外周血miR-299-5p和miR-127-3p的表达水平与非综合征型唇腭裂相关性研究

目的:研究外周血中miR-299-5p和miR-127-3p的表达水平与非综合征型唇腭裂(NSOC)的相关性。方法:选取于哈尔滨医科大学附属第一医院就诊的唇裂伴或不伴腭裂(CL/P)患儿、单纯腭裂(CPO)患儿和健康儿童各10例,采用实时荧光定量RT-PCR(qRT-PCR)检测各组外周血中miR-299-5p和miR-127-3p的表达水平,采用受试者工作特征(ROC)曲线和曲线下面积(AUC)分析其对NSOC的诊断效能。结果:与健康对照组相比,CL/P组和CPO组miR-299-5p和miR-127-3p的表达下调,差异有统计学意义(P<0.05)。miR-299-5p、miR-127-3p及其联合诊断NSOC的AUC分别为0.770(95%CI:0.558~0.982)、0.810(95%CI:0.651~0.969)、0.915(95%CI:0.754~0.985)。结论:NSOC患儿外周血miR-299-5p和miR-127-3p表达下调,可能与NSOC的发生有关,其可能成为诊断NSOC的潜在生物标记物,且二者联合诊断效能更优。     

miR-301b在调节脂肪细胞分化中的作用

摘要： 目的 研究 miR-301b 在调节间充质干细胞向脂肪细胞分化过程中的作用。方法 对小鼠骨髓基质细胞 ST2 进行脂肪细胞诱导分化, 并利用 qRT-PCR 检测细胞分化过程中成脂分化诱导组相对于阴性对照组的 miR-301b 表达变化。对 ST2 细胞转染 miR-301b mimics 并进行成脂诱导分化, 利用 qRT-PCR 和 Western blot 技术检测 miR- 301b mimics 转染组和阴性对照片段 （NC） 转染组细胞的脂肪特异性基因和蛋白表达水平的变化。结果 成脂分化诱导组 miR-301b 相对表达水平 （0.219±0.021） 较对照组 （1.000±0.425） 减少 （P＜0.05）。miR-301b mimics 转染组的脂肪细胞特异性转录因子过氧化物酶体增殖物激活受体 （PPARγ）、 CCAAT 增强子结合蛋白 （C/EBPα） 和脂肪型脂肪酸结合蛋白 （aP2） 的相对表达量均较 NC 转染组降低 （P＜0.05）。miR-310b mimics 转染组与 NC 转染组相比, 标志基因aP2、 转录因子PPARγ 和C/EBPα 蛋白的表达量均减少 （P＜0.05）。结论 miR-301b可抑制脂肪细胞分化。     

Euxanthone Impairs the Metastatic Potential of Osteosarcoma by Reducing COX-2 Expression

Osteosarcoma (OS) is one of the most common malignancies of bone. This study was aimed to explore the anti-metastatic effect of euxanthone on OS. Adhesion assay and Transwell assay were used to examine the effect of euxanthone on adhesion, migration and invasion of OS cells. COX-2-over-expressing plasmid was applied to transfect OS cells to assess whether COX-2 affects the anti-metastatic function of euxanthone. PDCD4 knockdown and miR-21 mimic were applied to assess whether euxanthone suppresses the transactivation of c-jun via modulating miR-21-PDCD4 signaling. The effect of euxanthone in vivo was also examined by lung metastasis assay. Euxanthone, a xanthone derivative extracted from Polygala caudata, has been found to exhibit anti-neoplastic activities. In present study, our results showed that euxanthone suppressed cell adhesion, migration, and invasion in OS cells. Our experimental data also showed that repression of COX-2 by euxanthone mediated its anti-metastatic activities. Moreover, our findings revealed that euxanthone modulated the COX-2 expression through the miR-21/PDCD4/c-jun signaling pathway. The anti-metastatic activities of euxanthone were also validated in a pulmonary metastasis model. Taken together, our results highlighted the potential of euxanthone to be used in the treatment of OS. Anat Rec, 302:1399–1408, 2019. © 2018 Wiley Periodicals, Inc.     

miR-142-5p regulates pancreatic cancer cell proliferation and apoptosis by regulation of RAP1A

Pancreatic cancer, one of the fatal and aggressive malignancies, leads the sixth cancer-associated death in China. microRNAs are believed to exert function in the diagnosis and treatment of pancreatic cancer. In the present study, we firstly found that miR-142-5p was downregulated in pancreatic cancer tumor tissues while Ras-related protein Rap-1 A (RAP1A) was upregulated compared with para-carcinoma non-tumor tissues. Then, we found that RAP1A could be a putative target gene of miR-142-5p by bioinformatics tool TargetScan. Furthermore, we conducted luciferase reporter assay, RT-qPCR, western blot and correlation analysis to demonstrate that miR-142-5p could negatively regulate RAP1A expression by binding to its 3′UTR. In addition, cell-counting kit 8 (CCK-8) and flow cytometry assays certified that miR-142-5p overexpression may inhibit pancreatic cancer cell proliferation but promote cell apoptosis; while the variation could be reversed by co-transfected with pcDNA3.1-RAP1A. Finally, miR-142-5p overexpression downregulated p-ERK1/2, phosphate p38 mitogen-activated protein kinases (p-p38); however, the variation induced by miR-142-5p mimic could be reversed by co-transfected with pcDNA3.1-RAP1A. In conclusion, our findings indicate that targeting miR-142-5p may provide a novel strategy for the treatment of pancreatic cancer.     

Inhibition of hsa_circ_0001313 (circCCDC66) induction enhances the radio-sensitivity of colon cancer cells via tumor suppressor miR-338-3p: Effects of cicr_0001313 on colon cancer radio-sensitivity

Circular RNA_0001313 (circ_0001313), also known as circCCDC66, is a novel circRNA that recently found to be upregulated in colon cancer tissues and promote colon cancer progression. However, the role of circ_0001313 in regulating radio-sensitivity of colon cancer and its molecular mechanism remain undetermined. Here we found circ_0001313 was significantly upregulated and miR-338-3p was downregulated in radio-resistant colon cancer tissues compared to radio-sensitive tissues. Radiation treatment in colon cells triggered a remarkable upregulation of circ_0001313 and a downregulation of miR-338-3p. Knockdown of circ_0001313 reduced cell viability, colony formation rate and increased caspase-3 activity in colon cancer cells under irradiation. Moreover, circ_0001313 act as a sponge for miR-338-3p in colon cancer cells. Furthermore, miR-338-3p could reverse the effects of circ_0001313 knockdown on cell viability, colony formation, and caspase-3 activity. These findings revealed that knockdown of circ_0001313 could induce radio-sensitivity of colon cancer cells by negatively regulating miR-338-3p.     

Upregulation of miR‑95-3p inhibits growth of osteosarcoma by targeting HDGF

Osteosarcoma is the most common bone malignancy and miR-95-3p plays an important role in multiple cancers. The purpose of this study was to explore the effect and potential mechanism of miR-95-3p on the growth of osteosarcoma. In vitro, the osteosarcoma cell lines, SAOS-2 and U2OS cells, were transfected with miR-95-agomir to assess the role of miR-95-3p in proliferation and apoptosis of osteosarcoma cells. We determined that overexpression of miR-95-3p significantly attenuated cell proliferation but enhanced apoptosis in SAOS-2 and U2OS cells. We also found that overexpression of miR-95-3p in osteosarcoma cells downregulated the expression of hepatoma-derived growth factor (HDGF). Next, knockdown of HDGF by siRNA targeting HDGF clearly inhibited cell proliferation and induced apoptosis in U2OS cells. In vivo, a tumor formation assay in BALB/c nude mice was conducted by injecting the pre-miR-95 or control vector lentivirus-infected U2OS cells to determine the effect of miR-95-3p on the growth of osteosarcoma. Results showed miR-95-3p overexpression inhibited the osteosarcoma growth and downregulated the HDGF expression in xenografted tumor. For mechanism study, we co-transfected HDGF/pcDNA3.1 plasmid and miR-95-agomir to U2OS cells, and we demonstrated that overexpression of HDGF could attenuate the effects of miR-95-3p on U2OS cell proliferation, apoptosis and migration. These findings indicated that miR-95-3p might act as a potential tumor suppressor in osteosarcoma by targeting HDGF. Thus, miR-95-3p may become a potential therapeutic in treatment of osteosarcoma.     

抑制miR-200c表达对糖尿病肾病大鼠肾组织的保护作用及机制研究

目的　探讨抑制miR-200c表达对糖尿病肾病（diabetic nephropathy,DN）SD大鼠肾的保护作用及机制。 方法　采用高糖高脂饮食联合链脲佐菌素（streptozotocin,STZ）腹腔注射诱导建立SD大鼠DN模型,将造模成功的30只DN大鼠随机分为模型组和观察组,每组15只,同时取15只正常健康的SD大鼠作为对照组,造模成功后每7 d给予观察组大鼠尾静脉注射antagomir-200c（30 mg/kg）,模型组和对照组给予尾静脉注射等量的生理盐水。8周后,检测大鼠血清肌酐（Cr）、尿素氮（BUN）和24 h尿蛋白定量水平,实时荧光定量PCR（qRT-PCR）检测肾组织中miR-200c的表达,HE染色观察肾组织病理学变化,活性氧簇（ROS）和丙二醛（MDA）试剂盒检测肾组织中ROS和MDA水平,Western blot检测肾组织中转化生长因子-β1（TGF-β1）、纤连蛋白（fibronectin）的水平。 结果　与对照组比较,模型组大鼠肾组织miR-200c表达、血清中BUN、Cr水平、24 h尿蛋白定量、肾间质损伤评分、ROS、MDA水平及TGF-β1、fibronectin蛋白表达均升高（P<0.05）。与模型组比较,观察组大鼠以上指标均降低（P<0.05）。 结论　miR-200c在STZ诱导的DN大鼠肾组织中表达升高,抑制miR-200c能够对DN大鼠的肾起到一定保护作用,可能与降低肾组织的氧化应激水平和对TGF-β1信号通路的抑制有关。     

男性不育症患者部分附睾来源的miRNA表达水平研究

目的研究精浆miR-888,miR-890和miR-891a在特发性少精症和特发性无精症患者精浆中的水平变化与疾病的关系。方法纳入诊断为特发性少精症和特发性无精症的患者各20例,同时纳入20例健康生育男性作为对照组,采用Real-time PCR方法检测精浆中miR-888,miR-890和miR-891a的相对含量,分析各组间指标表达水平的差异,采用ROC曲线分析三项指标在特发性少精症和特发性无精症中的诊断价值。结果特发性少精症的精浆miR-890表达水平较正常生育组高,miR-888水平两组间差异也具有统计学意义。特发性无精症组的miR-888和miR-890的精浆表达水平均较正常生育组高。特发性无精症组的精浆miR-891a水平显著高于少精症组。miR-890能够较好区分特发性少精症组及正常对照组,三项指标均能很好区分特发性无精症组和正常对照组。结论精浆中miR-888,miR-890和miR-891a的表达水平对于男性不育症研究和诊断具有一定的价值。     

过表达miR-218对舌鳞癌细胞系SCC-4和SCC-9的增殖和侵袭的影响

目的 探讨miR-218在舌鳞癌细胞中的表达,以及对细胞增殖、凋亡和侵袭的影响。方法 在人舌鳞癌细胞系SCC-4和SCC-9中分别转染negative control、miR-218模拟物和抑制剂,然后利用MTT法检测细胞增殖活性;通过流式细胞仪检测细胞周期和细胞凋亡;通过Transwell实验检测细胞侵袭能力,采用GraphPad 7.0软件包对数据进行统计学分析。结果 与对照组相比,转染miR-218 抑制剂的SCC-4和SCC-9细胞,细胞增殖、细胞周期、细胞凋亡和侵袭能力无显著改变;而转染miR-218 模拟物的细胞,增殖能力变弱,凋亡数增加,侵袭能力显著降低。结论 miR-218在口腔癌中的表达量和细胞增殖、细胞周期、细胞凋亡和侵袭能力相关,提示miR-218与口腔癌的发生、发展有一定关系。