The Smad3-miR-29b/miR-29c axis mediates the protective effect of macrophage migration inhibitory factor against cardiac fibrosis

Although macrophage migration inhibitory factor (MIF) is known to have antioxidant property, the role of MIF in cardiac fibrosis has not been well understood. We found that MIF was markedly increased in angiotension II (Ang-II)-infused mouse myocardium. Myocardial function was impaired and cardiac fibrosis was aggravated in Mif-knockout (Mif-KO) mice. Functionally, overexpression of MIF and MIF protein could inhibit the expression of fibrosis-associated collagen (Col) 1a1, COL3A1 and α-SMA, and Smad3 activation in mouse cardiac fibroblasts (CFs). Consistently, MIF deficiency could exacerbate the expression of COL1A1, COL3A1 and α-SMA, and Smad3 activation in Ang-II-treated CFs. Interestingly, microRNA-29b-3p (miR-29b-3p) and microRNA-29c-3p (miR-29c-3p) were down-regulated in the myocardium of Ang-II-infused Mif-KO mice but upregulated in CFs with MIF overexpression or by treatment with MIF protein. MiR-29b-3p and miR-29c-3p could suppress the expression of COL1A1, COL3A1 and α-SMA in CFs through targeting the pro-fibrosis genes of transforming growth factor beta-2 (Tgfb2) and matrix metallopeptidase 2 (Mmp2). We further demonstrated that Mif inhibited reactive oxygen species (ROS) generation and Smad3 activation, and rescued the decrease of miR-29b-3p and miR-29c-3p in Ang-II-treated CFs. Smad3 inhibitors, SIS3 and Naringenin, and Smad3 siRNA could reverse the decrease of miR-29b-3p and miR-29c-3p in Ang-II-treated CFs. Taken together, our data demonstrated that the Smad3-miR-29b/miR-29c axis mediates the inhibitory effect of macrophage migration inhibitory factor on cardiac fibrosis.     

Licochalcone A reverses NNK-induced ectopic miRNA expression to elicit in vitro and in vivo chemopreventive effects

BackgroundChemoprevention is the best cost-effective way regarding cancers. MicroRNAs (miRNAs) have been reported to be differentially expressed during the development of lung cancer. However, if lung cancer prevention can be achieved through modulating miRNAs expression so far remains unknown.PurposeTo discover ectopically expressed miRNAs in NNK-induced lung cancer and clarify whether Licochalcone A (lico A) can prevent NNK-induced lung cancer by modulating miRNA expression.Study design and methodsA/J mice were used to construct a lung cancer model by intraperitoneal injection with physiological saline NNK (100 mg/kg). Chemopreventive effects of lico A against lung cancer at 2 mg/kg and 20 mg/kg doses were evaluated in vivo. MicroRNA array and RT-qPCR were used to assess the expression levels of miRNAs. MLE-12 cells were treated with 0.1 mg/ml NNK, stimulating the ectopic expression pattern of miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p. miR-144-3p mimics and inhibitors were used to manipulate miR-144-3p levels. The effects of lico A (10 μM) on cell cycle distribution, apoptosis, and the expression of CK19, RASA1, miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p in NNK-treated MLE-12 cells were studied.ResultsThe expression levels of miR-144-3p, miR-20a-5p, and miR-29c-3p increased, while those of let-7d-3p and miR-328-3p decreased in both NNK-induced A/J mice and MLE-12 cells. Lico A could reverse the NNK-induced ectopic miRNA (miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p) expression both in vivo and in vitro and elicit in vivo lung cancer chemopreventive effect against NNK. In MLE-12 cells, the overexpression of miR-144-3p elicited the same effect as NNK regarding the expression of lung cancer biomarker CK19; the silencing of miR-144-3p reversed the effect of NNK on cell cycle distribution and apoptosis. Lico A could reverse the effect of NNK on the expression of miR-144-3p, CK19, and RASA1 (predicted target of miR-144-3p).ConclusionThe present study suggests that miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p were involved in the in vivo pathogenesis of NNK-induced lung cancer, and lico A could reverse the effect of NNK both in vivo and in vitro to elicit lung cancer chemopreventive effects through, at least partially, these five ectopically expressed miRNAs, especially miR-144-3p.     

环状RNA circMRPS35通过miR-130a-3p/ZNRF3轴调节胃癌细胞的增殖、凋亡、迁移和侵袭

摘要 目的：探讨环状RNA MRPS35（circMRPS35）对胃癌（GC）细胞增殖、凋亡、迁移和侵袭的调控机制。方法：体外培养人GC细胞系（HGC-27、MGC-803、MKN45和AGS）和正常胃上皮GES-1细胞,实时荧光定量PCR（RT-qPCR）检测circMRPS35、miR-130a-3p和锌环指蛋白3（ZNRF3）mRNA表达。另取MGC-803细胞,分为对照组、pc-NC组、pc-circMRPS35组、pc-circMRPS35+miR-NC组、pc-circMRPS35+miR-130a-3p组,采用Lipofectamine 3000进行质粒转染。RT-qPCR检测circMRPS35、miR-130a-3p和ZNRF3 mRNA表达,Western blot检测ZNRF3蛋白表达,CCK-8法、流式细胞术检测细胞增殖与凋亡,划痕实验和Transwell小室实验检测细胞迁移与侵袭能力,裸鼠移植瘤实验探究circMRPS35对GC细胞体内生长的影响。双荧光素酶报告基因检测miR-130a-3p与circMRPS35或ZNRF3的靶标关系。结果：GC细胞系中circMRPS35和ZNRF3 mRNA呈低表达,miR-130a-3p呈高表达（均P＜0.05）。过表达circMRPS35可降低miR-130a-3p,上调ZNRF3 mRNA和蛋白水平,抑制细胞增殖、迁移和侵袭,并促进细胞凋亡（均P＜0.05）;circMRPS35过表达对GC细胞恶性行为和裸鼠移植瘤生长的抑制作用可被miR-130a-3p mimic逆转（P＜0.05）。双荧光素酶实验结果显示,过表达miR-130a-3p可降低circMRPS35-WT和ZNRF3-WT的荧光素酶活性（P＜0.05）。结论：circMRPS35可能通过miR-130a-3p/ZNRF3轴抑制GC细胞的增殖、迁移和侵袭,并促进细胞凋亡。     

Oncosuppressive role of MicroRNA-205–3p in gastric cancer through inhibition of proliferation and induction of senescence: Oncosuppressive role of MicroRNA-205 in gastric cancer

BackgroundOur previous study showed that CXCL11 could play an immunomodulatory role. In this study, we investigated the regulator (miR-205–3p) of CXCL11 and the mechanism of miR-205–3p as a tumor suppressor gene in gastric cancer (GC).Materials and methodsA target relationship between miR-205–3p and CXCL11 was revealed by using the bioinformatics method. This study detected the expressions of miR-205–3p and CXCL11 through qRT-PCR and Western blotting. Moreover, the expressions of Akt, PD-L1, p16, p21, and senescence-associated secretory phenotype (SASP) factor were determined. The effects of miR-205 on proliferation, invasion, and senescence of GC cells were assessed by using methods, such as transfection, Transwell assay, tablet cloning, flow cytometry, and senescence-associated beta-galactosidase (SA-β-gal) staining. Furthermore, the effects were verified using methods, like immunohistochemistry, flow cytometry and SA-β-gal in animal experiments.ResultsBased on the study, it is found that the expression of miR-205–3p is down-regulated, while that of CXCL11 is up-regulated in GC cell lines. By regulating CXCL11, miR-205–3p inhibits Akt activation, reduces the proliferation and invasion of GC cells, promotes cell apoptosis, induces senescence of GC cells, and secretes immunostimulatory SASP factor. The animal experiments confirm that miR-205–3p promotes cell senescence, down-regulates the immunosuppressive signal induced by PD-L1, and promotes secretion of immunostimulatory SASP factor, so that more T cells are recruited in blood and tumors.ConclusionsThis study revealed the molecular mechanism of miR-205–3p in inhibiting proliferation and invasion and inducing senescence of GC cells by regulating CXCL11 and Akt pathways in animal and cell experiments.Key words: microRNA-205–3p, CXCL11, Gastric cancer, Senescence, Proliferation     

Bone marrow mesenchymal stem cells-derived exosomal microRNA-19b-1-5p reduces proliferation and raises apoptosis of bladder cancer cells via targeting ABL2

BackgroundExosomes are involved in intercellular communication via specialized molecular cargo, such as microRNAs (miRNAs). However, the mechanisms underlying exosomal miR-19b-1-5p in bladder cancer remain largely unknown, thus, we aim to investigate the effect of exosomal miR-19b-1-5p on bladder cancer with the involvement of non-receptor protein tyrosine kinase Arg (ABL2).MethodsmiR-19b-1-5p and ABL2 expression were tested in bladder cancer. miR-19b-1-5p inhibition/elevation assays were conducted to determine its role in bladder cancer. Exosomes were extracted from bone marrow mesenchymal stem cells (BMSCs). Exosomes and T24 cells were co-cultured to verify their function in biological characteristics of bladder cancer cells.ResultsmiR-19b-1-5p was down-regulated while ABL2 was upregulated in bladder cancer. Exosomal miR-19b-1-5p suppressed malignant behaviors of bladder cancer cells, and also inhibited tumor growth in vivo. Up-regulated ABL2 mitigated the effects of miR-19b-1-5p up-regulation on bladder cancer cells.ConclusionBMSCs-derived exosomal miR-19b-1-5p suppresses bladder cancer growth via decreasing ABL2.     

miR-326-Histone Deacetylase-3 Feedback Loop Regulates the Invasion and Tumorigenic and Angiogenic Response to Anti-cancer Drugs

Histone modification is known to be associated with multidrug resistance phenotypes. Cancer cell lines that are resistant or have been made resistant to anti-cancer drugs showed lower expression levels of histone deacetylase-3 (HDAC3), among the histone deacetylase(s), than cancer cell lines that were sensitive to anti-cancer drugs. Celastrol and Taxol decreased the expression of HDAC3 in cancer cell lines sensitive to anti-cancer drugs. HDAC3 negatively regulated the invasion, migration, and anchorage-independent growth of cancer cells. HDAC3 conferred sensitivity to anti-cancer drugs in vitro and in vivo. TargetScan analysis predicted miR-326 as a negative regulator of HDAC3. ChIP assays and luciferase assays showed a negative feedback loop between HDAC3 and miR-326. miR-326 decreased the apoptotic effect of anti-cancer drugs, and the miR-326 inhibitor increased the apoptotic effect of anti-cancer drugs. miR-326 enhanced the invasion and migration potential of cancer cells. The miR-326 inhibitor negatively regulated the tumorigenic, metastatic, and angiogenic potential of anti-cancer drug-resistant cancer cells. HDAC3 showed a positive feedback loop with miRNAs such as miR-200b, miR-217, and miR-335. miR-200b, miR-217, and miR-335 negatively regulated the expression of miR-326 and the invasion and migration potential of cancer cells while enhancing the apoptotic effect of anti-cancer drugs. TargetScan analysis predicted miR-200b and miR-217 as negative regulators of cancer-associated gene, a cancer/testis antigen, which is known to regulate the response to anti-cancer drugs. HDAC3 and miR-326 acted upstream of the cancer-associated gene. Thus, we show that the miR-326-HDAC3 feedback loop can be employed as a target for the development of anti-cancer therapeutics.     

MicroRNA-149 Inhibits Proliferation and Cell Cycle Progression through the Targeting of ZBTB2 in Human Gastric Cancer

An increasing body of evidence indicates that miR-149 can both suppress and promote tumor growth depending on the tumor type. However, the role of miR-149 in the progression of gastric cancer (GC) remains unknown. Here we report that miR-149 is a tumor suppressor in human gastric cancer. miR-149 expression is decreased in GC cell lines and clinical specimens in comparison to normal gastric epithelial cell and tissues, respectively. The expression levels of miR-149 also correlate with the differentiation degree of GC cells and tissues. Moreover, ectopic expression of miR-149 in gastric cancer cells inhibits proliferation and cell cycle progression by down-regulating ZBTB2, a potent repressor of the ARF-HDM2-p53-p21 pathway, with a potential binding site for miR-149 in its mRNA''s 3′UTR. It is also found that ZBTB2 expression increases in GC cells and tissues compared to normal gastric epithelial cell and tissues, respectively. Silencing of ZBTB2 leads to suppression of cell growth and cell cycle arrest in G0/G1 phase, indicating that ZBTB2 may act as an oncogene in GC. Furthermore, transfection of miR-149 mimics into gastric cancer cells induces down-regulation of ZBTB2 and HDM2, and up-regulation of ARF, p53, and p21 compared to the controls. In summary, our data suggest that miR-149 functions as a tumor suppressor in human gastric cancer by, at least partially through, targeting ZBTB2.     

MiR-376c Down-Regulation Accelerates EGF-Dependent Migration by Targeting GRB2 in the HuCCT1 Human Intrahepatic Cholangiocarcinoma Cell Line

MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.     

Genome-Wide Analyses of Radioresistance-Associated miRNA Expression Profile in Nasopharyngeal Carcinoma Using Next Generation Deep Sequencing

Background

Rapidly growing evidence suggests that microRNAs (miRNAs) are involved in a wide range of cancer malignant behaviours including radioresistance. Therefore, the present study was designed to investigate miRNA expression patterns associated with radioresistance in NPC.

Methods

The differential expression profiles of miRNAs and mRNAs associated with NPC radioresistance were constructed. The predicted target mRNAs of miRNAs and their enriched signaling pathways were analyzed via biological informatical algorithms. Finally, partial miRNAs and pathways-correlated target mRNAs were validated in two NPC radioreisitant cell models.

Results

50 known and 9 novel miRNAs with significant difference were identified, and their target mRNAs were narrowed down to 53 nasopharyngeal-/NPC-specific mRNAs. Subsequent KEGG analyses demonstrated that the 53 mRNAs were enriched in 37 signaling pathways. Further qRT-PCR assays confirmed 3 down-regulated miRNAs (miR-324-3p, miR-93-3p and miR-4501), 3 up-regulated miRNAs (miR-371a-5p, miR-34c-5p and miR-1323) and 2 novel miRNAs. Additionally, corresponding alterations of pathways-correlated target mRNAs were observed including 5 up-regulated mRNAs (ICAM1, WNT2B, MYC, HLA-F and TGF-β1) and 3 down-regulated mRNAs (CDH1, PTENP1 and HSP90AA1).

Conclusions

Our study provides an overview of miRNA expression profile and the interactions between miRNA and their target mRNAs, which will deepen our understanding of the important roles of miRNAs in NPC radioresistance.     

Down-Regulation of eIF4GII by miR-520c-3p Represses Diffuse Large B Cell Lymphoma Development

Deregulation of the translational machinery is emerging as a critical contributor to cancer development. The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described. Here, we established that elevated miR-520c-3p represses global translation, cell proliferation and initiates premature senescence in HeLa and DLBCL cells. Moreover, we demonstrate that miR-520c-3p directly targets translation initiation factor, eIF4GII mRNA and negatively regulates eIF4GII protein synthesis. miR-520c-3p overexpression diminishes cells colony formation and reduces tumor growth in a human xenograft mouse model. Consequently, downregulation of eIF4GII by siRNA decreases translation, cell proliferation and ability to form colonies, as well as induces cellular senescence. In vitro and in vivo findings were further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally up-regulated eIF4GII protein expression. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.     

miR-137 effects on gastric carcinogenesis are mediated by targeting Cox-2-activated PI3K/AKT signaling pathway

The discovery of microRNAs (miRNAs) provided a new avenue for early diagnosis and treatment of GC. MiR-137 has been reported to be under-expressed and involved in various cell processes. However, the role of miR-137 in GC is less known. In this study, we show that miR-137 is under-expressed in GC and functions as a tumor suppressor through targeting Cyclooxygenase-2 (Cox-2), which subsequently suppresses the activation of PI3K/AKT signaling pathway both in vitro and in vivo. Moreover, restored Cox-2 expression partially abolished the tumor suppressive effects of miR-137 in GC cells, suggesting miR-137 may suppress GC carcinogenesis by targeting Cox-2.     

MicroRNA-135b/CAMK2D Axis Contribute to Malignant Progression of Gastric Cancer through EMT Process Remodeling

There is a continued need for investigating the roles of microRNAs (miRNAs) and their targets on the progression of gastric cancer (GC), especially metastasis. Here, we performed an integrated study to identify dysregulated miRNAs critical for GC development and progression. miR-135b was determined as a promising biomarker for GC. The expression level of miR-135b was increased among GC cell lines, patient tumor tissues, serum samples, and correlation with aggravation of the GC patients. The in vitro functional assays demonstrated overexpression of miR-135b promoted cell proliferation, migration and invasion in GC, while miR-135b inhibition led to the opposite results. CAMK2D was found to be the direct target of miR-135b, serving as a tumor suppressor in GC cells. Based on our and public datasets, we confirmed the attenuation of CAMK2D expression in GC tissues. And, the expression levels of miR-135b and CAMK2D were closely associated with prognosis of GC patients. Ectopic expression of miR-135b resulted in the down-regulation of CAMK2D. Additionally, CAMK2D was a prerequisite for miR-135b to promote GC cells proliferation and migration by regulating the EMT process, which was confirmed by the in vivo experiments. Importantly, in vivo injection of miR-135b antagomir significantly repressed the tumor growth and metastasis of xenograft models, which suggested that the miR-135b antagomir were promising for clinical applications. Taken together, these results indicate that miR-135b/CAMK2D axis drives GC progression by EMT process remodeling, suggesting that miR-135b may be utilized as a new therapeutic target and prognostic marker for GC patients.     

P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells

Tumor growth and metastatic spreading are heavily affected by the P2X7 receptor as well as microvesicles and exosomes release into the tumor microenvironment. P2X7 receptor stimulation is known to trigger vesicular release from immune and central nervous system cells. However, P2X7 role in microvesicles and exosomes delivery from tumor cells was never analyzed in depth. Here we show that P2X7 is overexpressed in patients affected by metastatic malignant melanoma and that its expression closely correlates with reduced overall survival. Antagonism of melanoma cell-expressed P2X7 receptor inhibited in vitro anchorage-independent growth and migration and in vivo dissemination and lung metastasis formation. P2X7 stimulation triggered the release of miRNA-containing microvesicles and exosomes from melanoma cells, profoundly altering the nature of their miRNA content, as well as their dimensions and quantity. Among the more than 200 miRNAs that we found up-or-down-modulated for each vesicular fraction tested, we identified three miRNAs, miR-495-3p, miR-376c-3p, and miR-6730-3p, that were enriched in both the exosome and microvesicle fraction in a P2X7-dependent fashion. Interestingly, upon transfection, these miRNAs promoted melanoma cell growth or migration, and their vesicular release was minimized by P2X7 antagonism. Our data unveil an exosome/microvesicle and miRNA-dependent mechanism for the pro-metastatic activity of the P2X7 receptor and highlight this receptor as a suitable prognostic biomarker and therapeutic target in malignant melanoma.Subject terms: Ligand-gated ion channels, Metastasis, Melanoma, Ion channel signalling, miRNAs