MicroRNA‐145 as one negative regulator of astrogliosis

Astrogliosis occurs at the lesion site within days to weeks after spinal cord injury (SCI) and involves the proliferation and hypertrophy of astrocytes, leading to glia scar formation. Changes in gene expression by deregulated microRNAs (miRNAs) are involved in the process of central nervous system neurodegeneration. Here, we report that mir‐145, a miRNA enriched in rat spinal neurons and astrocytes, was downregulated at 1 week and 1 month after SCI. Our in vitro studies using astrocytes prepared from neonatal spinal cord tissues indicated that potent inflammagen lipopolysaccharide downregulated mir‐145 expression in astrocytes, suggesting that SCI‐triggered inflammatory signaling pathways could play the inhibitory role in astrocytic mir‐145 expression. To induce overexpression of mir‐145 in astrocytes at the spinal cord lesion site, we developed a lentivirus‐mediated pre‐miRNA delivery system using the promoter of glial fibrillary acidic protein (GFAP), an astrocyte‐specific intermediate filament. The results indicated that astrocyte‐specific overexpression of mir‐145 reduced astrocytic cell density at the lesion border of the injured spinal cord. In parallel, overexpression of mir‐145 reduced the size of astrocytes and the number of related cell processes, as well as cell proliferation and migration. Through a luciferase reporter system, we found that GFAP and c‐myc were the two potential targets of mir‐145 in astrocytes. Together, the findings demonstrate the novel role of mir‐145 in the regulation of astrocytic dynamics, and reveal that the downregulation of mir‐145 in astrocytes is a critical factor inducing astrogliosis after SCI. GLIA 2015;63:194–205     

氯吡格雷通过上调miR-145表达抑制VSMC增殖的机制研究

目的 本研究旨在探究miR-145是否对血管平滑肌细胞(VSMC)的增殖起调控作用,以及氯吡格雷如何通过调控CD40来发挥其消炎作用,以期为氯吡格雷的药用作用发挥提供新的理论依据.方法 实验分组为①DMSO组,即溶剂对照组;②TNF-α组;③miR-145抑制剂对照组;④氯吡格雷组;⑤miR-145抑制剂+氯吡格雷组.并通过EdU标记检测细胞增殖;qRT-PCR用于检测miR-145,CD40和VSMC中Calponin mRNA的表达;Western blot检测CD40的蛋白表达水平;通过ELISA检测细胞培养物上清液中IL-6的水平.结果 与氯吡格雷组相比,miR-145抑制剂+氯吡格雷组的Calponin mRNA水平降低(P <0.01);与DMSO组相比,TNF-α组的miR-145 mRNA水平下降(P<0.01);与TNF-α组相比,氯吡格雷组的miR-145 mRNA水平上升(P<0.01);与氯吡格雷组相比,miR-145抑制剂+氯吡格雷组的miR-145 mRNA水平下降(P<0.01).miR-145抑制剂对照组不影响CD40的水平;与DMSO组相比,TNF-α组的CD40 mRNA水平上升(P<0.01);与TNF-α组相比,氯吡格雷组的CD40 mRNA水平下降(P<0.01);与氯吡格雷组相比,miR-145 抑制剂+氯吡格雷组的CD40 mRNA水平上升(P<0.01).TNF-α组上清液中IL-6的水平高于DMSO组(P<0.01);氯吡格雷组中IL-6水平低于TNF-α组(P<0.01);miR-145抑制剂+氯吡格雷组IL-6的水平高于氯吡格雷组(P<0.01).结论 氯吡格雷通过抑制CD40的表达,诱导miR-145抑制VSMC细胞增殖并发挥消炎作用.     

miR-145抑制c-Myc基因表达对鼻咽癌细胞增殖的影响

目的：探究miR-145影响鼻咽癌细胞增殖可能的机制。方法：采用Real-time PCR法检测鼻咽癌细胞株CNE-1、CNE-2、CNE-2Z和鼻咽部永生化上皮细胞株NP69中miR-145和c-Myc的mRNA表达水平,Western blotting法检测c-Myc的蛋白表达水平,双萤光素酶报告基因实验检测miR-145与基因c-Myc的关系。分别将miR-Negative control、miR-145 mimics和si-NC、si-c-Myc转染进入CNE-1细胞,采用Real-time PCR及Western blotting法检测转染效果,CCK-8法检测转染后细胞的增殖情况,以及碘化丙啶（IP）染色流式细胞术检测细胞周期情况。结果： miR-145在鼻咽癌细胞系中明显低表达。转染miR-145后明显抑制CNE-1细胞的增殖\[3 d: （1.03±0.02） vs （1.21 ± 0.02）, P<0.05\];\[ 4 d: （1.79±0.02） vs （2.09±0.07）, P<0.01\]和导致G1期阻滞\[（79.57±1.47）% vs （69.98±1.16）%,P<0.05\]。miR-145可以直接作用于c-Myc的3’UTR区域,抑制c-Myc的转录和表达。c-Myc下调可明显抑制CNE-1细胞的增殖\[3 d: （0.80±0.02） vs （1.02±0.01）, P<0.01\];\[4 d: （1.68±0.4） vs （1.92±0.07）, P<0.01\],并致G1期阻滞\[（63.73± 1.81）% vs （54.10±2.26）%,P<0.05\]。结论： miR-145通过靶向作用于c-Myc的3’UTR区来抑制鼻咽癌细胞的增殖,对更深入探索鼻咽癌的诊断和治疗有着重要意义。     

Tumor‐suppressive microRNA‐145 induces growth arrest by targeting SENP1 in human prostate cancer cells

Prostate cancer (PCa) prevails as the most commonly diagnosed malignancy in men and the third leading cause of cancer‐related deaths in developed countries. One of the distinct characteristics of prostate cancer is overexpression of the small ubiquitin‐like modifier (SUMO)‐specific protease 1 (SENP1), and the upregulation of SENP1 contributes to the malignant progression and cell proliferation of PCa. Previous studies have shown that the expression of microRNA‐145 (miRNA‐145) was extensively deregulated in PCa cell lines and primary clinical prostate cancer samples. Independent target prediction methods have indicated that the 3′‐untranslated region of SENP1 mRNA is a potential target of miR‐145. Here we found that low expression of miR‐145 was correlated with high expression of SENP1 in PCa cell line PC‐3. The transient introduction of miR‐145 caused cell cycle arrest in PC‐3 cells, and the opposite effect was observed when miR‐145 inhibitor was transfected. Further studies revealed that the SENP1 3′‐untranslated region was a regulative target of miR‐145 in vitro. MicroRNA‐145 also suppressed tumor formation in vivo in nude mice. Taken together, miR‐145 plays an important role in tumorigenesis of PCa through interfering SENP1.     

微小RNA-27 a对黑色素瘤WM239细胞增殖和凋亡的影响

目的：探讨微小RNA-27a（ miR-27a）模拟物和抑制物转染黑色素瘤WM239细胞后对细胞增殖和凋亡的影响。方法将miR-27a模拟物、抑制物及其阴性对照转染WM239细胞,荧光显微镜观察转染效率,实时荧光定量PCR检测相应的微小RNA,四甲基偶氮唑盐（ MTT）法检测细胞增殖,流式细胞仪检测细胞凋亡和细胞周期。结果细胞转染效率为80%~90%。转染miR-27a模拟物后,细胞内miR-27a表达量明显上升（2-△△CT值为26.98±0.01）,与正常对照组相比差异有统计学意义（ t＝－1123.67,P＝0.00）;转染miR-27a抑制物后,细胞中miR-27a的表达量下降（2-△△CT值为0.96±0.02）,与正常对照组相比差异无统计学意义（t＝4.04,P＝0.06）。转染miR-27a模拟物后,细胞增殖受到明显抑制,与正常对照组相比差异具有统计学意义[72 h吸光度（0.45±0.02）∶（0.72±0.01）,F＝129.56,P﹤0.05]。miR-27a模拟物组G0-G1期的细胞比例升高[（74.83±1.46）∶（63.73±1.25）,F＝30.33,P﹤0.05],S期和G2-M期细胞比例减少[（21.33±1.75）∶（27.50±1.25）,F＝14.98,P﹤0.05;（3.90±1.31）∶（8.80±2.10）,F＝3.66,P﹤0.05];模拟物组细胞凋亡率与正常对照组相比明显增加[（29.67±0.91）%∶（1.44±0.85）%, F＝530.90,P﹤0.01];而抑制物组对细胞周期和凋亡无明显作用。结论 miR-27a抑制黑色素瘤细胞增殖,具有抑瘤作用,这与其促进细胞凋亡,阻滞细胞周期于G0-G1期相关。     

MicroRNA-21对食管鳞状上皮癌细胞TE-1生物学特性的影响

目的:食管鳞状上皮癌TE-1细胞中microRNA-21(miR-21)高表达,本研究探讨miR-21对TE-1细胞生物学特性的影响。方法:转染miRZip-21慢病毒,持久抑制TE-1细胞中高表达的miR-21,将由此获得的稳定细胞系命名为anti-miR-21细胞系,实时定量PCR方法验证,以TE-1细胞作为对照。细胞增殖实验检测anti-miR-21细胞增殖能力,Transwell侵袭实验观察细胞侵袭能力,Transwell迁移实验及划痕实验观察细胞迁移能力,克隆形成实验及细胞毒性实验分别观察放射线及药物敏感性变化。结果:Anti-miR-21细胞的相对增殖率较对照组TE-1细胞低,差异具有统计学意义(P<0.05)。Transwell侵袭实验及迁移实验显示穿过小室的anti-miR-21细胞较TE-1细胞分别减少51.97%及64.31%,差异具有统计学意义(P<0.05)。划痕实验显示anti-miR-21细胞较TE-1细胞的迁移率降低,差异具有统计学意义(P<0.01)。克隆形成实验分析显示anti-miR-21细胞的D0值(2.73Gy vs 3.60Gy)和Dq值(1.25Gy vs 2.75Gy)均低于对照组,差异具有统计学意义(P<0.05)。细胞毒性实验显示anti-miR-21细胞在顺铂及氟尿嘧啶各个梯度浓度下细胞存活率均降低,差异具有统计学意义(P<0.05)。结论:MiR-21影响食管鳞状上皮癌TE-1细胞的生物学特性。抑制miR-21表达后,降低了TE-1细胞的增殖、侵袭及迁移能力,同时增加了其对放射及化学药物的敏感性。MiR-21可能成为食管鳞癌治疗的潜在靶点。     

Targeting the microRNA-21/AP1 axis by 5-fluorouracil and pirarubicin in human hepatocellular carcinoma

MicroRNAs function as oncomiRs and tumor suppressors in diverse cancers. However, the utility of specific microRNAs in predicting the clinical benefit of chemotherapy has not been well-established. Here, we investigated the correlation between microRNA-21 expression and hepatic arterial infusion chemotherapy with 5-fluorouracil and pirarubicin (HAIC) for hepatocellular carcinoma (HCC). We found that HCC patients with low microRNA-21 levels in tumors tended to have a longer time to recurrence and disease-free survival. We demonstrated that microRNA-21 suppression in combination with 5-fluorouracil and pirarubicin treatment inhibited tumor growth in subcutaneous xenograft mice models. Mechanistically, the AP-1 and microRNA-21-mediated axis was verified to be a therapeutic target of cytotoxic drugs and deregulation of this axis led to an enhanced cell growth in HCC. Taken together, our findings demonstrate that microRNA-21 is a chemotherapy responsive microRNA and can serve as a prognostic biomarker for HCC patients undergoing HAIC. Targeting microRNA-21 enhances the effect of chemotherapeutic drugs, thereby suggesting that microRNA-21 suppression in combination with HAIC may be a novel approach for HCC treatment.     

miR-874 functions as a tumor suppressor by inhibiting angiogenesis through STAT3/VEGF-A pathway in gastric cancer

MicroRNAs are endogenously expressed, small non-coding RNAs that regulate gene expression by targeting mRNAs for translational repression or degradation. Our previous studies indicated that miR-874 played a suppressive role in gastric cancer (GC) development and progression. However, the role of miR-874 in tumor angiogenesis and the mechanisms underlying its function in GC remained to be clarified. Here, gain- and loss-of-function assays demonstrated that miR-874 inhibited the tumor angiogenesis of GC cells in vitro and in vivo. Through reporter gene and western blot assays, STAT3 was shown to be a direct target of miR-874. Overexpression of STAT3 rescued the loss of tumor angiogenesis caused by miR-874. Conversely, the STAT3-shRNA attenuated the increased tumor angiogenesis caused by the miR-874-inhibitor. Furthermore, the levels of miR-874 were inversely correlated with those of STAT3 protein in GC tissues. Taken together, these findings indicate that down-regulation of miR-874 contributes to tumor angiogenesis through STAT3 in GC, highlighting the potential of miR-874 as a target for human GC therapy.