宫颈癌中高危型HPV感染与miR-362-3p、Nemo样激酶表达的相关性研究

高危型人乳头瘤病毒(High-risk human papillomavirus,HPV)感染是宫颈癌发病的重要因素,但高危型HPV感染引起宫颈上皮恶变的机制尚不清楚.微小RNA (micro RNA,miR)-362-3p是具有抑癌活性的miR,在宫颈癌中表达降低;Nemo样激酶(Nemo-like kinase,NLK)是生物信息学预测得到的miR-362-3p靶基因,在宫颈癌中表达增加.但宫颈癌发病过程中高危型HPV感染与miR-362-3p、NLK的关系尚不清楚.本研究检测了miR-362-3p、NLK在宫颈癌组织及宫颈癌细胞株中的变化,并通过转染miR-362-3p模拟物、NLK表达质粒的方式验证了miR-362-3p靶向NLK调节高危型HPV阳性宫颈癌细胞增殖的作用.结果 显示:与癌旁组织比较,宫颈癌组织中miR-362-3p的表达明显降低、NLK的表达明显增加且与高危型HPV阴性的宫颈癌组织比较,高危型HPV阳性的宫颈癌组织中miR-362-3p的表达明显降低、NLK的表达明显增加;与正常宫颈上皮细胞比较,HPV16感染的SiHa细胞、HPV18感染的HeLa细胞及HPV阴性的C33A细胞中miR-362-3p的表达明显降低、NLK的表达明显增加且SiHa细胞中miR-362-3p表达降低、NLK表达增加最明显;在SiHa细胞中,过表达miR-362-3p能够降低细胞活力及NLK的表达,同时也使NLK双荧光素酶报告基因的荧光活力降低;过表达NLK能够逆转miR-362-3p降低细胞活力及NLK表达的作用.以上结果表明宫颈癌中高危型HPV感染与miR-362-3p低表达、NLK高表达有关,过表达miR-362-3p通过靶向抑制NLK降低高危型HPV感染宫颈癌细胞的增殖活力.本研究的创新点在于初步探索了高危型HPV感染促进宫颈癌细胞增殖的作用及机制,在高危型HPV感染的宫颈癌中miR-362-3p表达减少并靶向引起NLK表达增加,进而促进了宫颈癌细胞的增殖,这为将来深入认识高危型HPV引起宫颈癌发病的机制提供依据.     

miR-30d负性调节HeLa细胞的增殖

有证据显示,microRNA( miRNA)可作为癌基因或抑癌基因发挥功能,参与细胞增殖和凋等 调控;PI3K/Akt通路的持续活化与肿瘤的发生、发展密切相关．在本研究中,我们通过microarray技术在HeLa细胞中检测经ly294002作用24 h阻断PI3K/Akt通路后microRNA的表达变化．Taqman 实时 PCR实验结果显示,has-miR-30d的表达在PI3K/Akt通路被阻断后明显上调．萤光素酶报告基因转染结合Taqman 实时 PCR显示,稳定转染miR- 30d过表达质粒的HeLa细胞系中miR-30d的表达量升高并且有活性．MTT及流式细胞术分析显示,过表达miR-30d的HeLa细胞其增殖受到抑制．这些结果说明,has-miR-30d可抑制HeLa细胞增殖,负性调节细胞生长,是一个潜在的抑癌基因．     

miR-455慢病毒表达载体的构建与鉴定

目的：构建人源microRNA-455（miR-455）慢病毒载体,并鉴定成熟has-miR-455在细胞内的表达水平。方法：提取siHa细胞中的人基因组DNA,设计并合成人miR-455的上下游引物,PCR扩增目的基因,将其中表达miR-455的结构经酶切后插入慢病毒转移质粒pWPT-GFP,构建成pWPT-GFP-pri-miR-455,在293T细胞中与pMD2G、pSPAX2包装产生慢病毒,并用含慢病毒的上清感染SiHa细胞。结果：测序结果证明插入质粒载体中的miR-455前体序列完全正确,慢病毒载体构建成功并获得相应的慢病毒;重组慢病毒质粒pWPT-GFP-pri-miR-455感染SiHa细胞后上调miR-455的表达近40倍。结论：构建了miR-455的慢病毒载体,并能在293T细胞中表达,产生的慢病毒能成功感染SiHa细胞。为进一步研究miR-455的功能,以及利用慢病毒进行基因治疗奠定了基础。     

miR-9靶向调控PTEN在HPV16+及HPV18+宫颈癌细胞凋亡中的作用

研究微小RNA-9(microRNA-9,miR-9)靶向调控人第10号染色体缺失的磷酸酶及张力蛋白同源蛋白（Recombinantphosphataseandtensinhomolog,PTEN）在人乳头瘤病毒（Humanpapillomavirus,HPV）16+及HPV18+宫颈癌细胞凋亡中的作用。收集宫颈癌组织及正常宫颈组织，培养HPV16+SiHa细胞、HPV18+HeLa细胞、HPV阴性C33A细胞及正常宫颈上皮H8细胞，检测miR-9、PTEN的表达水平。SiHa细胞和HeLa细胞进行分组转染后检测增殖抑制率、凋亡率、PTEN及miR-9表达水平。结果显示高危型HPV阳性及阴性的宫颈癌组织中miR-9的表达水平高于正常宫颈组织、PTEN的表达水平低于正常宫颈组织（P<0.05）且高危型HPV阳性宫颈癌组织中miR-9、PTEN表达的变化更显著；SiHa细胞、HeLa细胞中miR-9的表达水平高于C33A细胞和H8细胞、PTEN的表达水平低于C33A细胞和H8细胞（P<0.05）；敲低miR-9表达后，SiHa细胞、HeLa细胞的PTEN表达水平、增殖抑制率、凋...     

抑癌基因miR-195对口腔癌细胞CAL27生物学特性的影响

为了探究抑癌基因miR-195对体外培养的口腔癌细胞CAL27生物学特性的影响,本研究在口腔癌细胞CAL27中,通过质粒转染的方法,构建miR-195过表达和mimic空载体的CAL27细胞株,在用RT-PCR检测质粒构建成功的基础上,运用MTT法和Western blotting法分别检测过表达miR-195的人舌鳞癌细胞CAL27细胞增殖和凋亡的生物学特性。结果显示,过表达miR-195的CAL27细胞株构建成功(p0.05);与mimic空载体的CAL27细胞株相比,过表达miR-195的细胞株增殖能力明显下降(p0.05);与mimic空载体的CAL27细胞株相比,过表达miR-195的细胞株促凋亡蛋白Bax表达明显增加(p0.05),抗凋亡蛋白Bcl-2表达明显降低(p0.05)。本研究表明过表达miR-195可有效抑制口腔癌细胞CAL27的生物学特性。     

miR-429与肿瘤

miR-429是miR-200家族成员之一。研究表明,miR-429异常表达与肿瘤的发生、发展、转移、凋亡和耐药等密切相关。但miR-429在肿瘤中所起的作用一直有争议,可作为肿瘤抑制剂或促进剂,具肿瘤细胞/组织特异性。其在骨肉瘤、肾癌、卵巢癌、乳腺癌、宫颈癌、胶质瘤、口腔鳞状细胞癌、胃癌、食管癌、胰腺癌中起抑癌作用,而在肺癌、前列腺癌和子宫内膜癌中起促癌作用,但在结肠癌、肝癌、膀胱癌中的作用尚不明确。本文综述了近年来miR-429在肿瘤发生、发展中的作用及潜在的调控机制,为其作为肿瘤诊断、治疗及预后的潜在生物标记分子提供新的启示和参考。     

miR-182在前列腺癌组织中的表达及其对前列腺癌细胞增殖和凋亡的影响

目的:观察前列腺癌组织及不同前列腺癌细胞系中miR-182的表达,并探讨下调其表达对前列腺癌细胞增殖和凋亡的影响及机制。方法:采用实时荧光定量PCR(q RT-PCR)检测30例前列腺癌组织和30例相应的癌旁组织以及前列腺正常上皮RWPE-1细胞、前列腺癌PC-3、LNCa P和DU145细胞中miR-182的表达,进一步采用Lipfectamine 2000脂质体转染miRNA-182 inhibitor和阴性对照miRNA于PC-3细胞后,通过噻唑蓝(MTT)比色法检测细胞增殖情况,流式细胞术检测细胞凋亡率,免疫印迹(Western blot)法检测转录因子FOXO1、血管内皮生长因子(VEGF)和抑癌基因p53蛋白的表达。结果:miR-182在前列腺癌组织中的表达明显高于癌旁组织(P0.05);miR-182在前列腺癌细胞系PC-3、LNCa P和DU145中的表达均高于前列腺正常上皮细胞RWPE-1(P0.05),其中PC-3细胞中miR-182表达水平最高。转染miRNA-182 inhibitor至PC-3细胞成功下调miR-182表达后,细胞的增殖能力明显受到抑制,细胞凋亡能力明显增强,FOXO1表达水平显著升高,VEGF和p53的表达明显降低,差异均具有统计学意义(P0.05)。结论:miR-182在前列腺癌组织及细胞中呈高表达,下调miR-182的表达可能通过增加FOXO1的表达并减少VEGF和p53的表达,抑制前列腺癌细胞增殖并诱导细胞凋亡。     

DNA损伤诱导的长链非编码RNA-UCA1促进HeLa细胞增殖

尿路上皮癌抗原1 (UCA1)是一种长链非编码RNA,在多种肿瘤内高表达.然而,其在宫颈癌细胞和组织中的表达报告颇不一致,且功能尚未确定.本文探索UCA1在宫颈癌HeLa细胞中的生物学功能.实时定量PCR（qRT-PCR）结果显示,UCA1、p21和p53 mRNA在阿霉素（doxorubicin,DOX）或γ射线照射的HeLa细胞中表达上调|相反,敲减p53表达则可抑制DOX诱导的UCA1上调.表明DNA损伤诱导的UCA1可能与p53有关.转染结合CCK8检测HeLa细胞增殖活力结果显示,与对照比较,过表达UCA1促进HeLa细胞增殖,干扰UCA1表达则减缓细胞增殖.此外,流式细胞术结果显示,过表达UCA1导致阿霉素诱导的凋亡率下降;siRNA抑制UCA1表达后引起细胞G2/M期比例上升,S期下降,且阿霉素诱导的细胞凋亡率上升.上述结果说明,DNA损伤诱导的UCA1可促进HeLa细胞增殖,减少细胞凋亡.然而,是否DNA损伤诱导的UCA1上调依赖p53尚需进一步实验证明.     

miR-125a-5p通过靶向调控GAB2抑制乳腺癌细胞的迁移能力

miR-125a-5p可负性调节GAB2表达,抑制胶质瘤细胞的侵袭和转移。本研究旨在证明miR-125a-5p抑癌作用的普遍性,即miR-125a-5p是否可通过靶向抑制GAB2抑制乳腺癌细胞的迁移。荧光素酶实验结果显示,miR-125a-5p可特异识别GAB2的3′-UTR,抑制报告酶的表达。荧光定量PCR结果揭示,与正常乳腺上皮细胞MCF-10A比较,miR-125a-5p在乳腺癌细胞MDA231和MCF-7中的表达明显降低;与迁移能力相对较低的MCF-7细胞比较,miR-125a-5p在迁移能力较高的MDA231细胞中的表达量更低。Western 印迹结果证明,与空载体（对照）和anti-miR125a 5p转染细胞比较,转染miR-125a-5p明显抑制GAB2蛋白在乳腺癌细胞中的表达。Transwell结果显示,与空载体转染的对照细胞比较,转染miR-125a-5p的乳腺癌细胞穿过基质胶的细胞数明显减少;相反,转染anti-miR125a-5p的细胞穿过基质胶的细胞数却明显增多。上述结果提示,miR-125a-5p在正常的乳腺细胞中高表达,而在乳腺癌细胞中低表达,其表达水平与癌细胞的迁移能力和GAB2表达呈反向关系。本研究结果还提示,miR-125a-5p通过靶向负调控GAB2抑制乳腺癌细胞的迁移能力。总之,本研究证明,miR-125a-5p在肿瘤中发挥抑癌作用。     

Protective effect of bicyclol against pulmonary fibrosis via regulation of microRNA-455-3p in rats

Idiopathic pulmonary fibrosis (IPF), a chronic, progressive and irreversible disease, needs long-term treatment. Bicyclol was found to play a great role in pulmonary fibrosis, and the present study is to explore how bicyclol affects IPF with the involvement of microRNA-455-3p (miR-455-3p) and Bax. Bleomycin (BLM) was used to induce the IPF model in Sprague-Dawley rats to detect the expression of miR-455-3p, Bax, and B-cell lymphoma factor 2 (Bcl-2). Moreover, to further investigate the mechanisms of bicyclol, the BLM-induced fibrotic cell model was used after the lung epithelial cells HPAEpiC received miR-455-3p knockout treatment. The rats were then treated with vehicle and bicyclol, respectively. The apoptosis of fibrotic cells and Bax/Bcl-2 were identified. Inhibition function of bicyclol was optimal at a dose of 150 mg/kg. Bicyclol inhibited cell apoptosis and reduced Bax/Bcl-2 expression in rats. miR-455-3p could potentially bind to Bax gene. Bicyclol reduced the levels of methylenedioxyamphetamine, superoxide dismutase, and glutathione in rat lung tissue, inhibited the apoptosis of rats with IPF and upregulated miR-455-3p expression. In vitro studies showed that bicyclol significantly promoted miR-455-3p expression in HPAEpiC fibrosis. Bicyclol inhibited fibrosis-induced apoptosis of HPAEpiC in alveolar epithelial cells through promoting miR-455-3p, which inhibited Bax expression in IPF. Bicyclol may suppress the apoptosis of alveolar epithelial cells by upregulating miR-455-3p. This study laid a theoretical foundation for further understanding of IPF and searching for new molecular therapeutic targets.     

RP4 通过miR-183-5p.1上调FOXO1抑制乳腺癌细胞增殖

近年来,越来越多的证据表明,长非编码RNAs在肿瘤发生发展中发挥重要作用。位于12号染色体的长非编码RNA RP4-816N1.7（简称RP4）在乳腺癌细胞中的作用未见报道。我们通过实时荧光定量PCR证实,RP4在乳腺癌细胞中的表达量普遍低于其在正常乳腺上皮细胞MCF-10A中的表达量。RP4在MCF-7和MDA-MB-231中表达量分别比其在MCF-10A中的表达量下调21.57%和91.33%。过表达RP4可明显抑制乳腺癌细胞增殖。敲低RP4可显著增加乳腺癌细胞的增殖能力。生物信息学预测,RP4可能与miR-183-5p.1结合,且叉头蛋白O1(FOXO1)可能是miR-183-5p.1的潜在靶标。实时荧光定量PCR结果提示,RP4可下调miR-183-5p.1,而miR-183-5p.1也可下调RP4和FOXO1的表达。双荧光素酶报告基因结果证实,miR-183-5p.1可与RP4结合,下调其表达,也能与FOXO1 3′UTR结合,抑制其mRNA和蛋白质水平的表达量。最后,本文通过BrdU实验证实,RP4通过FOXO1抑制乳腺癌细胞的增殖。总之,RP4通过内源性结合miR-183-5p.1,上调FOXO1表达,进而抑制乳腺癌细胞增殖。     

MicroRNA-192 targeting retinoblastoma 1 inhibits cell proliferation and induces cell apoptosis in lung cancer cells

microRNAs play an important roles in cell growth, differentiation, proliferation and apoptosis. They can function either as tumor suppressors or oncogenes. We found that the overexpression of miR-192 inhibited cell proliferation in A549, H460 and 95D cells, and inhibited tumorigenesis in a nude mouse model. Both caspase-7 and the PARP protein were activated by the overexpression of miR-192, thus suggesting that miR-192 induces cell apoptosis through the caspase pathway. Further studies showed that retinoblastoma 1 (RB1) is a direct target of miR-192. Over-expression of miR-192 decreased RB1 mRNA and protein levels and repressed RB1-3'-UTR reporter activity. Knockdown of RB1 using siRNA resulted in a similar cell morphology as that observed for overexpression of miR-192. Additionally, RB1-siRNA treatment inhibited cell proliferation and induced cell apoptosis in lung cancer cells. Analysis of miRNA expression in clinical samples showed that miR-192 is significantly downregulated in lung cancer tissues compared to adjacent non-cancerous lung tissues. In conclusion, our results demonstrate that miR-192 is a tumor suppressor that can target the RB1 gene to inhibit cell proliferation and induce cell apoptosis in lung cancer cells. Furthermore, miR-192 was expressed at low levels in lung cancer samples, indicating that it might be a promising therapeutic target for lung cancer treatment.     

过表达miR-455促进肝癌细胞诱导的管型形成

目的:构建miR-455前体的真核表达载体,探讨其对肝癌细胞诱导管型形成的影响。方法:采用PCR扩增miR-455前体序列,通过双酶切将其连接到真核表达载体pcDNA3中。连接产物转化入大肠杆菌内进行扩增,采用菌落PCR、双酶切和测序鉴定重组子。将构建的质粒转染SMMC-7721细胞,通过real-time PCR检测成熟miR-455的表达,采用ELISA方法检测培养液上清中VEGF的表达。用该上清处理人脐静脉内皮细胞HUVEC后,检测管型形成的情况。结果:本实验成功构建了miR-455前体的真核表达载体,将其瞬时转染SMMC-7721细胞后,real-time PCR检测结果显示miR-455表达水平显著升高(P0.01)。过表达miR-455后,SMMC-7721细胞上清VEGF表达水平呈时间依赖性升高(24 h P0.05,48 h和72 h P0.01)。人脐静脉内皮细胞分别用转染pcDNA3和pcDNA3-pre-455的肝癌细胞培养液上清(72 h)重悬,接种于基质胶Matrigel上,转染pcDNA3-pre-455组形成典型的微管,管样结构明显完整。结论:过表达miR-455可促进肝癌细胞诱导的微管形成。     

lncRNA CEBPA-AS1靶向miR-455-3p调控胃癌细胞生物学行为的分子机制研究

摘要 目的：探讨lncRNA CEBPA-AS1对胃癌细胞生物学行为的影响及其可能作用机制。方法：qRT-PCR法检测胃癌组织、癌旁组织与正常人胃上皮GES1细胞和人胃癌SNU-1、AGS、HS-746T细胞系中lncRNA CEBPA-AS1、miR-455-3p的表达量。si-NC、si-lncRNA CEBPA-AS1、miR-NC、miR-455-3p mimics、si-lncRNA CEBPA-AS1与anti-miR-NC、si-lncRNA CEBPA-AS1与anti-miR-455-3p分别转染至SNU-1细胞（分别命名为si-NC组、si-lncRNA CEBPA-AS1组、miR-NC组、miR-455-3p组、si-lncRNA CEBPA-AS1+anti-miR-NC组和si-lncRNA CEBPA-AS1+anti-miR-455-3p组）后,MTT实验与平板克隆形成实验分别检测细胞增殖及克隆形成能力,Transwell小室实验检测细胞迁移及侵袭能力,双荧光素酶报告基因实验与qRT-PCR实验验证lncRNA CEBPA-AS1与miR-455-3p的靶向调控关系,Western blot法检测MMP2、MMP9蛋白表达情况。结果：与癌旁组织比较,胃癌组织中lncRNA CEBPA-AS1的表达量显著升高,miR-455-3p的表达量显著降低,差异均有统计学意义（均P＜0.05）。与GES1细胞比较,SNU-1、AGS、HS-746T细胞中lncRNA CEBPA-AS1的表达量显著升高,miR-455-3p的表达量显著降低,其中SNU-1细胞的lncRNA CEBPA-AS1表达量最高,差异均有统计学意义（均P＜0.05）。与si-NC组比较,si-lncRNA CEBPA-AS1组细胞活力降低,细胞克隆形成数、迁移及侵袭细胞数减少,MMP2、MMP9蛋白表达水平降低,差异均有统计学意义（P＜0.05）。与miR-NC组比较,miR-455-3p组细胞活力降低,细胞克隆形成数、迁移及侵袭细胞数减少,MMP2、MMP9蛋白表达水平降低,差异均有统计学意义（P＜0.05）。lncRNA CEBPA-AS1可靶向结合miR-455-3p,并可负调控miR-455-3p的表达。与si-lncRNA CEBPA-AS1+anti-miR-NC组比较,si-lncRNA CEBPA-AS1+anti-miR-455-3p组细胞活力升高,细胞克隆形成数、迁移及侵袭细胞数增多,MMP2、MMP9蛋白表达水平升高,差异均有统计学意义（P＜0.05）。结论：干扰lncRNA CEBPA-AS1表达可通过靶向调控miR-455-3p而抑制胃癌细胞增殖、克隆形成、迁移及侵袭。     

Long noncoding RNA LINC00958 regulates cell sensitivity to radiotherapy through RRM2 by binding to microRNA-5095 in cervical cancer

Long noncoding RNAs (lncRNAs) have been implicated in the regulation of resistance to radiotherapy in cervical cancer, which is a type of gynecological disease with high mortality in women around the world. Hence, our purpose is to delineate the involvement of LINC00958 in regulating cell sensitivity to radiotherapy in cervical cancer. LINC00958 expression in cervical cancer was assayed, followed by verification of the relationship among LINC00958, microRNA-5095 (miR-5095) and ribonucleotide reductase subunit M2 (RRM2). Hela cells were transduced with up-/downregulation of miR-5095 or RRM2, or LINC00958 silencing, respectively, and then treated with or without a 6 Gy dose of X-ray irradiation. Then the cell proliferation, apoptosis, survival fraction rate, as well as sensitivity to radiotherapy, were assessed. Finally, xenograft tumor in nude mice was established by transplanting Hela cells transfected with sh-LINC00958 and irradiated with 6 Gy of X-ray. High expression of LINC00958 was revealed in The Cancer Genome Atlas and Gene Expression Profiling Interactive Analysis, as well as in radiation-resistant patients, which was associated with lower sensitivity to radiotherapy in cervical cancer. Moreover, cervical cancer patients with higher LINC00958 expression exhibited a shorter overall survival according to Kaplan–Meier analysis. In addition, LINC00958 could regulate the expression of RRM2 by competing for miR-5095. A combination of radiotherapy with LINC00958 silencing, RRM2 downregulation or miR-5095 overexpression was found to inhibit cervical cancer cell proliferation and tumor growth, while promoting cell apoptosis both in vitro and in vivo. Collectively, our results suggest that LINC00958 could regulate RRM2 by competing to miR-5095, which regulates cell sensitivity to radiotherapy in cervical cancer.     

LBX2-AS1 promotes ovarian cancer progression by facilitating E2F2 gene expression via miR-455-5p and miR-491-5p sponging

LBX2-AS1 is a long non-coding RNA that facilitates the development of gastrointestinal cancers and lung cancer, but its participation in ovarian cancer development remained uninvestigated. Clinical data retrieved from TCGA ovarian cancer database and the clinography of 60 ovarian cancer patients who received anti-cancer treatment in our facility were analysed. The overall cell growth, colony formation, migration, invasion, apoptosis and tumour formation on nude mice of ovarian cancer cells were evaluated before and after lentiviral-based LBX2-AS1 knockdown. ENCORI platform was used to explore LBX2-AS1-interacting microRNAs and target genes of the candidate microRNAs. Luciferase reporter gene assay and RNA pulldown assay were used to verify the putative miRNA-RNA interactions. Ovarian cancer tissue specimens showed significant higher LBX2-AS1 expression levels that non-cancerous counterparts. High expression level of LBX2-AS1 was significantly associated with reduced overall survival of patients. LBX2-AS1 knockdown significantly down-regulated the cell growth, colony formation, migration, invasion and tumour formation capacity of ovarian cancer cells and increased their apoptosis in vitro. LBX2-AS1 interacts with and thus inhibits the function of miR-455-5p and miR-491-5p, both of which restrained the expression of E2F2 gene in ovarian cancer cells via mRNA targeting. Transfection of miRNA inhibitors of these two miRNAs or forced expression of E2F2 counteracted the effect of LBX2-AS1 knockdown on ovarian cancer cells. LBX2-AS1 was a novel cancer-promoting lncRNA in ovarian cancer. This lncRNA increased the cell growth, survival, migration, invasion and tumour formation of ovarian cancer cells by inhibiting miR-455-5p and miR-491-5p, thus liberating the expression of E2F2 cancer-promoting gene.