MiR-429 is an independent prognostic factor in colorectal cancer and exerts its anti-apoptotic function by targeting SOX2

Emerging evidence has demonstrated that microRNAs (miRNAs) can act as oncogenes or tumor suppressors to participate in cancer development. In this study, we found that miR-429 expression was up-regulated in human colorectal cancer (CRC) tissues, and the high miR-429 expression was significantly associated with tumor size, lymph node metastasis and poor prognosis. Functionally, miR-429 overexpression suppressed cell apoptosis by directly targeting SOX2 in HT-29 cells. Taken together, our data suggest for the first time that miR-429 could play an oncogenic role in the cellular processes of CRC and represent a novel prognostic biomarker for CRC.     

RhoC is a major target of microRNA-93-5P in epithelial ovarian carcinoma tumorigenesis and progression

Background

An increasing amount of evidence has revealed that microRNAs regulate various biological processes, including cell differentiation, cell proliferation, apoptosis, drug resistance, and fat metabolism. Studies have shown that miR-93’s targetome in cancer has not been fully defined. Moreover, the role of miR-93 in epithelial ovarian carcinoma (EOC) remains largely unknown.

Methods

MIR-93 mRNA expression in normal ovarian tissue, benign tumors, borderline tumors, primary ovarian carcinomas, and metastatic omentum was quantified. The ovarian carcinoma cell lines OVCAR3, SKOV3/DDP, and HO8910-PM were transfected with miR-93-5P, after which cell phenotype and expression of relevant molecules were assayed. Dual-luciferase reporter assay and a xenograft mouse model were used to examine miR-93 and its target gene RHOC (Ras homolog gene family member C).

Results

MIR-93 mRNA expression was significantly lower in ovarian carcinomas and borderline tumors than in normal ovarian tissues (p < 0.05), and was lower in metastatic omentum than in relative primary ovarian carcinomas (p < 0.05). MIR-93 mRNA expression was also negatively associated with differentiation (well vs. poor and moderate) and International Federation of Gynecology and Obstetrics staging (FIGO stage I/II vs. stage III/IV) in ovarian carcinoma (p < 0.05), besides, miR-93 was higher expressed in mucinous adenocarcinoma than the other types (p < 0.05). MiR-93-5P overexpression reduced proliferation (p < 0.05); promoted G₁ or S arrest and apoptosis (p < 0.05); suppressed migration and invasion (p < 0.05); and reduced RhoC, P70S6 kinase, Bcl-xL, matrix metalloproteinase 9 (MMP9) mRNA or protein expression; conversely, it induced P53 and cleaved PARP expression (p < 0.05). Dual-luciferase reporter assay indicated that miR-93 directly targeted RhoC by binding its 3′ untranslated region. MiR-93-5P transfection also suppressed tumor development and RhoC expression (determined by immunohistochemistry) in vivo in the xenograft mouse model (p < 0.05).

Conclusions

This is the first demonstration that miR-93-5P may inhibit EOC tumorigenesis and progression by targeting RhoC. These findings indicate that miR-93-5P is a potential suppressor of ovarian cellular proliferation. The involvement of miR-93-5P–mediated RhoC downregulation in inhibiting EOC aggressiveness may provide extended insight into the molecular mechanisms underlying cancer aggressiveness.     

Plasma miR-183 predicts recurrence and prognosis in patients with colorectal cancer

Colorectal cancer (CRC) is one of the most common malignancies worldwide. The prognosis for this cancer is poor, and the development of novel biomarkers, particularly non-invasive surrogate biomarkers, is urgently needed. Recent studies have demonstrated that microRNAs (miRNAs) are stably detectable in the blood and can serve as useful biomarkers for various types of cancer. In this study, the miR-183 expression levels were found to be significantly overexpressed in plasma samples from CRC patients compared with controls, and the postoperative plasma miR-183 levels were significantly reduced compared with the preoperative levels. The value of the area under the receiver operating characteristic (ROC) curve obtained for miR-183 was 0.829, which was higher than those for carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9). High plasma miR-183 expression was significantly associated with lymph node metastasis, distant metastasis, higher pTNM stage (III-IV), and tumor recurrence. CRC patients with elevated miR-183 expression in plasma displayed shorter disease-free survival (DFS) and lower overall survival (OS). More importantly, plasma miR-183 was independently correlated with tumor recurrence and a lower OS. Collectively, our results suggested that the elevated miR-183 in the plasma could be a promising biomarker for predicting the risk of tumor recurrence and poor survival in CRC patients.     

子宫内膜癌组织中 miR-10a 及 let-7i 的表达及临床意义

目的 探讨 miR-10a 及 let-7i 在子宫内膜癌组织中的表达及意义。方法 选取子宫内膜癌组织40 例、子宫内膜不 典型增生组织20例及正常子宫内膜组织30例,采用多聚腺苷酸加尾实时荧光定量逆转录聚合酶链反应[poly（A）-RT-qPCR] 测定miR-10a 及 let-7i 在三种子宫内膜组织中的表达,并将结果与临床及病理资料进行分析。结果 miR-10a 在子宫内膜癌 组织中高表达（高于正常及不典型增生的子宫内膜组织,P＜0.01）;在病理分级为3级的患者中高表达（高于1+2级者）; 在Ⅲ+Ⅳ期患者中高表达（高于Ⅰ+Ⅱ期者）;在有淋巴结转移的患者中高表达,在深肌层浸润（浸润深度≥1/2）的患者 中高表达;差异显著（P ＜ 0.05）。在不同年龄、雌激素受体（ER）及孕激素受体（PR）是否阳性的子宫内膜癌患者中其 表达量差异无统计学意义（P ＞ 0.05）。let-7i 在子宫内膜癌组织低表达（低于正常和不典型增生的子宫内膜组织,P ＜ 0.01）。结论 在子宫内膜癌组织中miR-10a 表达上调,let-7i 表达下调,miR-10a 可能参与并促进了子宫内膜癌的侵袭 和转移。     

miR-3195对人喉癌Hep-2细胞增殖的抑制作用及其机制

目的：探讨miR-3195对喉癌Hep2细胞增殖的影响及其分子机制。方法：选取2008年1月至2012年8月期间在南华大学教学医院郴州市第一人民医院耳鼻喉科收治的29例喉癌患者的喉癌组织及其相应的癌旁组织标本,采用qPCR检测miR-3195在喉癌和癌旁组织中的表达;构建miR-3195在喉癌Hep-2细胞中稳定高表达的细胞株,采用MTT法观察miR-3195稳定高表达组和对照组的增殖情况;建立裸鼠移植瘤模型,观察miR-3195稳定高表达的人喉癌细胞株Hep-2在裸鼠体内增殖的情况。利用生物信息学预测 miR-3195 的靶基因,构建 TBX1 3''UTR 的荧光素酶载体,双荧光素酶检测系统检测其荧光素酶活性;Western blotting检测稳定高表达miR-3195组和对照组细胞株的TBX1蛋白表达水平。结果：miR-3195在喉癌组织中的表达明显低于癌旁组织（P<0.01）;miR-3195上调可抑制Hep-2细胞的增殖（P<0.01）,且可明显抑制裸鼠移植瘤瘤体的生长（P<0.05）;双荧光素酶报告基因检测结果表明miR-3195可能与TBX1靶向结合（P<0.05）,同时Western blotting法验证了miR-3195能够抑制TBX1蛋白的表达（P<0.05）。结论：miR-3195对Hep2细胞有明显的增殖抑制作用,其分子机制可能与负性调控TBX1的表达有关。     

miR-513a-3p靶向鼠双微体基因2对胃癌细胞增殖迁移侵袭的影响

目的探讨miR-513a-3p靶向鼠双微体基因2(MDM2)对胃癌细胞的增殖、迁移和侵袭的影响及其作用机制。方法采用脂质体法将miR-NC、miR-513a-3p、anti-miR-NC、anti-miR-513a-3p、si-NC、si-MDM2、miR-513a-3p+pcDNA3.1和miR-513a-3p+pcDNA3.1-MDM2转染至BGC-823细胞中,采用实时荧光定量聚合酶链反应(qRT-PCR)检测miR-513a-3p的表达水平,采用Western blot检测cyclin D1、MMP-2、p21、E-cadherin和MDM2蛋白的表达水平,四甲基偶氮唑蓝法检测各组胃癌细胞BGC-823的活性,Transwell法检测各组胃癌细胞BGC-823的迁移和侵袭能力,双荧光素酶报告基因检测实验检测miR-513a-3p与MDM2的靶向关系。结果胃癌细胞BGC-823、MGC-803中miR-513a-3p的表达水平分别为0.21±0.02和0.34±0.03,与胃上皮细胞GES-1(0.76±0.08)比较,差异均有统计学意义(均P<0.05)。培养24、48和72 h后,miR-NC组细胞的吸光度(A)值分别为0.57±0.05、1.03±0.10和1.43±0.14,miR-513a-3p组细胞的A值分别为0.36±0.03、0.48±0.05和0.63±0.06,差异均有统计学意义(均P<0.05);miR-NC组细胞的迁移和侵袭数分别为(130±11.80)个和(117±10.60)个,miR-513a-3p组细胞分别为(58±5.64)个和(50±5.13)个,差异均有统计学意义(均P<0.05)。培养24、48和72 h后,si-NC组细胞的A值分别为0.53±0.05、0.95±0.10和1.36±0.14,si-MDM2组细胞的A值分别为0.39±0.04、0.57±0.06和0.80±0.08;si-NC组细胞的迁移和侵袭数分别为(141±12.02)个和(109±10.60)个,si-MDM2组的迁移和侵袭数分别为(66±6.67)个和(61±6.18)个,差异均有统计学意义(均P<0.05)。培养24、48和72 h后,miR-513a-3p+pcDNA3.1组细胞的A值分别为0.34±0.03、0.46±0.05和0.61±0.06,miR-513a-3p+pcDNA3.1-MDM2组细胞的A值分别为0.48±0.05、0.82±0.08和1.17±0.12,差异均有统计学意义(均P<0.05);miR-513a-3p+pcDNA3.1组细胞的迁移和侵袭数分别为(56±5.71)个和(51±5.16)个,miR-513a-3p+pcDNA3.1-MDM2组分别为(113±10.28)个和(104±10.02)个,差异均有统计学意义(均P<0.05)。结论miR-513a-3p可抑制胃癌细胞的增殖、迁移和侵袭能力,其机制可能与靶向调控MDM2的表达有关,可为胃癌的预防和治疗提供新靶点。     

miR-424对非小细胞肺癌A549细胞生长和侵袭的影响及分子机制

背景与目的已有的研究表明miR-424可抑制肾癌细胞增殖,抑制宫颈鳞癌细胞的迁移和侵袭能力,而其对非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞的影响目前尚无系统研究。本研究探讨miR-424对NSCLC A549细胞生长和侵袭迁移能力的影响并进一步研讨其分子机制。方法应用CCK8检测过表达及抑制miR-424的表达对A549细胞增殖的影响。应用Transwell检测过表达及抑制miR-424的表达对A549细胞侵袭能力的影响。应用Western blot检测过表达及抑制miR-424的表达对A549细胞中MMP9和MMP2蛋白水平的影响。构建E2F63’UTR区的荧光素酶报告载体,验证miR-424对E2F6的靶向作用。采用Western blot检测过表达及抑制miR-424的表达后,A549细胞中E2F6的表达。结果过表达miR-424后,A549的生长和侵袭能力显著降低。过表达miR-424后,A549细胞的MMP-2和MMP-9表达下降。荧光素酶活性检测表明miR-424能够抑制E2F6的荧光素酶活性。过表达miR-424后,细胞内E2F6的表达降低。结论 miR-424能够通过调控E2F6而抑制A549的生长和侵袭能力。     

CD44、CD133耦连miR-425装载纳米脂质体对结直肠癌干细胞PDL-1表达的下调作用

目的 探究CD44、CD133耦连miR-425装载纳米脂质体对结直肠癌干细胞PDL-1表达的下调作用.方法选择结直肠癌细胞系HCT116分选出CD44、CD133/1+细胞,进行miR-425装载纳米脂质体转染,分为对照组(转染con-trol载体)、实验组(转染miR-425慢病毒载体),空白组以纳米脂质体代替,测定干细胞生长状态,检测PDL-1表达水平.结果分选的CD 133/1+细胞呈圆形,12 h后开始贴壁生长,状态良好.qRT-PCR检测显示实验组的miR-425的相对表达水平为(182.44±22.19),对照组与空白组分别为(1.84±1.11)和(1.67±0.98),实验组明显高于对照组与空白组(P<0.05).MTT检测结果表明,与空白组(0.78±0.22)、对照组(0.81±0.17)比较,实验组(0.24±0.15)的HCT116细胞生长速度明显减慢(P<0.05).空白组与对照组的细胞增殖率分别为(0.48±0.14)与(0.51±0.18),都明显高于实验组的(0.30±0.11).Western blot检测分析显示实验组、对照组、空白组的PDL-1相对表达量分别为(0.33±0.13)、(0.76±0.21)和(0.82±0.15),实验组明显低于对照组与空白组(P<0.05).结论CD44、CD133耦连miR-425装载纳米脂质体能抑制结直肠癌干细胞PDL-1的表达,从而显著抑制干细胞的生长、增殖能力.     

miR-10a-5p基因甲基化对卵巢癌铂类耐药的影响

目的 卵巢癌是死亡率最高的妇科肿瘤,较强的化疗耐药性是其预后差的主要原因之一,为了阐明卵巢癌对铂类药物的耐药机制,本研究探讨miRNA基因的甲基化水平对卵巢癌铂类耐药的影响.方法 将卵巢癌组织分为敏感组和耐药组,每组各3例;采用基因芯片技术,对比分析了两组微RNA(microRNA,miRNA)的表达差异;采用实时荧光定量PCR,分别在6例敏感和3例耐药组织、铂类药物敏感(CoC1)和耐药的卵巢癌细胞系(CoC1/DDP),检测了候选miRNA的表达差异;应用Massarray技术,检测敏感组织(15例)与耐药组织(6例)中miRNA基因启动子的甲基化差异;应用生物信息学分析,鉴定目标miRNA的潜在靶基因.结果 以铂类药物敏感的卵巢癌组织样本为对照,利用基因芯片筛选,鉴定了6条在耐药组织样本中出现表达上调的miRNA(miR-493-3p、miR-10a-5 p、miR-16-2-3p、miR-1248、miR-451a、miR-628-3p)和6条表达下调的miRNA(miR-509-3p、miR-1197、miR-376a-3p、miR-1273a、miR-550a-3p、miR-19b-3p).组织验证发现,miR-509-3p、miR-493-3p、miR-10a-5p、miR-16-2-3p和miR-451a,与芯片结果一致;培养细胞研究发现,4条miRNA的表达调控方式与组织芯片结果一致,miR-10a-5p、miR-16-2-3p、miR-1248和miR-628-3p在耐药细胞系中高表达.进一步研究发现,与敏感肿瘤组织相比,耐药组织中miR-10a-5p基因启动子的甲基化水平出现显著降低,P=0.04.结合生物信息学预测HOXA1和USF2为miR-10a-5p与耐药相关的靶基因.结论 与敏感组相比,耐药组miR-10a-5p基因启动子甲基化水平显著降低,miR-10a-5p表达升高,通过抑制 HOXA1和USF2,抑制细胞凋亡,导致铂类化疗药物耐受.