 expression in NSCLC cells. SRSF5 silencing increased the apoptotic rate, upregulated Bax and Cyt C, and decreased Bcl-2 level in NSCLC cells. Moreover, Knockdown of SRSF5 repressed glycolysis in NSCLC cells via reducing PKM2 expression. Enhanced glycolysis by PKM2 overexpression attenuated the effects of SRSF5 silencing on NSCLC cell proliferation and apoptosis. Overall, knockdown of SRSF5 inhibited proliferative ability and induced apoptosis by suppressing PKM2 expression in NSCLC cells.