Profilin 1, negatively regulated by microRNA-19a-3p,serves as a tumor suppressor in human hepatocellular carcinoma

Profilin 1 (PFN1) is a critical actin-regulatory protein; however, its functional role in hepatocellular carcinoma (HCC) progression remains to be further elucidated. In the present study, we observed that the expression levels of PFN1 were significantly decreased in HCC tissues and cell lines. Low PFN1 expression was significantly correlated with aggressive clinicopathological characteristics and poor prognosis of HCC patients. Further in vitro experiments demonstrated that overexpression of PFN1 remarkably inhibited the proliferation, migration, invasion and EMT of HCC cells. Moreover, we also found that PFN1 was a direct target gene of miR-19a-3p, and in HCC tissues, and there was a significantly inverse correlation between PFN1 mRNA and miR-19a-3p expression. Collectively, our results showed that PFN1 functions as a tumor suppressor in HCC, and might serve as a diagnostic and therapeutic target for HCC patients.     

miR-499 rs3746444多态性抑制宫颈癌细胞的增殖作用及分子机制

目的探讨miR-499 rs3746444多态性与宫颈癌临床特征相关性及对宫颈癌细胞增殖的影响作用与机制。方法应用病例对照研究方法,选取2012年6月至2018年12月于内蒙古自治区人民医院就诊的宫颈癌病人857例作为实验组,同期873例健康志愿者作为对照组,两组年龄均在23~62岁,采用TaqMan探针法对rs3746444位点的多态性进行基因分型;构建包含pGL3-Sox63'UTR和miR-499的GG或AA的海肾荧光素酶载体(Renilla luciferase vector)于prl-sV40质粒中,并采用lipo2000转染人宫颈上皮永生化细胞H8和人宫颈癌细胞系:腺癌样SiHa、上皮样HeLa和C33A,CCK-8法检测细胞增殖;双荧光素报告基因检测法分析Frefly和Renilla荧光素酶活性;Western blot检测Hela和SiHa细胞Cyclin D1、CyclinE、CDK4、CDK6和Sox6蛋白表达。结果AG和GG基因型宫颈癌发生风险分别是AA基因型个体的1.09倍和2.41倍,携带G等位基因的个体患宫颈癌的风险是AA基因型个体的1.28倍,GG基因型宫颈癌患者III期及以上的比例显著高于AA基因型。GG表型肿瘤高度分化比例明显高于AA表型组,rs3746444不同基因型对正常宫颈H8细胞的增殖无显著影响。miR-499 GG+Sox6-3'UTR处理组Hela、C33A和SiHa细胞72 h时OD450显著低于miR-499 AA+Sox6-3'UTR组,且高于Sox6-3'UTR组(P<0.05)。miR-499 GG+Sox6-3'UTR处理组Hela和SiHa细胞Cyclin D1、CyclinE、CDK4和CDK6显著高于miR-499 AA+Sox6-3'UTR组,低于Sox6-3'UTR组(P<0.05)。与miR-499 AA+Sox6-3'UTR处理组相比,miR-499 GG+Sox6-3'UTR处理组Sox6的表达水平增加(P<0.05),且均低于Sox6-3'UTR对照组(P<0.05)。结论miR-499的rs3746444突变(A>G)与宫颈癌肿瘤生长和分化程度密切相关,其通过上调Sox6介导的CyclinD1高表达具有明显的促进宫颈癌细胞生长增殖作用。     

胰腺导管腺癌中microRNA-21与TGF-β1信号通路相关

目的：对胰腺导管腺癌样本进行基因分析,筛选与胰腺导管腺癌相关的microRNA(miRNA),并初步分析目标miRNA与胰腺癌转化生长因子-β1(TGF-β1)信号通路的相关性。方法：收集在海南省中医院住院的胰腺导管癌患者术前外周静脉血血清标本19份,健康体检人群的外周静脉血血清标本21份作为非胰腺导管癌对照组。利用GCBI(Gene-Cloud Biotechnology Information)数据平台筛选与胰腺导管腺癌样本有关的基因并对其进行生物信息学分析,将筛选出的基因作为研究对象,用real-time PCR和蛋白质印迹法验证该基因在胰腺导管腺癌中的表达。通过改变胰腺导管腺癌细胞中该基因的表达水平观察其与TGF-β1之间的关系。结果：通过聚类分析和基因功能富集分析筛选出miRNA-21为胰腺导管癌相关基因。MiRNA-21在胰腺导管腺癌患者体内高表达。在miRNA-21过表达的PANC-1细胞中,TGF-β1表达受到抑制;但当miRNA-21的表达受到抑制时,TGF-β1的表达明显上升。结论：MiRNA-21在胰腺导管腺癌患者体内高表达,可调控TGF-β1的表达,从而参与胰腺导管腺癌的发生发展。     

MiR-30a-5p通过泛素水解酶22抑制人宫颈癌细胞上皮-间质转化功能

【目的】研究microRNA-30a-5p（miR-30a-5p）对人宫颈癌Hela细胞上皮-间质转化功能的影响及其相关机制。【方法】宫颈癌Hela细胞株分别转染目的mir的模拟物和阴性对照模拟物,分别以30a-5p组、NC组命名并标记细胞。同时,以未经过处理的Hela细胞作为对照（Control组）。分别用逆转录-聚合酶链反应法检测各组宫颈癌细胞的miR-30a-5p含量。Transwell实验检测3组细胞迁移能力和侵袭能力。Western-blot法检测3组细胞神经-钙粘素（N-cadherin）、α-连环蛋白（α-Catenin）和泛素水解酶22（USP22）表达水平。运用生物信息学方法预测miR-30a-5p的靶基因。采用Western blot法检测USP22过表达对miR-30a-5p抑制EMT的拮抗作用。双荧光素酶实验检测miR-30a-5p与USP22的关系。建立皮下移植瘤模型观察miR-30a-5p的体内作用。【结果】30a-5p组宫颈癌细胞miR-30a-5p的表达水平明显上调,表达水平为Control组的853.82（862.26~843.11）倍（P＜0.01）。30a-5p组侵袭细胞数量8.17（8.32~8.03）明显低于Control组（P＜0.01）。30a-5p组细胞N-cadherin蛋白的细胞内含量明显下降,α-Catenin蛋白的细胞内含量明显上升,USP22蛋白表达量明显降低。合并USP22过表达处理的30a-5p组宫颈癌细胞中N-cadherin蛋白表达量明显升高,α-Catenin蛋白表达量明显降低。双荧光素酶检验结果显示USP22为miR-30a-5p的下游靶基因（P＜0.01）。30a-5p组皮下移植瘤明显小于Control组（P＜0.01）。与Control组肿瘤组织相比,30a-5p组肿瘤组织miR-30a-5p的相对含量升高,USP22蛋白含量降低,N-cadherin蛋白的含量降低,α-Catenin蛋白含量升高。【结论】miR-30a-5p在宫颈癌Hela细胞中,可能通过靶向识别下游靶基因USP22,进而抑制其翻译。最终实现对宫颈癌细胞EMT过程的抑制。     

微小RNA-29a通过沉默信息调节因子2相关酶类1基因调控肝细胞脂肪变性的机制

目的探讨人肝细胞株L02脂肪变性时微小RNA-29a(miR-29a)的表达变化及其靶向沉默信息调节因子2相关酶类1(silent mating type information regulation 2 homolog-1,Sirt1)调节脂肪肝细胞脂肪沉积的机制。方法采用油酸和棕榈酸混合物诱导建立非酒精性脂肪肝细胞模型,验证模型成功后,PCR检测miR-29a和Sirt1的表达变化;生物学预测miR-29a的靶基因;分别转染miR-29a模拟物和抑制剂,过表达miR-29a和抑制miR-29a后再建立人脂肪肝细胞模型。油红O染色观察细胞中脂质蓄积情况并测定甘油三酯含量,荧光定量PCR和免疫印迹法检测Sirt1基因和蛋白表达变化。结果脂肪肝细胞模型组miR-29a相对表达量和甘油三酯含量显著高于对照组(P<0.01),Sirt1相对表达量显著低于对照组(P<0.01);生物学预测Sirt1是miR-29a的靶基因,过表达miR-29a后,细胞内脂滴明显增多,脂肪沉积加重,甘油三酯含量显著增加(P<0.05),细胞中miR-29a的表达显著上调(P<0.01),而Sirt1 mRNA表达显著下调(P<0.05),Sirt1蛋白表达呈下降趋势;与之相反,抑制miR-29a后,细胞中脂滴相对减少,脂肪沉积减轻,甘油三酯含量显著下降(P<0.05),细胞中miR-29a的表达被有效抑制(P<0.01),而Sirt1 mRNA的表达显著上调(P<0.05),Sirt1蛋白表达较对照组呈上升趋势。结论 miR-29a在非酒精性脂肪肝细胞中表达显著上调,miR-29a通过表达上调负调控Sirt1表达从而促进脂肪肝细胞中脂肪沉积。     

Dysregulation of microRNA-106a-5p-RUNX1 axis associates with clinical progression and prognosis of osteosarcoma patients

MicroRNA-106a-5p (miR-106a-5p) functions as a tumor suppressor in osteosarcoma cells. Here, we aimed to identify novel target genes of miR-106a-5p in osteosarcoma, as well as to investigate their prognostic value and the biological functions. At first, the mammalian runt-related factor 1 (RUNX1) was identified as one of the target genes of miR-106a-5p in osteosarcoma cells by luciferase reporter gene assay, real-time quantitative RT-PCR and Western blot analysis. Then, the expression levels of miR-106a-5p and RUNX1 in osteosarcoma tissues were detected, and their associations with clinicopathological features and patients' prognosis were statistically analyzed. Compared with adjacent non-cancerous tissues, miR-106a-5p and RUNX1 mRNA/protein expression in osteosarcoma tissues were significantly decreased and increased, respectively (all P < 0.01). Low miR-106a-5p, high RUNX1 and miR-106a-5p-low/RUNX1-high expression in osteosarcoma tissues were all significantly associated with advanced Enneking stage, positive metastasis and shorter overall survival (all P < 0.05). Moreover, miR-106a-5p and RUNX1 expression, alone or in combination, were identified as independent prognostic factors for osteosarcoma patients' overall survival. Functionally, the enforced expression of miR-106a-5p significantly suppressed proliferation and invasion of osteosarcoma cells, while the overexpression of RUNX1 effectively reversed its suppressive roles. In conclusion, our findings show the dysregulation of miR-106a-5p-RUNX1 axis in human osteosarcoma tissues and suggest its crucial roles in cancer progression and patients' prognosis. More interestingly, miR-106a-5p may function as a tumor suppressor in osteosarcoma cells via regulating its target gene RUNX1.     

miR-372调控血管生成因子AGGF1的表达并抑制血管生成

目的:研究miR-372(miR-372)对新血管生成因子AGGF1基因的表达调控?方法:通过生物信息学预测找到可能靶向调控AGGF1基因的候选miRNA;通过荧光素酶报告基因实验?荧光定量聚合酶链反应(qPCR)和Western blot检测等实验证实miR-372对AGGF1基因表达的靶向调控;通过体外血管内皮细胞成管实验检测miR-372对血管生成的影响?结果:生物信息学预测显示miR-372靶向AGGF1;荧光素酶报告基因实验证实miR-372可通过结合AGGF1基因3′-非翻译区(3′-untranslational region,3′-UTR)而抑制其表达;qPCR和Western blot实验分别表明miR-372在mRNA水平和蛋白水平下调AGGF1表达;体外血管生成实验表明miR-372所介导的AGGF1表达下调可明显抑制血管生成?结论:miR-372可通过靶向结合AGGF1基因的3′-UTR下调其表达,并抑制内皮细胞血管生成能力?