miR-142-3p通过调控ELAVL1表达影响过氧化氢诱导的心肌细胞损伤的分子机制

目的探讨微小RNA-142-3p（miR-142-3p）对过氧化氢诱导的心肌细胞损伤的影响及其作用机制。 方法构建氧化应激损伤模型,以H9C2心肌细胞为研究对象,实验将心肌细胞转染后分为正常对照组、H₂O₂组、H₂O₂+miR-142-3p组、H₂O₂+miR阴性对照组、H₂O₂+?si-?ELAVL1组、H₂O₂+siRNA对照组、H₂O₂+miR-142-3p+pcDNA-ELAVL1组、H₂O₂+miR-?142-3p+pcDNA组。分别采用qRT-PCR与Western Blot检测细胞中miR-142-3p与ELAVL1表达;检测各组活性氧（ROS）生成水平;MTT检测细胞存活率,流式细胞术检测细胞凋亡。双荧光素酶报告实验验证miR-142-3p与ELAVL1的靶向作用。Western Blot检测细胞中Cleaved Caspase-3、STAT3、Caspase-3、p-STAT3蛋白表达。两组间比较采用两样本t检验;多组间比较采用单因素方差分析,两两比较采用LSD-t检验。 结果H₂O₂组心肌细胞中miR-142-?3p（0.26±0.06）、p-STAT3表达水平（0.36±0.04）、细胞存活率（61.73±6.48）﹪与正常对照组相比下降（P均< 0.01）,而ROS水平（1?566.38±121.57）、细胞凋亡率（27.46±1.73）﹪、Cleaved Caspase-3（0.68±0.08）及ELAVL1表达水平（4.23±0.31）均升高（P均< 0.01）;双荧光素酶报告实验证实ELAVL1是miR-142-3p的靶基因;miR-142-3p过表达或沉默ELAVL1表达可明显促进心肌细胞存活、上调p-STAT3表达,而抑制细胞凋亡及Cleaved Caspase-3表达;ELAVL1过表达可逆转miR-142-3p对过氧化氢处理H9C2细胞的保护作用。 结论miR-142-?3p可通过抑制ELAVL1表达进而减轻过氧化氢诱导的心肌细胞损伤,其可能通过影响STAT3信号通路而保护心肌细胞。     

MIR663AHG as a competitive endogenous RNA regulating TGF-β-induced epithelial proliferation and epithelial–mesenchymal transition in benign prostate hyperplasia

Benign prostate hyperplasia (BPH) is the most commonly seen disease among aging males. Transforming growth factor(TGF)-β-mediated epithelial–mesenchymal transition (EMT) and epithelial overproliferation might be central events in BPH etiology and pathophysiology. In the present study, long noncoding RNA MIR663AHG, miR-765, and FOXK1 formed a competing endogenous RNAs network, modulating TGF-β-mediated EMT and epithelial overproliferation in BPH-1 cells. miR-765 expression was downregulated in TGF-β-stimulated BPH-1 cells; miR-765 overexpression ameliorated TGF-β-mediated EMT and epithelial overproliferation in BPH-1 cells. MIR663AHG directly targeted miR-765 and negatively regulated miR-765; MIR663AHG knockdown also attenuated TGF-β-induced EMT and epithelial overproliferation in BPH-1 cells, whereas miR-765 inhibition attenuated MIR663AHG knockdown effects on TGF-β-stimulated BPH-1 cells. miR-765 directly targeted FOXK1 and negatively regulated FOXK1. FOXK1 knockdown attenuated TGF-β-induced EMT and epithelial overproliferation and promoted autophagy in BPH-1 cells, and partially attenuated miR-765 inhibition effects on TGF-β-stimulated BPH-1 cells. In conclusion, this study provides a MIR663AHG/miR-765/FOXK1 axis modulating TGF-β-induced epithelial proliferation and EMT, which might exert an underlying effect on BPH development and act as therapeutic targets for BPH treatment regimens.     

LncRNA MIR4435-2HG potentiates the proliferation and invasion of glioblastoma cells via modulating miR-1224-5p/TGFBR2 axis

Glioblastoma (GBM) belongs to the high-grade (IV) gliomas with extremely poor prognosis. Accumulating evidence uncovered the key roles of long non-coding RNAs (lncRNAs) in GBM development. This study aimed to determine the biological actions and the clinical relevance of lncRNA MIR4435-2 Host Gene (MIR4435-2HG) in GBM. Data from GEPIA database showed that MIR4435-2HG was up-regulated in GBM tissues and high expression of MIR4435-2HG correlated with shorter overall survival of GBM patients. Further experimental assays verified the up-regulation of MIR4435-2HG in GBM tissues and cell lines. In vitro cell studies and in vivo animal studies showed that knockdown of MIR4435-2HG resulted in the inhibition of GBM cell proliferation and invasion and in vivo tumour growth, while MIR4435-2HG overexpression driven GBM progression. Furthermore, MIR44435-2HG was found to sponge miR-1224-5p and suppress miR-1224-5p expression; overexpression of miR-1224-5p attenuated the enhancement in GBM cell proliferation and invasion induced by MIR4435-2HG overexpression. In a subsequent study, miR-1224-5p was found to target transforming growth factor-beta receptor type 2 (TGFBR2) and repressed TGFBR2 expression, and in vitro assays showed that miR-1224-5p exerted tumour-suppressive effects via targeting TGFBR2. More importantly, TGFRB2 knockdown antagonized hyper-proliferation and invasion of GBM cells with MIR4435-2HG overexpression. Clinically, the down-regulation of miR-1224-5p and up-regulation of TGFBR2 were verified in the GBM clinical samples. Taken together, the present study suggests the oncogenic role of MIR4435-2HG in GBM and underlies the key function of MIR4435-2HG-driven GBM progression via targeting miR-1224-5p/TGFBR2 axis.     

LncRNA NEAT1 promotes docetaxel resistance in prostate cancer by regulating ACSL4 via sponging miR-34a-5p and miR-204-5p

Docetaxel resistance remains one of the main problems in clinical treatment of metastatic prostate cancer (PCa). Previous studies identified differently expressed lncRNAs in docetaxel-resistant PCa cell lines, while the potential mechanisms were still unknown. In the present study, we found NEAT1 was expressed at high levels in docetaxel-resistant PCa clinical samples and related cell lines. When knockdown NEAT1, cell proliferation and invasion in docetaxel-resistant PCa cells in vitro and in vivo were downregulated. Our further researches explained that NEAT1 exerts oncogenic function in PCa by competitively ‘sponging’ both miR-34a-5p and miR-204-5p. Inhibition of miR-34a-5p or miR-204-5p expression mimics the docetaxel-resistant activity of NEAT1, whereas ectopic expression of miR-34a-5p or miR-204-5p attenuates the anti-drug function of NEAT1 in PCa cells. Besides, we also found ACSL4 is a target of both miR-34a-5p and miR-204-5p, and ACSL4 was also inhibited by miR-34a-5p and miR-204-5p. Moreover, suppression of miR-34a-5p or/and miR-204-5p greatly restrained the expression of ACSL4 upon NEAT1 overexpression. Joint expression of miR-34a-5p and miR-204a-5p synergistically decreased the expression of ASCL4, indicating miR-34a-5p and miR-204a-5p collaboratively inhibit the expression of ACSL4. Innovatively, we concluded that NEAT1 contributes to the docetaxel resistance by increasing ACSL4 via sponging miR-34a-5p and miR-204-5p in PCa cells.     

MicroRNA-1296-5p suppresses the proliferation,migration, and invasion of human osteosarcoma cells by targeting NOTCH2

Osteosarcoma (OS) is a highly aggressive bone tumor with a poor prognosis. MicroRNAs are revealed to exerts essential roles in the carcinogenesis and tumor invasion of OS. But, the function of miR-1296-5p and its related mechanism in OS progression have not yet been studied. This study discovered the levels of miR-1296-5p in OS and corresponding noncancerous tissues, and we demonstrated that miR-1296-5p level was markedly downregulated in tumor specimens as compared with nontumor tissues. In addition, we discovered that miR-1296-5p was also underexpressed in OS cells compared with the hFOB1.19 osteoblast cells. Interestingly, the reduced expression of miR-1296-5p was confirmed to associated with large tumor size, advanced tumor stages, and distance metastasis, respectively. Patients with OS low-expressing miR-1296-5p showed a prominent shorter survival. In addition, gain-of-function assays verified that miR-1296-5p overexpression remarkably repressed OS cell proliferation, migration, and invasion. Conversely, depletion of miR-1296-5p facilitated the growth and mobility of OS cells. Notably, miR-1296-5p inversely modulated notch receptor 2 (NOTCH2) in OS cells. The level of NOTCH2 messenger RNA was negatively correlated with miR-1296-5p level in OS samples. NOTCH2 knockdown markedly suppressed the abilities of MG-63 cell proliferation and mobility. More importantly, the restoration of NOTCH2 prominently rescued miR-1296-5p-induced tumor-suppressive effects on MG-63 cells. In conclusion, our study identified the reduced expression of miR-1296-5p, which contributed to OS progression. miR-1296-5p might be a promising prognostic marker and therapeutic target in OS.     

miRNA-877-5p inhibits malignant progression of prostate cancer by directly targeting SSFA2

In this study, we aimed to investigate the role of miR-877-5p in the malignant phenotypes of prostate cancer (PCa) cells and its underlying mechanism. RT-qPCR analysis was performed to examine the expression of miR- 877-5p and sperm-specific antigen 2 (SSFA2) in PCa tissues and cells. Cell counting kit-8 (CCK-8) assay, 5- ethynyl-20-deoxyuridine (EdU) assay, flow cytometry, wound-healing assay, and Transwell invasion assay were performed to determine the functional roles of miR-877-5p in PCa cells. The association of miR-877-5p with SSFA2 was determined by luciferase reporter and RNA pull-down assays. In this study, we found that the expression level of miR-877-5p was decreased in PCa tissues and cells. Functionally, overexpression of miR- 877-5p exerted tumor suppressor properties in PCa cells. Mechanistically, SSFA2 was identified as a target gene of miR-877-5p, while overexpression of SSFA2 could abrogate the anti-tumor effects of miR-877-5p in PCa cells. These findings demonstrated that miR-877-5p/SSFA2 axis functioned as a potential target for PCa treatment.Key words: prostate cancer, miR-877-5p, proliferation, migration, invasion     

IKKα activation of NOTCH links tumorigenesis via FOXA2 suppression

Proinflammatory cytokine TNFα plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFα-mediated tumor development remains unclear. Here, we show that IKKα, an important downstream kinase of TNFα, interacts with and phosphorylates FOXA2 at S107/S111, thereby suppressing FOXA2 transactivation activity and leading to decreased NUMB expression, and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKα, pFOXA2 (S107/111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKα and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFα/IKKα-associated FOXA2 inhibition likely contributes to inflammation-mediated cancer pathogenesis. Here, we report a TNFα/IKKα/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer.     

Long noncoding RNA LEF1-AS1 silencing suppresses the initiation and development of prostate cancer by acting as a molecular sponge of miR-330-5p via LEF1 repression

Prostate cancer (PCa) is one of the major cancers affecting males with high mortality around the world. Recent studies have found that some long noncoding RNAs play a critical part in the cellular processes of PCa. In our study, aberrant expressed lymphoid enhancer-binding factor-1 antisense RNA 1 (LEF1-AS1), microRNA-330-5p (miR-330-5p), and lymphoid enhancer-binding factor-1 (LEF1) were screened out from a microarray database, the role of the novel noncoding RNA regulatory circuitry in the initiation and development of PCa was investigated. LEF1-AS1 and LEF1 were highly expressed while miR-330-5p was poorly expressed in PCa. Following that, the PCa PC-3 cell line was adopted for subsequently experiments, in which the expression of LEF1-AS1 and miR-330-5p was subsequently altered by means of exogenous transfection. After that, the effects of up- or downregulation of LEF1-AS1 and miR-330-5p on epithelial–mesenchymal transition (EMT) and the cell ability for proliferation, invasion, migration in vitro, and tumorigenesis and lymph node metastasis (LNM) in vivo were evaluated. RNA crosstalk revealed that LEF1-AS1 bound to miR-330-5p and LEF1 was the target gene of miR-330-5p. Silenced LEF1-AS1 or elevated miR-330-5p exhibited inhibited EMT processes, reduced ability of proliferation, invasion and migration, coupling with decreased tumorigenesis and LNM in nude mice. The key findings of this study collectively propose downregulation of LEF1-AS1 competing with miR-330-5p to inhibit EMT, invasion and migration of PCa by LEF1 repression.     

MicroRNA-93 Promotes Epithelial–Mesenchymal Transition of Endometrial Carcinoma Cells

MicroRNA-93, derived from a paralog (miR-106b-25) of the miR-17-92 cluster, is involved in the tumorigenesis and progression of many cancers such as breast, colorectal, hepatocellular, lung, ovarian, and pancreatic cancer. However, the role of miR-93 in endometrial carcinoma and the potential molecular mechanisms involved remain unknown. Our results showed that miR-93 was overexpressed in endometrial carcinoma tissues than normal endometrial tissues. The endometrial carcinoma cell lines HEC-1B and Ishikawa were transfected with miR-93-5P, after which cell migration and invasion ability and the expression of relevant molecules were detected. MiR-93 overexpression promoted cell migration and invasion, and downregulated E-cadherin expression while increasing N-cadherin expression. Dual-luciferase reporter assay showed that miR-93 may directly bind to the 3′ untranslated region of forkhead box A1 (FOXA1); furthermore, miR-93 overexpression downregulated FOXA1 expression while miR-93 inhibitor transfection upregulated FOXA1 expression at both mRNA and protein level. In addition, transfection with the most effective FOXA1 small interfering RNA promoted both endometrial cancer cell migration and invasion, and downregulated E-cadherin expression while upregulating N-cadherin expression. Therefore, we suggest that miR-93 may promote the process of epithelial–mesenchymal transition in endometrial carcinoma cells by targeting FOXA1.     

Genistein Up-Regulates Tumor Suppressor MicroRNA-574-3p in Prostate Cancer

Genistein has been shown to inhibit cancers both in vitro and in vivo, by altering the expression of several microRNAs (miRNAs). In this study, we focused on tumor suppressor miRNAs regulated by genistein and investigated their function in prostate cancer (PCa) and target pathways. Using miRNA microarray analysis and real-time RT-PCR we observed that miR-574-3p was significantly up-regulated in PCa cells treated with genistein compared with vehicle control. The expression of miR-574-3p was significantly lower in PCa cell lines and clinical PCa tissues compared with normal prostate cells (RWPE-1) and adjacent normal tissues. Low expression level of miR-574-3p was correlated with advanced tumor stage and higher Gleason score in PCa specimens. Re-expression of miR-574-3p in PCa cells significantly inhibited cell proliferation, migration and invasion in vitro and in vivo. miR-574-3p restoration induced apoptosis through reducing Bcl-xL and activating caspase-9 and caspase-3. Using GeneCodis software analysis, several pathways affected by miR-574-3p were identified, such as ‘Pathways in cancer’, ‘Jak-STAT signaling pathway’, and ‘Wnt signaling pathway’. Luciferase reporter assays demonstrated that miR-574-3p directly binds to the 3′ UTR of several target genes (such as RAC1, EGFR and EP300) that are components of ‘Pathways in cancer’. Quantitative real-time PCR and Western analysis showed that the mRNA and protein expression levels of the three target genes in PCa cells were markedly down-regulated with miR-574-3p. Loss-of-function studies demonstrated that the three target genes significantly affect cell proliferation, migration and invasion in PCa cell lines. Our results show that genistein up-regulates tumor suppressor miR-574-3p expression targeting several cell signaling pathways. These findings enhance understanding of how genistein regulates with miRNA in PCa.     

NOTCH1 regulates the proliferation and migration of bladder cancer cells by cooperating with long non-coding RNA HCG18 and microRNA-34c-5p

In recent years, the NOTCH signaling pathway has been gradually studied in human malignancies. Inactivation of the NOTCH signaling pathway was uncovered to be correlated with the carcinogenesis of bladder cancer (BCa). Nevertheless, the specific molecular mechanism of NOTCH1 (one of the core factors of the NOTCH signaling pathway) is not well elucidated in BCa. This study focused on the mechanism by which NOTCH1 affects the biological behaviors of BCa cells. According to the experimental results of quantitative real-time polymerase chain reaction, NOTCH1 was dysregulated in BCa tissues and cell lines. The prognostic value of NOTCH1 for the patients with BCa was determined using the Kaplan-Meier method. Mechanism investigations revealed that NOTCH1 is a target of miR-34c-5p in BCa. Furthermore, microarray analysis was used to find the dysregulated long noncoding RNAs (lncRNA), which can bind with miR-34c-5p. Mechanism experiments further demonstrated the rationality of the HCG18-miR-34c-5p-NOTCH1 pathway. Functional assays were then applied to validate the inhibitory influences of NOTCH1 on the proliferation and migration of BCa cells. Furthermore, the inhibitory effects of NOTCH1 could be affected by miR-34c-5p or lncRNA HCG18. All findings in this study revealed that NOTCH1 suppresses the BCa progression by cooperating with lncRNA HCG18 and miR-34c-5p.     

MiRNA-296-3p-ICAM-1 axis promotes metastasis of prostate cancer by possible enhancing survival of natural killer cell-resistant circulating tumour cells

Natural killer (NK) cells are important in host to eliminate circulating tumour cells (CTCs) in turn preventing the development of tumour cells into metastasis but the mechanisms are very poorly defined. Here we find that the expression level of miR-296-3p is much lower in the non-metastatic human prostate cancer (PCa) cell line P69 than that in the highly metastatic cell line M12, which is derived from P69. We demonstrate that miR-296-3p directly targets and inhibits the expression of intercellular adhesion molecule 1 (ICAM-1) in the malignant M12. The data from clinical tissue microarrays also show that miR-296-3p is frequently upregulated and ICAM-1 is reversely downregulated in PCa. Interestingly, ectopic expression of miR-296-3p in P69 increases the tolerance to NK cells whereas knockdown of miR-296-3p in M12 reduces the resistance to NK cells, which both phenotypes can be rescued by re-expression or silencing of ICAM-1 in P69 and M12, respectively. These results are also manifested in vivo by the decrease in the incidence of pulmonary tumour metastasis exhibited by knockdown of miR-296-3p in M12 when injected into athymic nude mice via tail vein, and consistently down-expression of ICAM-1 reverses this to increase extravasation of CTCs into lungs. Above results suggest that this newly identified miR-296-3p-ICAM-1 axis has a pivotal role in mediating PCa metastasis by possible enhancing survival of NK cell-resistant CTC. Our findings provide novel potential targets for PCa therapy and prognosis.     

MicroRNA-487a-3p functions as a new tumor suppressor in prostate cancer by targeting CCND1

Prostate cancer (PCa) is one of the major health problems of the aging male. The roles of dysregulated microRNAs in PCa remain unclear. In this study, we mined the public published data and found that miR-487a-3p was significantly downregulated in 38 pairs of clinical prostate tumor tissues compared with the normal tissues. We further verified this result by in situ hybridization on tissue chip and quantitative real-time polymerase chain reaction (qRT-PCR) in PCa/normal cells. miR-487a-3p targeting of cyclin D1 (CCND1) was identified using bioinformatics, qRT-PCR and western blot analyses. The cellular proliferation, cell cycle, migration, and invasion were assessed by cell counting kit-8, flow cytometry analysis and transwell assay. We discovered that overexpression of miR-487a-3p suppressed PCa cell growth, migration, invasion by directly targeting CCND1. Knockdown of CCND1 in PCa cells showed similar results. Meanwhile, the expression level of CCND1 was significantly upregulated in the PCa tissues and cell lines, which presented negative correlation with the expression of miR-487a-3p. More important, we demonstrated significantly reduced growth of xenograft tumors of stable miR-487a-3p-overexpressed human PCa cells in nude mice. Taken together, for the first time, our results revealed that miR-487a-3p as a tumor suppressor of PCa could target CCND1. Our finding might reveal miR-487a-3p could be potentially contributed to the pathogenesis and a clinical biomarker or the new potential therapeutic target of PCa.     

miR-449c-5p availability is antagonized by circ-NOTCH1 for MYC-induced NOTCH1 upregulation as well as tumor metastasis and stemness in gastric cancer

Gastric cancer (GC), identified as the most common gastrointestinal malignancy, is one of the primary causes of cancer-related mortality in the world. Although surgery and chemotherapy for GC treatment have been improved, the 5-year overall survival rate is still unsatisfactory. Circ-NOTCH1 is a novel circular RNA derived from its host gene NOTCH1, and has not been studied in any cancers. Here we explored the potential role and mediatory mechanism of circ-NOTCH1 in GC. In this study, circ-NOTCH1 exhibited increased expression in GC tissues and cells. Suppression of circ-NOTCH1 inhibited cell migration, invasion, tumor spheroids number, and side population ratio. Circ-NOTCH1 also promoted GC growth and metastasis in vivo. Additionally, it was found that circ-NOTCH1 could bind to miR-449c-5p. Circ-NOTCH1 promoted metastasis and stemness in GC through sponging miR-449c-5p. Subsequently, MYC was identified as a downstream gene of miR-449c-5p. MYC could bind to the promoter of NOTCH1 to regulate GC progression. Furthermore, rescue assays demonstrated that NOTCH1 knockdown reversed the effects of overexpression of MYC in metastasis and stemness in AGS cells/sh-circNOTCH1. Above findings explained that circ-NOTCH1 promoted metastasis and stemness in GC by targeting miR-449c-5p/MYC/NOTCH1 axis, suggesting the possibility of circ-NOTCH1 as a therapeutic marker for GC.