靶向PRMT2基因的micro RNA对雌激素介导的乳腺癌MCF7细胞增殖的影响

目的:构建靶向人蛋白质精氨酸甲基转移酶 2 (protein arginine methyltransferase2,PRMT2)基因的microRNA真核表达载体,鉴定其稳定转染乳腺癌细胞株MCF7后对细胞增殖的影响。方法:根据PRMT2mRNA序列设计合成pre-microRNA片段,定向克隆至pcDNATM6.2-GW/EmGFP-miR真核表达载体,并稳定转染入MCF7细胞。采用间接免疫荧光法和蛋白质印迹法检测重组体对PRMT2表达的干扰效果以确定其生物活性。采用结晶紫实验和平板克隆形成实验检测重组体转染对MCF7细胞体外增殖和细胞克隆形成能力的影响。FCM法检测重组体转染对MCF7细胞周期的影响。结果:构建的重组体插入片段的碱基序列完全正确,并且重组体稳定转染MCF7细胞后可成功干扰PRMT2基因的表达。在无处理因子的情况下,重组体转染对细胞的生长速度无明显影响;与转染空载体的MCF7细胞相比,稳定转染重组体的MCF7细胞对雌激素的敏感性增强,但其对雌激素拮抗剂4-OHT处理的敏感性无明显差异。平板克隆形成实验表明,重组体能够增强雌激素介导的MCF7细胞的克隆形成能力。FCM法检测表明,转染重组体的MCF7细胞中G2期细胞比例明显升高(P<0.05)。结论:成功构建靶向PRMT2基因的pcDNATM6.2-GW/EmGFP-miR真核表达载体,且在稳定转染MCF7细胞后具有生物学活性。抑制内源性PRMT2基因的表达能够提高雌激素介导的MCF7细胞的增殖和克隆形成能力,上调雌激素受体α目标基因cyclinD1和c-myc的表达,促进细胞周期进程。     

miR-16表达载体的构建

目的构建miR-16表达载体,为研究miR-16对HBV的干扰作用打下基础。方法根据miRNA的成熟序列以及附近约100多碱基共270个碱基序列,设计PCR引物,PCR扩增,退火后用T4联接到线性化的pSUPER质粒中,并对重组质粒（命名为pmiR-16）进行酶切及测序鉴定。结果成功构建了重组质粒PmiR-16。结论成功构建miR-16表达载体。     

miR-34a靶向调控Akt/Bcl-2对乳腺癌细胞多柔比星耐药性的影响

目的：探讨miR-34a通过下调AKT/BCL2信号通路对乳腺癌细胞多柔比星耐药性的影响及其分子机制?方法：通过实时定量PCR法检测miR-34a-3p在乳腺癌细胞（MCF-7）和乳腺耐药细胞株（MCF-7/ADR）中的表达,并成功构建miR-34a mimics/inhibitor调控其表达水平;通过CCK8法分别筛选多柔比星处理MCF-7及MCF-7/ADR细胞的IC50值并处理细胞;使用CCK8,流式细胞技术以及免疫印迹实验验证在miR-34a过表达及干扰组中,乳腺癌细胞存活率,凋亡细胞百分比以及AKT/BCL2信号通路的改变。结果：miR-34a在MCF-7/ADR中的表达显著低MCF-7细胞,同时成功构建miR-34a过表达及敲减模型;多柔比星处理MCF-7及MCF-7/ADR细胞的IC50分别为0.89 μg/mL、13.61 μg/mL。当MCF-7及MCF-7/ADR细胞中miR-34a表达水平降低时,经多柔比星处理后,细胞存活率显著升高（P<0.05）,凋亡细胞比例显著减少（P<0.01）,同时下游AKT/BCL2信号表达上调（P<0.01）。而当MCF-7/ADR细胞中miR-34a表达升高时相应的观察到相反的细胞表型。结论：miR-34a在乳腺癌细胞中的表达下调,可能通过减少对AKT/BCL2信号的负向调控,减少细胞凋亡,进而增强细胞对多柔比星的耐药性。     

皮肤恶性肿瘤microRNA的研究进展

MicroRNA(miRNA)分子广泛参与调控机体细胞的增生、分化、代谢、凋亡等生理病理活动,通过各种复杂的机制调控肿瘤的发生和发展。近来研究发现,皮肤肿瘤中存在miRNA的异常表达,提示miRNA亦参与皮肤肿瘤的发生和发展过程,同时人们对二者的关系及其具体机制进行了研究。该文对基底细胞癌(basal cell carcinoma,BCC)、鳞状细胞癌(squamous cell carcinoma,SCC)、恶性黑素瘤(malignant melanoma,MM)miRNA研究进展作一概述。     

Inhibition of miR-93 promotes interferon effector signaling to suppress influenza A infection by upregulating JAK1

Type I interferons play a critical role in host defense against influenza virus infection. Interferon cascade induces the expression of interferon-stimulated genes then subsequently promotes antiviral immune responses. The microRNAs are important regulators of innate immunity, but microRNAs-mediated regulation of interferon cascade during influenza infection remains to be fully identified. Here we found influenza A virus (IAV) infection significantly inhibited miR-93 expression in alveolar epithelial type II cells through RIG-I/JNK pathway. IAV-induced downregulation of miR-93 was found to upregulate JAK1, the target of miR-93, and then feedback promote antiviral innate response by facilitating IFN effector signaling. Importantly, in vivo administration of miR-93 antagomiR markedly suppressed IAV infection, protecting mice form IAVs -associated death. Hence, the inducible downregulation of miR-93 feedback suppress IAV infection by strengthening IFN-JAK-STAT pathway via JAK1 upregulation, and in vivo inhibition of miR-93 bears considerable therapeutic potential for suppressing IAV infection.     

Long Noncoding RNA 3632454L22Rik Contributes to Corneal Epithelial Wound Healing by Sponging miR-181a-5p in Diabetic Mice

PurposeThis work explores the abnormal expression of long noncoding RNAs (lncRNAs), microRNAs (miRNAs) and messenger RNAs (mRNAs) in diabetic corneal epithelial cells (CECs) and constructs an associated competitive endogenous RNA (ceRNA) network. Moreover, we revealed that Rik may exert advantageous effects on diabetic corneal epithelial wound closure by sponging miR-181a-5p.MethodsWe obtained the profiles of differentially expressed lncRNAs (DELs) of CECs of type 1 diabetic versus control corneas by microarray and summarized the differentially expressed miRNAs (DEmiRs) and differentially expressed genes (DEGs) data by published literature. Subsequently, the ceRNA network was constructed using bioinformatics analyses. The levels of lncRNA ENSMUST00000153610/3632454L22Rik (Rik) and miR-181a-5p were verified. The localization of Rik was identified with fluorescence in situ hybridization (FISH), and dual-luciferase assays proved the targeted relationship between Rik and miR-181a-5p. Furthermore, we validated the functional impact of Rik in vitro.ResultsOverall, 111 upregulated and 117 downregulated DELs were detected in diabetic versus control CECs. The level of Rik located in both the cytoplasm and the nucleus was clearly downregulated, whereas miR-181a-5p was upregulated in vitro and in vivo in the diabetic group versus the control group. Rik can act as a ceRNA to bind to miR-181a-5p, thus promoting diabetic corneal epithelial wound healing in vitro.ConclusionsThis work investigated the expression profile of DELs and constructed ceRNA networks of diabetic CECs for the first time. Furthermore, we revealed that Rik may positively impact diabetic corneal epithelial wound healing by sponging miR-181a-5p, providing a novel potential therapeutic target of diabetic keratopathy (DK).     

超声微泡介导微RNA治疗肝细胞癌的研究进展

基因治疗正逐渐成为肝细胞癌(HCC)、尤其晚期HCC的治疗方案.足够剂量的特定微RNA(miRNA)可抑制HCC细胞生长,超声微泡介导基因和药物递送治疗策略可促使miRNA于肿瘤区域靶向释放,从而达到治疗效果.本文对超声微泡介导miRNA治疗HCC研究进展进行综述.