miR-29a levels are elevated in the db/db mice liver and its overexpression leads to attenuation of insulin action on PEPCK gene expression in HepG2 cells

MicroRNAs comprise a class of small (～22 nucleotide) non-coding RNA species and they bind to their complementary sequence on the 3'UTR of target genes and cause translational repression. In the present study, we report that miR-29a levels are significantly elevated in the diabetic db/db mice liver. Further, we report the effects of such elevation on insulin action in HepG2 cells. Overexpression of miR-29a narrowed down insulin mediated Akt phosphorylation without altering the total Akt levels presumably due to another upstream mediator being directly targeted by miR-29a. This hunt led us to the discovery that the p85α subunit of PI3K (phosphoionositide-3-kinase), the upstream molecule in the insulin signaling cascade harbors the miR-29a binding site on its 3'UTR and a marked inhibition of PI3Kp85α was observed by this microRNA. This was consequently accompanied by attenuation of insulin inhibition of PEPCK gene expression. All these events could be significantly prevented in the presence of the miR-29a inhibitor. Our results, for the first time, show the effect of miR-29a in counteracting insulin action on PEPCK gene expression by primarily targeting PI3K and abrogating downstream insulin signaling in HepG2 cells.     

Regulation of angiogenesis through a microRNA (miR-130a) that down-regulates antiangiogenic homeobox genes GAX and HOXA5

Angiogenesis is critical to tumor progression. The homeobox gene GAX inhibits angiogenesis in vascular endothelial cells (ECs). We have identified a microRNA (miR-130a) that regulates GAX expression and hypothesized that it plays a major role in modulating GAX activity in ECs. A 280-bp fragment from the GAX 3'-untranslated region (3'-UTR) containing 2 miR-130a targeting sites was observed to be required for the rapid down-regulation of GAX expression by serum and proangiogenic factors, whereas the activity of the GAX promoter did not vary with exposure to serum or proangiogenic factors. This same 280-bp sequence in the GAX 3'-UTR cloned into the psiCHECK2-Luciferase vector mediated serum-induced down-regulation of the reporter gene when placed 3' of it. Finally, forced expression of miR-130a inhibits GAX expression through this specific GAX 3'-UTR sequence. A genome-wide search for other possible miR-130a binding sites revealed an miR-130a targeting site in the 3'-UTR of the antiangiogenic homeobox gene HOXA5, the expression and antiangiogenic activity of which are also inhibited by miR-130a. From these data, we conclude that miR-130a is a regulator of the angiogenic phenotype of vascular ECs largely through its ability to modulate the expression of GAX and HOXA5.     

MiR-200a and miR-200b target PTEN to regulate the endometrial cancer cell growth in vitro

Objective:To study whether miR-200a and miR-200b target PTEN gene expression to regulate the endometrial cancer cell growth in vitro. Methods:Endometrial cancer cells ECC-1 were cultured and transfected with the miR-200a and miR-200b mimics and inhibitors as well as the negative control mimics and inhibitors,and then the cell proliferation activity as well as the expression of PTEN and downstream genes in cells was determined; after transfection of miR-200a and miR-200b mimics as well as PTEN-3'UTR luciferase report gene plasmids,the fluorescence activity of luciferase reporter gene was determined. Results:12 h,24 h and 48 h after transfection,the cell proliferation activity of miR-200a mimics group and miR-200b mimics group were significantly higher than those of NC mimics group while the cell proliferation activity of mi R-200 a inhibitor group and miR-200b inhibitor group were significantly lower than those of NC inhibitor group; 48 h after transfection,PTEN expression in cells and PTEN-3'UTR luciferase reporter gene fluorescence activity of miR-200 a mimics group and miR-200b mimics group were significantly lower than those of NC mimics group while p-PI3K and p-Akt expression were significantly higher than those of NC mimics group; PTEN expression in cells and PTEN-3'UTR luciferase reporter gene fluorescence activity of miR-200 inhibitor group and miR-200b inhibitor group were significantly higher than those of NC inhibitor group while p-PI3K and p-Akt expression were significantly lower than those of NC inhibitor group. Conclusion:miR-200 a and miR-200b can promote the endometrial cancer cell growth in vitro by targeted inhibition of PTEN gene expression.     

MicroRNA miR-24 inhibits erythropoiesis by targeting activin type I receptor ALK4

MicroRNAs have been suggested to modulate a variety of cellular events. Here we report that miR-24 regulates erythroid differentiation by influencing the expression of human activin type I receptor ALK4 (hALK4). Ectopic expression of miR-24 reduces the mRNA and protein levels of hALK4 by targeting the 3'-untranslated region of hALK4 mRNA and interferes with activin-induced Smad2 phosphorylation and reporter expression. Furthermore, miR-24 represses the activin-mediated accumulation of hemoglobin, an erythroid differentiation marker, in erythroleukemic K562 cells and decreases erythroid colony-forming and burst-forming units of CD34+ hematopoietic progenitor cells. ALK4 expression is inversely correlated with miR-24 expression during the early stages of erythroid differentiation, and the forced expression of miR-24 leads to a delay of activin-induced maturation of hematopoietic progenitor cells in liquid culture. Thus, our findings define a regulation mode of miR-24 on erythropoiesis by impeding ALK4 expression.     

微核糖核酸506在人结肠癌细胞中的靶基因预测和鉴定

目的 预测并验证微核糖核酸506(miR-506)的真实靶基因.方法 通过多个生物信息学软件对miR-506可能调控的靶基因进行预测,结合其生物学功能和前期基因芯片结果,确定候选靶基因.根据miR-506结合候选靶基因的3'UTR位点,构建相应的报告基因载体及突变体.将所构建的报告基因载体或突变体、β半乳糖苷酶对照报告酶载体和miR-506前体共转染至SW1116细胞中,24 h后检测荧光报告酶变化.结果 过氧化物酶体增殖物激活受体(PPAR)α和维甲酸受体(RXR)α皆为has-miR-506的可能靶基因,miR-506作用于PPARα基因3'UTR区可能存在3个位点,RXRa基因3'UTR区可能存在2个位点.针对于PPARα基因的三组报告基因载体中,位点7930-7936组变化最为明显[(2.68±0.55)倍],位点3547-3553组和位点8335-8341组的荧光报告酶表达变化为其突变体的(1.43±0.22)倍和(1.34±0.20)倍.针对于RXRa基因的两组报告基因载体的荧光报告酶变化则并不显著.结论 PPARα为miR-506的真实靶基因,而RXRα非miR-506的靶基因.     

miR-206在大鼠肺动脉高压模型中的表达和意义

目的 探讨miR-206在大鼠肺动脉高压（pulmonary hypertension，PH）模型中的表达和意义。 方法 构建肺动脉高压大鼠模型，按缺氧暴露时间分别标记为(1、7、14、21和28 d)组，并将常压常氧（FIO₂为0.21）作为对照组（n = 5），提取大鼠右下肺叶组织及外周血，实时定量PCR检测miR-206表达；分离大鼠原代肺动脉平滑肌细胞（pulmonary arterial smooth muscle cell，PASMC），以miR-206 mimics转染细胞并进行缺氧暴露，检测细胞增殖能力。生物信息学分析发现并选择缺氧诱导因子（hypoxia-inducible factor，HIF）-1α作为miR-206的候选靶基因，以双荧光素酶报告实验验证miR-206是否可直接调控HIF-1α，共转染miR-206mimics与HIF-1α过表达质粒，检测PASMC细胞增殖能力变化。 结果 与对照组相比，大鼠肺组织中miR-206的表达在缺氧暴露后即可出现miR-206表达降低（P < 0.05），大鼠血清miR-206亦明显降低（P < 0.05）；缺氧暴露后的大鼠肺组织分离培养的PASMC miR-206表达与对照组相比也明显降低（P < 0.05），miR-206低表达时PASMC增殖能力明显增加（P < 0.05）；miR-206 mimics转染可拮抗因缺氧暴露引起的PASMC增殖能力增加（P < 0.05）。生物信息学发现HIF-1α的3’非编码区（3’ untranslated region，3'UTR）具有miR-206的结合位点，用miR-206和HIF-1α（野生型3'UTR）共转染的PASMC中荧光素酶报告基因活性较对照组显著下调（P < 0.05），而miR-206 mimics和HIF-1α（突变型3'UTR）共转染组则较对照组无明显统计学差异。共转染miR-206 mimics后，HIF-1α上调引起的细胞增殖被逆转（P < 0.05）。 结论 缺氧诱导的miR-206下调通过靶向PASMC中的HIF-1α途径来促进PH，miR-206可能是缺氧诱导PH早期的触发因素。     

A miR-24 microRNA binding-site polymorphism in dihydrofolate reductase gene leads to methotrexate resistance

MicroRNAs are predicted to regulate approximately 30% of all human genes by targeting sequences in their 3' UTR. Polymorphisms in 3' UTR of several genes have been reported to affect gene expression, but the mechanism is not fully understood. Here, we demonstrate that 829C-->T, a naturally occurring SNP, near the miR-24 binding site in the 3' UTR of human dihydrofolate reductase (DHFR) affects DHFR expression by interfering with miR-24 function, resulting in DHFR overexpression and methotrexate resistance. miR-24 has a conserved binding site in DHFR 3' UTR. DHFR with WT and 3' UTR containing the 829C-->T mutation were expressed in DG44 cells that lack DHFR. Overexpression of miR-24 in cells with WT DHFR resulted in down-regulation of DHFR protein, whereas no effect on DHFR protein expression was observed in the mutant 3' UTR-expressing cells. Inhibition of endogenous miR-24 with a specific inhibitor led to up-regulation of DHFR in WT and not in mutant cells. Cells with the mutant 3' UTR had a 2-fold increase in DHFR mRNA half-life, expressed higher DHFR mRNA and DHFR protein, and were 4-fold more resistant to methotrexate as compared with WT cells. SNP-829C-->T, therefore, leads to a decrease in microRNA binding leading to overexpression of its target and results in resistance to methotrexate. We demonstrate that a naturally occurring miRSNP (a SNP located at or near a microRNA binding site in 3' UTR of the target gene or in a microRNA) is associated with enzyme overproduction and drug resistance.     

人乳腺癌上皮细胞miR-217对EZH2表达的调控作用

目的 验证乳腺癌上皮细胞中miR-217是否通过直接作用于homolog2 zeste基因增强子（EZH2）3＇UTR调控EZH2表达.方法 利用RT-PCR技术检测人正常乳腺上皮细胞系HBL-100及乳腺癌上皮细胞系MCF-7、MDA-MB-231细胞株中的miR-217,在低表达miR-217细胞株中转染miR-217模拟物（miR-217 minics）,在高表达miR-217细胞株中转染miR-217抑制剂（miR-217 inhibitor）;Western blot法检测各实验细胞中的EZH2蛋白.用含有miR-217野生型及突变型识别位点的EZH2 3＇UTR载体（UTREZH2-UTR-WT和EZH2-UTR-MUT）质粒分别转染人胚肾细胞293T,检测其相对荧光素酶活性.结果 miR-217在MDA-MB-231细胞中低表达,转染miR-217 minics后,其EZH2蛋白表达下降;miR-217在HBL-100中高表达,转染miR-217 inhibitor后,其EZH2蛋白表达增高.人胚肾细胞293T中EZH2-UTR-WT的相对荧光素酶活性低于EZH2-UTR-MUT,两者相比,P＜0.05.结论 在乳腺癌上皮细胞中,miR-217对EZH2的表达具有调控作用,且miR-217通过直接靶向EZH2 3＇UTR调控EZH2表达.     

Tumor-suppressor p53 specifically binds to miR-29c-3p and reduces ADAM12 expression in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is an extremely aggressive malignant tumor associated with high migratory and invasive potential. The present study intends to explore regulatory mechanism of p53/microRNA (miR)-29c-3p/A disintegrin and metalloproteinase 12 (ADAM12) axis in HCC based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology.MethodsPutative miR-29c-3p binding sites on ADAM12 3′UTR were verified by a luciferase assay. The binding affinity of p53 to miR-29c-3p was assessed based on CRISPR/Cas9 technology to construct a p53 knockout (p53^?/?) HCCLM3 cell line. Furthermore, the effect of p53/miR-29c-3p/ADAM12 was assessed on maligant phenotypes in vitro and tumor formation and metastasis in nude mice.ResultsADAM12 was highly expressed but miR-29c-3p was poorly expressed in HCC. miR-29c-3p inhibited migratory and invasive abilities of HCC cells by targeting ADAM12 expression. p53 was found to target and upregulate miR-29c-3p, thus downregulating ADAM12 and conferring inhibitory effect on HCC cell activities. Moreover, ADAM12 knockout or p53 overexpression reduced HCC tumor formation and metastasis, which were reversed by further silencing of miR-29c-3p.ConclusionThe identification of the p53/miR-29c-3p/ADAM12 axis in migration and invasion of HCC may potentially further our understanding of mechanisms underpinning HCC, and also bear translational value as novel molecular targets.     

Functional SNP in the microRNA-367 binding site in the 3'UTR of the calcium channel ryanodine receptor gene 3 (RYR3) affects breast cancer risk and calcification

We have evaluated and provided evidence that the ryanodine receptor 3 gene (RYR3), which encodes a large protein that forms a calcium channel, is important for the growth, morphology, and migration of breast cancer cells. A putative binding site for microRNA-367 (miR-367) exists in the 3'UTR of RYR3, and a genetic variant, rs1044129 A→G, is present in this binding region. We confirmed that miR-367 regulates the expression of a reporter gene driven by the RYR3 3'UTR and that the regulation was affected by the RYR3 genotype. A thermodynamic model based on base pairing and the secondary structure of the RYR3 mRNA and miR-367 miRNA showed that miR-367 had a higher binding affinity for the A genotype than for the G genotype. The rs1044129 SNP was genotyped in 1,532 breast cancer cases and 1,600 healthy Chinese women. The results showed that compared with the AA genotype, G was a risk genotype for breast cancer development and was also associated with breast cancer calcification and poor survival. Thus, rs1044129 is a unique SNP that resides in a miRNA-gene regulatory loop that affects breast cancer risk, calcification, and survival.     

miR-140靶向调控SOX2在结肠癌中的作用机制研究

目的 分析miR-140在结肠癌中的作用机制.方法 qRT-PCR检测miR-140在结肠癌细胞和组织中的表达;CCK8法、细胞划痕实验检测miR-140对结肠癌HT29细胞增殖和迁移的影响.TargetScan筛选miR-140的潜在靶基因并通过双荧光素酶报告基因试验进行验证;采用qRT-PCR和Western bl...     

miR-519 reduces cell proliferation by lowering RNA-binding protein HuR levels

Gene expression is potently regulated through the action of RNA-binding proteins (RBPs) and microRNAs (miRNAs). Here, we present evidence of a miRNA regulating an RBP. The RBP HuR can stabilize and modulate the translation of numerous target mRNAs involved in cell proliferation, but little is known about the mechanisms that regulate HuR abundance. We identified two putative sites of miR-519 interaction on the HuR mRNA, one in its coding region (CR), one in its 3′-untranslated region (UTR). In several human carcinoma cell lines tested, HeLa (cervical), HCT116 and RKO (colon), and A2780 (ovarian), overexpression of a miR-519 precursor [(Pre)miR-519] reduced HuR abundance, while inhibiting miR-519 by using an antisense RNA [(AS)miR-519] elevated HuR levels. The influence of miR-519 was recapitulated using heterologous reporter constructs that revealed a greater repressive effect on the HuR CR than the HuR 3′-UTR target sequences. miR-519 did not alter HuR mRNA abundance, but reduced HuR biosynthesis, as determined by measuring nascent HuR translation and HuR mRNA association with polysomes. Modulation of miR-519 leading to altered HuR levels in turn affected the levels of proteins encoded by HuR target mRNAs. In keeping with HuR's proliferative influence, (AS)miR-519 significantly increased cell number and [³H]-thymidine incorporation, while (Pre)miR-519 reduced these parameters. Importantly, the growth-promoting effects of (AS)miR-519 required the presence of HuR, because downregulation of HuR by RNAi dramatically suppressed its proliferative action. In sum, miR-519 represses HuR translation, in turn reducing HuR-regulated gene expression and cell division.     

构建psiCHECK-2-解耦联蛋白2双荧光素酶报告基因及其荧光素酶活性的分析

目的:构建含解耦联蛋白2(UCP2)-3’非翻译区(UTR)序列的野生型和突变型质粒的双荧光素酶报告基因载体,探讨微小RNA(miR)-15b对UCP2基因表达的调控作用。方法:应用生物信息软件预测miR-15b的靶基因,分别将预测靶基因UCP2的3’UTR及其突变体克隆到荧光素酶载体psiCHECK-2骨架中,构建UCP2野生型和突变型质粒。并采用测序方法鉴定psiCHECK-2-UCP2载体是否构建成功。将UCP2野生型和突变型质粒分别与miR-15b模拟物、miR-15b模拟物正常对照、miR-15b抑制剂、miR-15b抑制剂正常对照在293T细胞中共转染。通过双荧光素酶报告基因检测分析miR-15b对UCP2基因表达的调控作用。结果:测序鉴定证实psiCHECK-2-UCP2双荧光素酶报告基因载体构建成功。双荧光素酶报告基因检测显示转染UCP2野生型和UCP2突变型报告基因的293T细胞过表达miR-15b后,UCP2野生型报告基因的荧光素酶活性明显下降,下调31%(P=0.003),过表达miR-15b抑制剂后,UCP2野生型报告基因的荧光素酶活性明显增加,上调46%(P=0.01)。而miR-15b模拟物、miR-15b模拟物正常对照、miR-15b抑制剂、miR-15b抑制剂正常对照对UCP2突变型的表达均无明显影响(P>0.05)。结论:UCP2是miR-15b直接作用的靶基因,且miR-15b结合于UCP2基因3’UTR区域,转录后水平对UCP2有直接的抑制作用。