Activation of NFKB-JMJD3 signaling promotes bladder fibrosis via boosting bladder smooth muscle cell proliferation and collagen accumulation

Chronic cystitis is characterized by the hyperplasia and fibrosis of the bladder wall as well as attenuated compliance of the bladder. To further unravel its underlying molecular mechanism, the role of NFκB-JMJD3 signaling pathway in cystitis induced bladder fibrosis was investigated. Jmjd3 and Col1/3 expression was detected in a cystitis mouse model that was developed by intraperitoneal injection of cyclophosphamide (CYP). Human bladder smooth muscle cells (hBSMCs) were stimulated in vitro with lipopolysaccharide (LPS), and the cell proliferation and collagen accumulation were detected using EdU, CCK8, flow cytometry, qPCR, western blotting and immunofluorescence assays. Furthermore, the effects of NFκB and JMJD3 on cell proliferation and collagen accumulation were investigated using its selective antagonists, JSH23 and GSK-J4, respectively. CYP induced cystitis significantly increased Jmjd3, Col1 and Col3 expression in the bladder muscle cells. Furthermore, LPS stimulation markedly activated NFκB signaling and elevated JMJD3 expression in hBSMCs, and the activation of NFκB-JMJD3 signaling significantly promoted cell proliferation and collagen accumulation by upregulating CCND1 and COL1/3 expression, respectively. Our study reveals the critical role of NFκB-JMJD3 signaling in cystitis induced bladder reconstruction by regulating hBSMC proliferation and extracellular matrix (ECM) deposition, and these findings provide an avenue for effective treatment of patients with cystitis.     

Low expression of miR-186-5p regulates cell apoptosis by targeting toll-like receptor 3 in high glucose–induced cardiomyocytes

To investigate the effect and mechanism of microRNA-186-5p (miR-186-5p) on the apoptosis in high glucose (HG)–treated cardiomyocytes. Diabetic cardiomyopathy model was established in cardiomyocytes by stimulating with HG. The expressions of miR-186-5p and toll-like receptor 3 (TLR3) were detected by quantitative polymerase chain reaction or Western blot analysis, respectively. Apoptosis was detected in HG-treated cardiomyocytes by flow cytometry and Western blot analysis. The interaction between miR-186-5p and TLR3 was explored by bioinformatics analysis and luciferase activity assay. Results showed that miR-186-5p expression was downregulated in HG-treated cardiomyocytes and its overexpression reversed HG-induced apoptosis and cleaved caspase-3 protein expression. Moreover, TLR3 was indicated as a target of miR-186-5p and regulated by miR-186-5p. Knockdown of TLR3 suppressed HG-induced apoptosis and cleaved caspase-3 protein expression. Besides, restoration of TLR3 ablated the effect of miR-186-5p on cell apoptosis. Collectively, miR-186-5p attenuated HG-induced apoptosis by regulating TLR3 in cardiomyocytes, providing novel biomarker for treatment of diabetic cardiomyopathy.     

PKM2-regulated STAT3 promotes esophageal squamous cell carcinoma progression via TGF-β1-induced EMT

Recent studies have demonstrated pleiotropic roles of pyruvate kinase isoenzyme type M2 (PKM2) in tumor progression. However, the precise mechanisms underlying the effects of PKM2 on esophageal squamous cell carcinoma (ESCC) metastasis and transforming growth factor β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) remain to be established. In this study, we observed upregulation of PKM2 in ESCC tissues that was markedly associated with lymph node metastasis and poor prognosis. High PKM2 expression in tumor tissues frequently coincided with the high pSTAT3Tyr705 expression and low E-cadherin expression. Furthermore, altered PKM2 expression was significantly associated with proliferation, migration, and invasion of ESCC cells, in addition to expression patterns of EMT markers (Snail, E-cadherin, and vimentin) and pSTAT3^Tyr705/STAT3 ratio. Overexpression of STAT3 significantly attenuated the effects of PKM2 knockdown on cell proliferation and motility as well as expression of pSTAT3 ^Tyr705 and EMT markers. Consistently, stable short hairpin RNA (shRNA)-mediated silencing of PKM2 reversed the effects of TGF-β1 treatment, specifically, upregulation of PKM2, phosphorylation of STAT3 at Tyr705, and increased EMT, migration, and invasion. We propose that PKM2 regulates cell proliferation, migration, and invasion via phosphorylation of STAT3 through TGF-β1-induced EMT. Our findings collectively provide mechanistic insights into the tumor-promoting role of PKM2, supporting its prognostic value and the therapeutic utility of PKM2 inhibitors as potential antitumor agents in ESCC.     

Gamma-secretase inhibitor suppressed Notch1 intracellular domain combination with p65 and resulted in the inhibition of the NF-κB signaling pathway induced by IL-1β and TNF-α in nucleus pulposus cells

In this experiment, the cross-talk betweenNotch and the NF-κB signaling pathway was examined to reveal the mechanism of slowing down the type II collagen (ColII) and aggrecan degeneration affected by inflammatory cytokines. The expression levels of ColII and aggrecan in the intervertebral disc were observed through immunohistochemistry and hematoxylin-eosin staining+alcian blue staining, respectively. The expression levels of ColII, aggrecan, Runx2, and NF-κB in the nuclei of human nucleus pulposus cells (hNPCs) in each group, as well as the phosphorylation and acetylation levels of p65, were examined through Western blot analysis. The 293T cells were transfected with a plasmid containing the overexpressed relative domain of Notch1 intracellular domain (NICD1), and immunoprecipitation (IP) was performed to observe the combination of NICD1 and p65. HNPCs were transfected with a lentiviral-contained overexpression lacking the ANK region of NICD1, and IP was performed to observe the combination of NICD1 and p65. The expression of ColII and aggrecan in the intervertebral disc culture increased when γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-Sphenylglycine t-butyl ester (DAPT) was added to the disc culture medium. Western blot revealed that DAPT inhibited p65 phosphorylation and acetylation, and the p65 and p50 levels in the nucleus decreased. NICD1 was found to be combined with p65 in contrast to the reverse consequences after ANK domain deletion in hNPCs. In nucleus pulposus cells, the combination of p65 and the ANK domain of NICD1 is a critical procedure for the degeneration related to the NF-κB signaling pathway activation induced by IL-1β and TNF-α.     

Prognostic evaluation of NANOG and OCT4 expression for posttransplantation hepatocellular carcinoma recurrence

Postoperative hepatocellular carcinoma (HCC) recurrence and metastasis throw great threaten to its overall survival (OS). This paper focus on exploring the prognostic significance of NANOG and OCT4 expression in HCC recurrence and OS after liver transplantation. Eighty-six patients who meet University of California San Francisco (UCSF) criteria and underwent liver transplantation in Tianjin First Central Hospital between August 2010 and August 2013 were included. Expression of NANOG and OCT4 was determined by immunohistochemistry. The relationships between NANOG and OCT4 expression with tumor recurrence, tumor count, histology stage, lymph node metastasis (LNM) and microvascular invasion (MVI) were explored through the χ² test and Cox regression analysis. We found that 19/26 and 20/24 patients with positive expression of NANOG and OCT4 relapsed. Combination of NANOG and OCT4 expression was indicated as valuable prognostic signature for HCC recurrence prediction (P < 0.0011). Besides, we identified other key factors with significant correlations with recurrence, such as LNM (P = 0.011) and MVI (P = 0.024). Strikingly, recurrence sites could significantly affect recurrence time (P = 0.0062) and patients with recurrence in transplanted liver have longer recurrence time. In conclusions, we analyzed the relationships between NANOG/OCT4 expression, clinicopathology features, HCC recurrence, and OS after liver transplantation for the first time. Combination of NANOG, OCT4, LNM, histopathological stage, and MVI may be predictor for HCC recurrence posttransplantation. Comprehensive of histopathological stage grade and LNM were considered as prognosis factor for OS after liver transplantation. This should be helpful for treatment method selection for HCC patients after liver transplantation.     

MicroRNA-329-mediated PTTG1 downregulation inactivates the MAPK signaling pathway to suppress cell proliferation and tumor growth in cholangiocarcinoma

Cholangiocarcinoma (CCA) is a severe malignancy usually producing a poor prognosis and high mortality rate. MicroRNAs (miRNAs) have been reported in association with CCA; however, the role miR-329 plays in the CCA condition still remains unclear. Therefore, this study was conducted to explore the underlying mechanism of which miR-329 is influencing the progression of CCA. This work studied the differential analysis of the expression chips of CCA obtained from the Gene Expression Omnibus database. Next, to determine both the expression and role of pituitary tumor transforming gene-1 (PTTG1) in CCA, the miRNAs regulating PTTG1 were predicted. In the CCA cells that had been intervened with miR-329 upregulation or inhibition, along with PTTG1 silencing, expression of miR-329, PTTG1, p-p38/p38, p-ERK5/ERK5, proliferating cell nuclear antigen (PCNA), Cyclin D1, Bcl-2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and caspase-3 were determined. The effects of both miR-329 and PTTG1 on cell proliferation, cell-cycle distribution, and apoptosis were also assayed. The miR-329 was likely to affect the CCA development through regulation of the PTTG1-mediated mitogen-activated protein kinase (MAPK) signaling pathway. The miR-329 targeted PTTG1, leading to inactivation of the MAPK signaling pathway. Upregulation of miR-329 and silencing of PTTG1 inhibited the CCA cell proliferation, induced cell-cycle arrest, and subsequently promoted apoptosis with elevations in Bax, cleaved caspase-3, and total caspase-3, but showed declines in PCNA, Cyclin D1, and Bcl-2. Moreover, miR-329 was also found to suppress the tumor growth by downregulation of PTTG1. To summarize, miR-329 inhibited the expression of PTTG1 to inactivate the MAPK signaling pathway, thus suppressing the CCA progression, thereby providing a therapeutic basis for the CCA treatment.     

Downregulation of microRNA-214 improves therapeutic potential of allogeneic bone marrow–derived mesenchymal stem cell by targeting PIM-1 in rats with acute liver failure

Acute liver failure (ALF) is a disease resulted from diverse etiology, which generally leads to a rapid degenerated hepatic function. However, transplantation bone marrow–derived mesenchymal stem cells (BMSCs) transplantation has been suggested to relieve ALF. Interestingly, microRNA-214 (miR-214) could potentially regulate differentiation and migration of BMSCs. The present study aims to inquire whether miR-214 affects therapeutic potential of BMSCs transplantation by targeting PIM-1 in ALF. 120 male Wistar rats were induced as ALF model rats and transplanted with BMSCs post-alteration of miR-214 or PIM-1 expression. Further experiments were performed to detect biochemical index (alanine aminotransferase [ALT], aspartate transaminase [AST], total bilirubin [TBiL]), and expression of miR-214, PIM-1, hepatocyte growth factor (HGF), caspase 3, tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in rat serum. Apart from the above detection, apoptosis of hepatocytes and Ki67 protein expression in hepatic tissues of rats were additionally assessed. After BMSCs transplantation with miR-214 inhibition, a decreased expression of ALT, AST, and TBiL yet an increased expression of HGF was shown, coupled with a decline in the expression of caspase 3, TNF-α, and IL-10. Meanwhile, alleviated hepatic injury and decreased apoptotic index of hepatic cells were observed and the positive rate of Ki67 protein expression was significantly increased. Moreover, miR-214 and caspase 3, TNF-α, and IL-10 decreased notably, while PIM-1 was upregulated in response to miR-214 inhibition. Strikingly, the inhibition of PIM-1 reversed effects triggered by miR-214 inhibition. These findings indicated that downregulation of miR-214 improves therapeutic potential of BMSCs transplantation by upregulating PIM-1 for ALF.