The homeostatic malfunction of a novel feedback pathway formed by lncRNA021545, miR-330-3p and epiregulin contributes in hepatocarcinoma progression via mediating epithelial-mesenchymal transition

A better understanding of tumor metastasis is urgently required for the treatment and prognosis of hepatocarcinoma patients. Current work contributes a novel ceRNA feedback regulation pathway composed of epiregulin (EREG), microRNA-330-3p (miR-330-3p) and long non-coding RNA 021545 (lncRNA021545) in regulating hepatocarcinoma malignancy via epithelial-mesenchymal transition (EMT) process. Closely correlated, the deficiencies of EREG and lncRNA021545 and the overexpression of miR-330-3p were involved in the clinical progression of hepatocarcinoma. In vitro results showed that 1) lncRNA021545 downregulation promoted, 2) miR-330-3p dysexpression positively correlated, and 3) EREG dysexpression reversely correlated with the migratory and invasive properties of hepatocarcinoma HCCLM3 and Huh7 cell lines. By directly binding to EREG and lncRNA021545, miR-330-3p expression change reversely correlated with their expressions in HCCLM3 and Huh7 cells, which was also confirmed in primary tumors from HCCLM3-xenograft mice in responding to miR-330-3p change. LncRNA021545 and EREG positively regulated each other, and lncRNA021545 negatively regulated miR-330-3p, while, EREG dysregulation unchanged miR-330-3p expression in hepatocarcinoma cells. Furthermore, systemic in vitro cellular characterizations showed that the malfunctions of the three molecules mediated the invasiveness of hepatocarcinoma cells via EMT process through affecting the expressions of E-cadherin, N-cadherin, vimentin, snail and slug, which was further confirmed by in vivo miR-330-3p promotion on the tumorigenicity and metastasis of HCCLM3 bearing nude mice and by in vitro miR-330-3p promotion on the migration and invasion of hepatocarcinoma cells to be antagonized by EREG overexpression through acting on EMT process. Our work indicates, that by forming a circuit signaling feedback pathway, the homeostatic expressions of lncRNA021545, miR-330-3p and EREG are important in liver health. Its collapse resulted from the downregulations of lncRNA021545 and EREG together with miR-330-3p overexpression promote hepatocarcinoma progression by enhancing the invasiveness of tumor cells through EMT activation. These discoveries suggest that miR-330-3p/lncRNA021545/EREG axis plays a critical role in hepatocarcinoma progression and as a candidate for its treatment.     

替罗非班通过上调miR-22保护ox-LDL诱导的脐静脉内皮细胞损伤

目的 探讨替罗非班对氧化型低密度脂蛋白(ox-LDL)诱导的脐静脉内皮细胞(EA.hy926)损伤的影响和可能机制.方法 采用低、中、高剂量的替罗非班作用于ox-LDL诱导的EA.hy926细胞,采用细胞计数法(CCK-8)、流式细胞术分别检测细胞活力和凋亡.实时荧光定量PCR(qRT-PCR)检测miR-22表达水平...     

干扰lncRNA RHPN1-AS1表达通过靶向miR-339-5p对肝癌细胞增殖和凋亡的影响

目的:探讨长链非编码RNA PCNA1(lncRNA RHPN1)反义AS1(RHPN1-AS1)对肝癌细胞增殖和凋亡的影响及其作用机制.方法:将si-NC(lncRNA RHPN1-AS1阴性对照组)、si-RHPN1-AS1(沉默lncRNA RHPN1-AS1组)、pcDNA(真核表达载体组)、pcDNA-RHP...     

miR-221与miR-222通过靶基因TIMP3调控胶质瘤细胞侵袭能力

目的 探讨miR-221和miR-222在胶质瘤细胞侵袭过程中的作用及其机制.方法 采用反义寡核苷酸下调miR-221和miR-222表达,Transwell、Western blot、荧光素酶实验及体内实验分别检测细胞侵袭能力、体内肿瘤生长、相关基因蛋白表达变化及靶基因鉴定.结果 下调miR-221和miR-222表达能明显抑制胶质瘤细胞侵袭能力,同时相关侵袭蛋白MMP2和MMP9表达下降.miRNA靶基因预测软件分析、Western blot、荧光素酶实验证实TIMP3是miR-221和miR-222的靶基因.裸鼠皮下肿瘤模型显示下调miR-221和miR-222表达抑制体内肿瘤生长.免疫组化发现TIMP3表达上调,MMP2和MMP9表达下调.结论 在胶质瘤细胞中,侵袭相关蛋白TIMP3是miR-221和miR-222的一个新靶基因.

Abstract：

Objective To investigate the role and mechanism of miR -221 and miR -222 in glioma cell invasion. Method After reduction of miR -221 and miR -222 by antisense oligonucleotides, cell invasion,invasion related - gene expression and target identification were determined by Transwell assay, Western blot analysis and luciferase reporter assay,respectively. Moreover,the effects of miR -221 and miR -222 on xenograft tumors in nude mice were also observed. Results Down - regulation of miR - 221 and miR - 222 decreased glioma cell invasion in vitro, and inhibited glioma growth in a subcutaneous mouse model. Furthermore,down -regulation of miR -221 and miR - 222 resulted in obvious inactivation of MMP2 and MMP9. Western blot analysis and luciferase reporter assay showed that the reduction of miR - 221 and miR -222 repressed TIMP3 protein expression and TIMP3 mRNA 3'UTR existed the biding sites of miR -221 and 222. Conclusions TIMP3 is a novel target of miR -221 and miR -222 in glioma cell invasion.     

人脑胶质瘤中miR-125b和Gli1的表达及临床意义

目的探讨miR-125b在胶质瘤中的表达及其与Gli1的关系。方法收集不同病理级别胶质瘤标本25例,以荧光定量PCR方法检测miR-125b和Gli1的表达,分析miR-125b和Gli1表达水平与胶质瘤病理级别及预后的关系。结果①miR-125b在各级别胶质瘤[星形细胞瘤(0.754±0.085)、间变星形细胞瘤(0.545±0.075)和胶质母细胞瘤(0.340±0.056)]中的表达较正常脑组织(1.000)下调(均P0.05),miR-125b的表达水平与肿瘤的恶性程度呈负相关;②Gli1表达水平在胶质瘤中[星形细胞瘤(1.034±0.093)、间变星形细胞瘤(1.203±0.113)和胶质母细胞瘤(1.578±0.087)]较正常脑组织(1.000)略高,肿瘤的恶性程度与Gli1表达水平呈负相关,间变星形细胞瘤与胶质母细胞瘤比较,差异有统计学意义(P0.05);③miR-125b低表达和Gli1过表达与患者的预后呈负相关。结论 miR-125b低表达和Gli1过表达与恶性胶质瘤的发生、发展密切相关。     

反义miR-21通过调控hTERT表达抑制胶质瘤细胞生长

目的探讨敲低miR-21表达对人胶质瘤细胞系U87细胞功能的影响以及相关作用机制。方法脂质体介导转染miR-21反义寡聚核苷酸（miR-21 inhibitor）敲低U87细胞miR-21的表达。使用实时荧光定量PCR鉴定转染后U87细胞miR-21表达水平;MTT法检测转染后细胞增殖水平,流式法评价转染后细胞周期分布及凋亡变化,并结合Western印迹及RT-PCR验证在U87细胞中miR-21和hTERT间关系。结果体外转染反义miR-21寡聚核苷酸能明显抑制U87细胞生长,诱导其凋亡,并且能够明显下调hTERT表达。结论反义miR-21可能通过下调hTERT表达抑制胶质细胞生长,miR-21可作为胶质瘤基因治疗的靶点。     

儿童原发免疫性血小板减少症miR-106b-5p的表达及其与T细胞的相关性研究

目的探讨血浆miR-106b-5p在原发免疫性血小板减少症（primary immune thrombocytopenia，ITP）中的表达及其与辅助性T细胞17（T helper cells 17，Th17）、调节性T细胞（T regulatory cell，Treg）、Th17/Treg的相关性。方法选取79例ITP患儿（ITP组）和40例健康儿童（对照组）为研究对象。79例ITP患儿根据治疗效果分为完全有效组（40例）、有效组（18例）、无效组（21例）。采用实时荧光定量PCR技术检测miR-106b-5p表达水平，流式细胞技术检测Th17和Treg，计算Th17/Treg，分析血浆miR-106b-5p表达水平与Th17、Treg及Th17/Treg的相关性。结果ITP组miR-106b-5p、Th17及Th17/Treg水平高于对照组（P<0.05），Treg水平低于对照组（P<0.05）。ITP组患儿治疗后miR-106b-5p、Th17、Th17/Treg水平低于治疗前（P<0.05），Treg水平高于治疗前（P<0.05）。完全有效组miR-106b-5p、Th17、Th17/Treg水平低于有效组和无效组（P<0.05），Treg水平高于有效组和无效组（P<0.05）。相关分析显示，ITP患儿血浆miR-106b-5p表达水平与Th17、Th17/Treg均呈正相关（分别r=0.730、0.816，均P<0.001），与Treg呈负相关（r=-0.774，P<0.001）。结论ITP患儿存在miR-106b-5p高表达和Th17/Treg比例失衡，在ITP患儿治疗过程中检测miR-106b-5p、Th17、Treg及Th17/Treg水平，对ITP患儿的疗效判断具有较好的指导作用。     

姜黄素下调miR-155抑制缺血性脑卒中炎性应答的实验研究

目的 分析姜黄素对缺血性脑卒中的抗炎作用,并探讨miR-155、Toll样受体4(TLR4)信号通路是否参与该抗炎机制. 方法 线栓法建立Sprague Dawley大鼠大脑中动脉闭塞模型(MCAO),将大鼠分为假手术组(8例)、MCAO模型组(8例)、姜黄素治疗组(8例),检测大鼠梗死体积、miR-155 mRNA相...     

miR-128真核表达载体的构建及功能的初步研究

目的构建miR-128真核表达载体,研究其在神经胶质瘤细胞株U343表达及对U343细胞增殖的影响。方法以人神经胶质瘤细胞株U343的DNA为模板扩增miR-128-1和miR-128-2的前体序列,将人miR-128-1(hsamiR128-1)和人miR-128-2(hsa-miR128-2)基因克隆到真核表达载体pCR3.1,将pCR3.1-miR-128-1(p-128-1)和pCR3.1-miR-128-2(p-128-2)表达载体瞬时转染U343细胞,采用Northern印迹检测成熟的miR-128表达。将U343细胞接种96孔板,MTT法检测过表达miR-128对细胞增殖的影响。结果p-128-1和p-128-2表达载体经酶切及测序鉴定正确;二者转染细胞后,反转录-聚合酶链反应(RT-PCR)扩增结果显示,其能有效表达miR-128的前体;Northern印迹结果均能产生成熟态的miR-128。MTT检测结果在无血清培养和维甲酸处理的情况下,细胞的增殖能力明显下降。结论成功构建了p-128-1和p-128-2的真核表达载体,转染神经胶质瘤细胞后能有效表达,miR-128基因过表达能抑制U343细胞的增殖。     

miR-195在乳腺癌患者血浆中的表达及其临床意义

目的：探讨mtR-195在乳腺浸润性导管癌患者术前、术后2周血浆中的表达及其与乳腺癌临床病理特征的相关性。方法：以miR-16为内参,采用茎环RT-qPCR方法检测48例术前、术后2周乳腺浸润性导管癌患者及35例健康对照者血浆中miR-195的表达,并分析其表达与乳腺浸润性导管癌临床病理指标的关系。结果：乳腺浸润性导管癌患者血浆中miR-195的表达较健康对照者明显升高（P〈0．05）,术后2周表达明显降低至健康对照者水平。ROC曲线显示,血浆miR-195评价乳腺浸润性导管癌的敏感性和特异性可达94．4％和68．8％。与临床特征相关性分析统计未见血浆miR-195表达与乳腺浸润性导管癌肿块大小、雌激素受体（ER）、孕激素受体（PR）、人类表皮生长因子受体-2（Her-2）状态及淋巴结转移状态等临床病理特征有明显相关（P〉0．05）。结论：血浆中miR-195的异常表达可作为乳腺癌诊断的新的肿瘤标志物。