LncRNA HIF1A-AS2 accelerates malignant phenotypes of renal carcinoma by modulating miR-30a-5p/SOX4 axis as a ceRNA

Objective: Several reports have proposed that lncRNAs, as potential biomarkers, participate in the progression and growth of malignant tumors. HIF1A-AS2 is a novel lncRNA and potential biomarker, involved in the genesis and development of carcinomas. However, the molecular mechanism of HIF1A-AS2 in renal carcinoma is unclear.Methods:The relative expression levels of HIF1A-AS2 and miR-30a-5p were detected using RT-qPCR in renal carcinoma tissues and cell lines. Using loss-of-function and overexpression, the biological effects of HIF1A-AS2 and miR-30a-5p in kidney carcinoma progression were characterized. Dual luciferase reporter gene analysis and Western blot were used to detect the potential mechanism of HIF1A-AS2 in renal carcinomas.Results:HIF1A-AS2 was upregulated in kidney carcinoma tissues when compared with para-carcinoma tissues (P < 0.05). In addition, tumor size, tumor node mestastasis stage and differentiation were identified as being closely associated with HIF1A-AS2 expression (P < 0.05). Knockdown or overexpression of HIF1A-AS2 either restrained or promoted the malignant phenotype and WNT/β-catenin signaling in renal carcinoma cells (P < 0.05). MiR-30a-5p was downregulated in renal cancers and partially reversed HIF1A-AS2 functions in malignant renal tumor cells. HIF1A-AS2 acted as a microRNA sponge that actively regulated the relative expression of SOX4 in sponging miR-30a-5p and subsequently increased the malignant phenotypes of renal carcinomas. HIF1A-AS2 showed a carcinogenic effect and miR-30a-5p acted as an antagonist of the anti-oncogene effects in the pathogenesis of renal carcinomas.Conclusions:The HIF1A-AS2-miR-30a-5p-SOX4 axis was associated with the malignant progression and development of renal carcinoma. The relative expression of HIF1A-AS2 was negatively correlated with the expression of miR-30a-5p, and was closely correlated with SOX4 mRNA levels in renal cancers.     

miR-211对高糖诱导下晶状体上皮细胞氧化应激反应的调控作用

目的 探讨miR-211对高糖诱导下晶状体上皮细胞氧化应激反应的调控作用及其可能存在的作用机制.方法 将HLEC-B3细胞在含不同浓度(0、10、20、40 mmol/L)葡萄糖的培养基中培养48 h,RT-PCR检测细胞miR-211表达,Western blot检测细胞SIRT1蛋白表达,试剂盒检测总抗氧化能力(T...     

miR-146b对急性呼吸窘迫综合征大鼠肺组织中ICAM-1表达的调控作用

目的:探讨miR-146b对急性呼吸窘迫综合征(ARDS)大鼠肺组织中细胞间黏附分子1(ICAM-1)表达的影响,阐明miR-146b治疗ARDS的分子机制.方法:采用尾静脉注射油酸法建立ARDS大鼠模型,并将建模成功的大鼠分为模型组(只给予油酸)、agomir阴性对照组和miR-146b agomir组,每组15只,...     

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.     

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.     

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。     

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。