Up/down-regulation of miR-21 changes biological function of colon can-cer cells and sensitivity to cetuximab

AIM: To explore the effects of miR-21 on biological behavior of colon cancer cells and their sensitivity to epidermal growth factor receptor monoclonal antibody cetuximab. METHODS: Lentiviral vectors were constructed to generate up- and down-regulations of miR-21 lentiviruses (LV-miR-21 and LV-anti-miR-21, respectively), and the corresponding negative control viruses (LV-miR-21 NC and LV-anti-miR-21 NC, respectively) were also constructed. The viruses were used to infect human colon cancer RKO cells. The changes of the miR-21 expression level, the cell proliferation, the colony-forming ability, the cell apoptosis and the sensitivity of the cells to cetuximab were detected by real-time PCR, MTT assay, soft agar colony assay, flow cytometry and CCK-8 assay. RESULTS: The lentivirus titers of LV-miR-21, LV-miR-2 NC, LV-anti-miR-21 and LV-anti-miR-21 NC were 3.0×1012 TU/L, 6.0×1011 TU/L, 2.0×1012 TU/L and 8.0×1011 TU/L, respectively. The infection efficiency was over 80% by the observation of green fluorescence. The miR-21 expression level, the cell proliferation, and the colony-forming ability in LV-miR-21 group were significantly higher than those in LV-anti-miR-21 group. The early apoptotic rate and the inhibitory rate of cetuximab for the cells in LV-anti-miR-21 group were higher than those in LV-miR-21 group. CONCLUSION: miR-21 promotes the proliferation of colon cancer cells. Down-regulation of miR-21 enhances the sensitivity of the colon cancer cells to the targeted therapy drug cetuximab.     

鸡miR-1749生物信息学分析及真核表达载体构建

为研究鸡miR-1749功能及其前体rs15860000(C>T)不同基因型对成熟miRNA生成的影响,采用Mfold软件预测C>T突变对miR-1749前体二级结构能值的影响;选用pcDNA3.1-EG-FP质粒,构建miR-1749前体不同等位基因的真核表达载体并转染DF1细胞,检测成熟miR-1749的表达;利用 TargetScan 和 miRDB 软件预测 miR -1749的靶基因,并将靶基因的并集在DAVID数据库进行GO功能富集和KEGG信号转导通路分析。 Mfold预测结果显示,miR-1749前体C>T突变影响其前体二级结构自由能值;成功构建pcDNA3.1-EGFP-pre-miR-1749-C和pcDNA3.1-EGFP-pre-miR-1749-T真核表达载体,转染DF1细胞后,不同基因型重组质粒miR-1749的表达量均显著高于空质粒组( P<0.01), T等位基因成熟miR-1749的表达量显著高于C等位基因(P<0.05)。2个软件预测结果显示,miR-1749靶基因的并集有250个基因,这些基因显著富集于Ras蛋白信号转导调控、GTPase介导信号转导调控、细胞稳态及胚后发育的生物学过程和GnRH、MAPK及细胞黏附分子的信号转导通路。     

microRNA-146a通过靶向MAPK4和Myosin Va基因调控羊驼黑色素细胞增殖、迁移

旨在研究microRNA-146a(miR-146a)对羊驼黑色素细胞增殖和迁移的调控及其分子机制.本研究使用双荧光素酶试验验证MAPK4和Myosin Va是miR-146a的靶基因;利用荧光定量PCR和蛋白质免疫印迹试验检测在羊驼黑色素细胞中过表达miR-146a对相关下游基因表达的影响;利用CCK8和Transw...     

IRIDIUM-192: A LITERATURE REVIEW FOR FURTHER REFERENCING THE ISOTOPE, ITS ACTIVITY UNITS, AND DOSIMETRY TECHNIQUES

The individual embarking on an iridium-192 brachytherapy program is faced with an abundance of concepts, terms and dosimetry techniques. This article introduces the beginning brachytherapist to the basic physical parameters of iridium-192, to the potential units for source strength and to the general concepts of the various dosimetry techniques. Multiple literature references are listed for more in-depth study.     

TECHNIQUES OF AFTERLOADING IRIDIUM-192 INTERSTITIAL BRACHYTHERAPY IN VETERINARY MEDICINE

Continuous low-dose-rate irradiation of animal tumors has become feasible through the implantation of iridium-192 by an afterloading technique. ¹⁹²Ir is an ideal radioisotope for interstitial brachytherapy in veterinary medicine because of its 74.2-day half-life, medium-energy gamma radiation (0.35 MeV), moderate shielding requirements, low cost, and adaptability to most tumor configurations. The afterloading technique can easily be applied to veterinary patients. The effectiveness of interstitial brachytherapy may be enhanced by combination with surgery and/or hyperthermia. Computer-assisted dose calculations make afterloaded ¹⁹²Ir implantation an accurate, precise method of interstitial brachytherapy.     

miR-127和miR-136在转基因克隆绵羊胎盘中的表达分析及其靶基因的鉴定

克隆动物胎盘发育缺陷是造成动物克隆效率低下的一个重要原因。目前认为克隆胎盘发育异常通常是由于一些基因表达的异常所致,与表观遗传修饰有关。microRNA是一种重要的表观遗传修饰方式,对动物胚胎发育和胎盘的形成有着重要的调控作用。为研究miR-127和miR-136在克隆动物胎盘中的表达情况及其与克隆动物发育缺陷的关系,本实验运用荧光定量PCR分析了死亡克隆绵羊胎盘和同期普通绵羊胎盘组织中miR-127和miR-136的相对表达量,并鉴定了miR-127和miR-136的靶基因及靶基因在胎盘中的表达情况。结果显示,miR-127和miR-136在克隆绵羊胎盘中的表达量分别增加了3.1和2.8倍。EGFP荧光敲除实验证实,胎盘发育相关基因Rtl1是miR-127和miR-136的靶基因,同时定量PCR分析发现Rtl1基因在克隆绵羊胎盘中的表达量降低了3/5。结果说明,miR-127和miR-136在克隆胎盘中的异常表达可导致Rtl1基因的低表达,这很可能是导致克隆动物死亡的一个重要原因。     

中国荷斯坦奶牛乳脂中miR-2285f生物信息学分析及靶基因验证

旨为探索bta-miR-2285f和宿主基因TANK结合激酶1(TANK binding kinase 1,TBK1)的生物学功能,以及bta-miR-2285f与靶基因互作调节奶牛乳脂代谢的潜在机制,本研究通过生物信息学方法分析bta-miR-2285f在不同物种间的保守性,并通过上游序列CPG岛分析、启动子预测、进一步进行组织表达谱分析,预测靶基因并进行GO功能和KEGG通路富集分析。利用双荧光素酶报告试验验证bta-miR-2285f调控的下游靶基因钙结合蛋白39(Calcium binding protein 39,CAB39),并在乳腺上皮细胞水平探究bta-miR-2285f的功能作用。结果表明：1)bta-miR-2285f是牛特异性miRNA,属于内含子miRNA;2)bta-miR-2285f与宿主基因TBK1上游序列2 kb处均不含有CPG岛。bta-miR-2285f在上游586 bp处为转录起始位点。宿主基因TBK1在上游105 bp处为转录起始位点;3)TargetScan和miRWalk预测miR-2285f有172个交集靶基因,主要参与新陈代谢、遗传信息传...     

绵羊甲状腺miR-370-3p靶向COL4A3基因调控繁殖力性状的初步研究

为探讨小尾寒羊甲状腺组织中与繁殖功能相关的miR-370-3p与COL4A3的靶向关系,本研究利用miRDB与Targetscan网站预测了miR-370-3p的靶基因,并取其交集进行功能富集分析,通过RNAhybrid与Targetscan网站预测绵羊miR-370-3p与候选靶基因的结合位点;随后检测了miR-370-3p与COL4A3基因在小尾寒羊甲状腺组织中的相对表达量。构建psiCHECK2-COL4A3-3′UTR-野生型/突变型双荧光素酶载体,并将其与miR-370-3p mimics/mimics NC共转染至HEK293T细胞并检测荧光活性。结果显示：1)miR-370-3p的靶基因可富集到孕酮介导的卵母细胞成熟、卵母细胞减数分裂、HIF-1信号通路、MAPK信号通路、mTOR信号通路、Ras信号通路和FoxO信号通路等与动物繁殖有关的信号通路中。2)miR-370-3p可与COL4A3基因3′UTR区域结合,且两者在小尾寒羊甲状腺组织中表达呈相反趋势。3)共转染psiCHECK2-COL4A3-3′UTR-野生型和miR-370-3p mimics组的荧光素酶活性显...     

miR-18a通过靶向结合CTGF调控猪颗粒细胞凋亡

为探究miR-18a对猪卵巢颗粒细胞凋亡的调控作用,利用生物信息学分析、荧光素酶活性检测和体外培养颗粒细胞试验验证miR-18a对CTGF的靶向作用及其在猪颗粒细胞中对CTGF基因表达的影响,并通过流式细胞技术检测其对猪卵巢颗粒细胞凋亡的调控作用。生物信息学分析结果表明,CTGF是miR-18a的潜在靶基因,荧光素酶活性检测进一步验证了miR-18a与CTGF的结合。在培养的颗粒细胞中转染miR-18a模拟物后,qRT-PCR和Western blot结果显示CTGF的mRNA和蛋白水平均显著降低,流式细胞技术检测表明miR-18a显著促进颗粒细胞的凋亡。而转染miR-18a抑制剂后,猪颗粒细胞的凋亡率显著降低,共转染miR-18a抑制剂和CTGF的干扰RNA后,颗粒细胞的凋亡率呈现回升。试验结果表明miR-18a通过靶向结合CTGF基因调控猪颗粒细胞的凋亡。