miR-193 regulates proliferation of rat marrow mesenchymal stem cells by cell cycle-related proteins

AIM: To investigate the effects of microRNA-193 (miR-193) on the proliferation and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS: Cultured rat MSCs were transfected with pre-miR-193 or anti-miR-193 to regulate the expression of miR-193. The proliferation of the MSCs after transfection was evaluated by MTS assay, colorimetric BrdU cell proliferation assay and Ki-67 immunostaining. Cell apoptosis was analyzed by flow cytometry with Annexin V/PI staining. The effect of miR-193 on the expression of cell cycle-related proteins was evaluated by qRT-PCR. RESULTS: Transfection of pre-miR-193 or anti-miR-193 regulated the expression of miR-193 in MSCs effectively. Over-expression of miR-193 significantly promoted the proliferation of MSCs (P<0.05), and inhibition of miR-193 reduced the proliferation of MSCs (P<0.05). miR-193 had no significant effect on the apoptosis of MSCs (P>0.05). The result of qRT-PCR indicated miR-193 promoted the expression of cyclin-dependent kinase 2 (CDK2) significantly (P<0.01). CONCLUSION: miR-193 promotes the proliferation of MSCs possibly through the CDK2 pathway.     

Antisense oligonucleotides targeting seed sequence of miR-155 and its applications in multiple myeloma

AIM：To investigate the role of tiny antisense nucleic acid against miR-155 (tiny antimiR-155, t-antimiR-155) in multiple myeloma cells. METHODS：According to the seed sequence of miR-155, t-antimiR-155 was designed and synthesized. t-antimiR-155 was transfected by LipofectamineTM 2000 into RPMI-8266 cells. The cells were divided into t-antimiR-155 group, scrambled control (SCR) group and blank control group. The growth-inhibitory potencies were measured by MTT assay. The ability of cell colony formation was detected by cell colony formation assay. The cell apoptosis was assessed by flow cytometry with annexin V/PI double staining. RESULTS：The best concentration and time were 0.4 μmol/L and 48 h, respectively. The cell colony forming experiment showed that the circumstances of forming cell community in t-antimiR-155 group was weaker than that in SCR group, and the colony formation inhibitory rate of former was significant higher than the latter. Compared with SCR group, the cell apoptosis in t-antimiR-155 group significantly increased. CONCLUSION：The t-antimiR-155 inhibits the progression of multiple myeloma cells by interfering with miR-155. miR-155 may serve as a potential target in gene therapy for treating multiple myeloma.     

miR-138对小鼠乳腺发育及泌乳功能的影响

microRNAs(miRNAs)在各种类型细胞增殖、分化和凋亡中发挥了重要作用。miR-138参与乳腺发育周期过程中细胞增殖分化的调控。试验以小鼠为动物模型,尾静脉注射miR-138基因抑制剂,应用幼鼠体重称重法,检测miR-138抑制后乳腺泌乳量变化;应用电子显微镜等技术观测乳腺组织形态变化,抑制miR-138后,小鼠乳腺上皮细胞增加,乳汁分泌量增加;抑制miR-138后,观察小鼠乳腺组织超微结构可发现,乳腺细胞的代谢活动增强;收集尾静脉注射miR-138 抑制剂的小鼠乳汁,检测发现其乳糖、乳中酪蛋白含量均有所增高。研究认为,miR-138可刺激乳腺上皮细胞增殖,增加乳腺发育泌乳过程中乳的分泌,并且调控乳汁中重要成分含量。     

miRNA-148a-3p的生物信息学分析及其在小鼠不同组织中的表达水平检测

旨在研究小鼠不同组织中miR-148a-3p的表达情况,并对其进行生物信息学分析,构建miR-148a-3p基因相关网络,进一步阐明其在机体中的作用.本研究以6周龄雄性C57BL/6 J小鼠胃、肺、脾、皮肤、肝、心和肾7个组织为研究材料,每组3个重复,进行3次平行试验.利用qRT-PCR技术比较小鼠不同组织中miR-1...     

miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene

MicroRNAs (miRNAs) have been widely identified in porcine testicular tissues and implicated as crucial regulators of proliferation, apoptosis, and differentiation in porcine spermatogenesis related cells. However, the function roles of most of the miRNAs that have been identified in Sertoli cells are poorly understood. In the present study, six experiments were conducted to study the regulatory role of miR-10b in porcine immature Sertoli cells. In experiment 1, the results showed that the relative mRNA expression level of miR-10b in porcine testicular tissues decreased quadratically (P<0.001) with increasing age, while the relative mRNA expression level of DAZAP1 gene increased (P<0.001). In addition, the mRNA expression of miR-10b was negatively (P<0.01) correlated with DAZAP1 mRNA expression (r=?0.550). In experiment 2, the results from the bioinformatic analysis and a luciferase reporter assay demonstrated that miR-10b directly targeted the DAZAP1 gene in porcine immature Sertoli cells. DAZAP1 mRNA and protein expressions were both regulated (P<0.05) by miR-10b. In experiments 3 to 5, the over-expression of miR-10b or the siRNA-mediated knockdown of the DAZAP1 gene promoted (P<0.05) porcine immature Sertoli cell proliferation, as determined by the Cell Counting Kit-8 (CCK-8) assay and the 5-Ethynyl-2'-deoxyuridine (EdU) assay. However, an annexin V-FITC/PI staining assay and the expression of cell survival-related genes indicated that over-expression of miR-10b or knockdown of DAZAP1 had no effect (P>0.05) on porcine immature Sertoli cell apoptosis. In experiment 6, the co-transfection treatment results showed that miR-10b promoted (P<0.05) porcine immature Sertoli cell proliferation by targeting DAZAP1 gene. Overall, these experiments demonstrated that miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene.     

KLF3基因3'-UTR区双荧光素酶报告载体及其突变载体的构建与活性鉴定

试验旨在构建锌指蛋白3（KLF3）基因3'-UTR区双荧光素酶基因报告载体及其突变载体,初步分析可能调控KLF3基因表达的miRNAs。首先通过PCR方法扩增KLF3基因的3'-UTR序列,将其克隆到经Xho Ⅰ、Not Ⅰ双酶切的双荧光素酶报告载体中;运用Targetscan软件预测可能与KLF3基因3'-UTR相互作用的miRNA;使用脂质体2000转染试剂将miRNAs mimics与构建好的KLF3基因3'-UTR段双荧光素酶报告载体或突变载体共转染于常规培养的293T细胞中,检测荧光素酶活性。结果表明,KLF3基因3'-UTR可能是miR-21的作用靶位点;双荧光报告显示,miR-21 mimics组（0.6900±0.0144）比突变组（1.000±0.0688）和空白对照组（1.000±0.0159）KLF3基因3'-UTR双荧光素酶基因报告载体和突变载体的活性降低了31%（P<0.01）。本试验成功构建了含有KLF3基因3'-UTR段双荧光素酶基因报告载体与突变载体,初步证实miR-21对KLF3基因有调控作用。     

草鱼miR-23a在嗜水气单胞菌感染过程中的调控机制

为探索嗜水气单胞菌感染后草鱼肾脏细胞(Ctenopharyngodon idella kidney,CIK)中miR-23a的调控机制,实验通过实时荧光定量PCR(qPCR)测定了嗜水气单胞菌感染CIK细胞后miR-23a的表达量变化,使用RNAhybrid软件预测miR-23a的靶基因并用双萤光素酶报告基因检测系统进...     

Effect of low expression of miR-21 on apoptosis of HepG2 cells induced by matrine

AIM: To explore whether miR-21 low expression enhances the effect of matrine (MAT) on the apoptosis of hepatocellular carcinoma cells.METHODS: Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of miR-21 in the HepG2 cells treated with different concentrations of MAT. The effect of miR-21 on MAT-induced HepG2 cell apoptosis was analyzed by flow cytometry. The mRNA and protein expression of Bcl-2 and Bax in the HepG2 cells treated with MAT was determined by RT-qPCR and Western blot.RESULTS: The expression of miR-21 increased with the increasing concentration of MAT. Low expression of miR-21 promoted MAT-induced apoptosis, and enhanced the expression of Bax at mRNA and protein levels (P<0.05), while inhibited the expression of Bcl-2 at mRNA and protein levels (P<0.05).CONCLUSION: Low expression of miR-21 enhances MAT-induced HepG2 cell apoptosis by inhibiting the expression of Bcl-2 and promoting Bax expression.     

miR-15a-5p inhibits proliferation and migration of human hepatocellular carcinoma SMMC-7721 cells

AIM: To observe the influence of high expression of miR-15a-5p on the proliferation and migration of human hepatocellular carcinoma SMMC-7721 cells.METHODS: The miR-15a-5p oligonucleotide, which was reconstructed with additional restriction sites of EcoR Ⅰ and Hind Ⅲ, was chemically synthesized and confirmed by sequencing. The miR-15a-5p eukaryotic expression system was constructed by pcDNA6.2-GW/Em-GFP-pre-miR-15a-5p plasmid. The miR-15a-5p was transfected into the SMMC-7721 cells transiently by plasmid, and quantified by quantitative real-time PCR at the mRNA level. The cell viability was measured by CCK-8 assay, and the living cell counting was performed by the method of Trypan blue exclusion. The migration ability of the SMMC-7721 cells with high expression of miR-15a-5p was detected by wound healing test.RESULTS: The sequence of miR-15a-5p oligonucleotide 100% matched the designed sequence. Compared with control group, the miR-15a-5p expression was increased significantly (P<0.05). The viability, the living cell number and the migration ability of the SMMC-7721 cells were decreased in high expression of miR-15a-5p group with statistically significant difference (P<0.05).CONCLUSION: The abilities of proliferation and migration in human hepatocellular carcinoma SMMC-7721 cells are decreased by high expression of miR-15a-5p.     

Ccr-lncRNA172145靶向miR-206在锦鲤体色调控中的作用初探

基于前期对锦鲤皮肤组织的转录组测序数据,筛选到4条在3种皮肤（黑色、红色、白色）中显著差异表达的lncRNA;通过RNAhybrid和TargetScan靶基因软件,发现lncRNA172145与黑色素合成通路中miR-206之间存在靶向结合位点。基于CPC、CPAT及CNIT软件,对lncRNA172145进行编码能力分析,证实该序列为lncRNA,不具备编码蛋白能力。然后,利用qRT-PCR技术对该序列时空表达水平进行了检测,发现在眼睛、黑色皮肤、鳍条及血液中的表达量显著高于其他组织;自原肠胚时期表达量开始显著性上升,高水平趋势一直持续到孵化后20 d。借助双荧光素酶报告实验,进一步证实lncRNA172145与miR-206之间存在靶向调控关系。最后,通过合成miR-206拮抗剂,对miR-206进行了体内沉默,发现与注射阴性对照拮抗剂组和PBS组相比,miR-206拮抗剂组的lncRNA172145表达水平显著性升高。研究结果说明lncRNA172145可能通过靶向到miR-206,参与到黑色素合成通路的调控,这为后续深入挖掘二者在黑色素合成通路中具体的分子作用机制提供了基础资料。