miR-183 inhibits invasion of gastric cancer by targeting Ezrin

miR-183, a member of an evolutionarily conserved miRNA cluster (miR-96, miR-182, and miR-183), has been demonstrated to act as both a tumor suppressor and oncogene in various type of human cancer. However, the biological role of miR-183 in gastric cancer (GC) still remains unclear. In the present study, miR-183 expression was significantly decreased in gastric cancer tissues compared with its’ adjacent normal tissues, and down-regulation of miR-183 was significantly associated with lymph node metastasis and pathological TNM stage. Furthermore, Erzin, which was reported to be up-regulated in gastric cancer, was identified as an efficient target of miR-183. Overexpression of miR-183 markedly suppressed cells invasion by downregulation of Ezrin expression. However, miR-183 expression didn’t affect cells proliferation and cell cycle distribution of GC. In conclusion, our study demonstrated that miR-183 acts as a tumor suppressor in GC, partially at least via regulation of Ezrin. Therefore, miR-183 may be a potential target for the treatment of gastric cancer.     

Down-regulation of miR-183 promotes migration and invasion of osteosarcoma by targeting Ezrin

Recent studies have emphasized causative links between aberrant microRNA expression patterns and cancer progression. miR-183 is dysregulated in certain types of human cancers. The expression pattern, clinical significance, and biological role of miR-183 in osteosarcoma, however, remain largely undefined. In this paired analysis, we found that miR-183 was markedly down-regulated in osteosarcoma cells and tissues compared with matching normal bone tissues using RT-qPCR. Statistical analyses revealed that the expression levels of miR-183 significantly correlated with lung metastasis as well as with local recurrence of osteosarcoma. miR-183 expression was inversely correlated with Ezrin mRNA and protein expression levels in osteosarcoma cells as well as in a subset of primary osteosarcoma. Ectopically expressed miR-183 inhibited migratory and invasive abilities of osteosarcoma cells, whereas knockdown of endogenous miR-183 significantly enhanced these abilities. Using a luciferase reporter carrying the 3'-untranslated region (3'-UTR) of Ezrin, we identified Ezrin as a direct target of miR-183. Moreover, ectopic expression of Ezrin could significantly rescue miR-183-suppressed migration and invasion. Of interest, suppression of Ezrin by miR-183 caused a reduction of phosphorylated p44/42 (p-p44/42). Finally, suppression of Ezrin by RNAi mimicked miR-183 action in the suppression of migration and invasion, which was associated with down-regulation of p-p44/42. Taken together, these results suggest that as a tumor suppressor miRNA, miR-183 plays an important role in the aggressiveness of osteosarcoma.     

miR-132 靶向调控Ezrin 抑制卵巢癌细胞增殖、促进凋亡的实验研究

目的:探讨微小核糖核酸分子(miRNA)-132 在卵巢癌中生物学作用和作用靶点。方法:收集22 例卵巢癌及癌旁非肿瘤组织标本,RT-PCR 检测miR-132 表达量;RT-PCR 检测人正常卵巢上皮细胞和卵巢癌细胞系中miR-132 表达量;选择miR-132 表达量最高或最低的卵巢癌细胞株,分别转染阴性对照质粒(NC)和miR-132 mimic 质粒,RT-PCR 检测转染后miR-132 表达量;CCK-8 法检测细胞增殖,流式细胞仪检测细胞凋亡,Western blot 检测Ezrin 蛋白表达。结果:卵巢癌组织中miR-132 表达量显著低于癌旁非肿瘤组织,而卵巢癌细胞系中miR-132 表达量显著低于正常卵巢上皮细胞,差异均有统计学意义(P<0.05);选择卵巢癌细胞株中miR-132 表达量最低卵巢癌SKOV3 细胞株进行基因转染,与转染阴性对照质粒相比,转染miR-132 mimic 质粒后miR-132 表达量显著上升,细胞增殖显著降低,细胞凋亡显著增加,差异均有统计学意义(P<0.05);Western blot 结果显示,上调miR-132 表达后,卵巢癌SKOV3 细胞株中Ezrin 蛋白表达显著上升(P<0.05)。结论:在卵巢癌中,miR-132 可能作为一种抑癌基因通过靶向调控Ezrin 抑制卵巢癌细胞增殖、促进凋亡。     

LncRNA OIP5-AS1通过下调miR-421表达促进骨肉瘤细胞增殖

目的 探讨lncRNA OIP5-AS1通过调节miR-421对骨肉瘤癌细胞增殖能力的影响.方法 通过生物信息学网站预测miR-421是否与lncRNA OIP5-AS1结合,双荧光素酶报告基因实验进行验证.构建lncRNA OIP5-AS1过表达载体,转染细胞骨肉瘤细胞,Real time-qPCR检测miR-421...     

miR-129-5p靶向调控VCP抑制骨肉瘤细胞体外迁徙侵袭

目的 探讨miR-129-5p是否靶向调控VCP基因抑制骨肉瘤细胞迁徙侵袭.方法 构建miR-129-5p过表达及低表达的慢病毒载体,转染骨肉瘤细胞U2-OS;采用实时荧光定量PCR检测上调及下调miR-129-5p的U2-OS细胞中miR-129-5p的表达量;采用RT-PCR和Western blot技术检测VCP mRNA和蛋白表达;采用划痕实验和Transwell侵袭实验检测细胞迁徙、侵袭情况.结果 实时荧光定量PCR结果显示U2-OS细胞中miR-129-5p表达被明显上调或下调;RT-PCR和Western blot检测结果显示:miR-129-5p上调U2-OS细胞组中VCP mRNA和蛋白表达水平显著低于阴性对照细胞组(阴性慢病毒转染);miR-129-5p下调U2-OS细胞组中VCP mRNA和蛋白表达水平显著高于阴性对照细胞组;miR-129-5p上调U2-OS细胞迁徙和侵袭力显著低于阴性对照细胞,miR-129-5p下调的U2-OS细胞迁徙和侵袭力显著高于阴性对照细胞.结论 miR-129-5p靶向调控VCP的表达而抑制骨肉瘤细胞迁徙和侵袭能力.     

CISD2基因沉默通过AKT通路抑制人骨肉瘤细胞U2OS的增殖

目的检测CDGSH铁硫结构域2 (CDGSH iron sulfur domain 2, CISD2)基因对骨肉瘤细胞增殖能力的影响。方法应用化学合成小干扰RNA技术沉默人骨肉瘤细胞系U2OS中CISD2基因的表达,分别通过免疫荧光染色与Western blot实验检测基因沉默效率;通过MTT实验与克隆形成实验,研究沉默CISD2基因对细胞增殖能力的影响;流式细胞术检测细胞周期;Western blot实验检测AKT,p-AKT蛋白的表达。结果设计的siRNA抑制人类骨肉瘤细胞系U2OS细胞CISD2基因表达。沉默CISD2基因抑制U2OS细胞的增殖能力与克隆形成能力;流式细胞术实验结果表明沉默CISD2可以诱导U2OS细胞G_0/G_1期抑制;Western blot实验显示沉默CISD2可以抑制p-AKT蛋白的表达。结论沉默CISD2基因抑制骨肉瘤细胞系U2OS的增殖能力与抑制AKT信号传导通路相关。     

miR-496过表达抑制结肠癌细胞生长和转移

目的:研究miR-496过表达对结肠癌细胞生长和转移的影响及其分子机制。方法:运用生物信息学软件筛选miR-496靶向相互作用蛋白;real-time PCR和Western blot法测定结肠癌细胞系HT29、HCT116、SW480以及正常结肠上皮细胞NCM460中miR-496、CTNNB1 mRNA和β-catenin蛋白的表达;运用Lipofectamine 2000将miR-496 mimics转染HT29、HCT116和SW480细胞,分别命名为HT29-miR-496 mimics、HCT116-miR-496 mimics和SW480-miR-496 mimics细胞,转染scramble为阴性对照;运用MTT法、乳酸脱氢酶(LDH)试剂盒法、克隆形成实验和Transwell分别测定细胞活力、LDH漏出率、克隆形成能力和转移能力;萤光素酶报告基因实验测定miR-496启动子活性;Western blot法测定β-catenin、真核细胞翻译起始因子4E结合蛋白1(4E-BP1)、p-4E-BP1、低密度脂蛋白受体相关蛋白6(LRP6)、p-LRP6、MMP-7、MMP-9、MMP-13以及TIMP-2的蛋白水平。结果:miR-496与β-catenin内源性相互作用;miR-496在HT29、HCT116和SW480细胞中低表达,而在NCM460高表达;β-catenin在HT29、HCT116和SW480细胞中高表达,而在NCM460低表达;培养24 h、48 h、72 h、96 h的HT29-miR-496 mimics、HCT116-miR-496 mimics和SW480-miR-496 mimics细胞活力、LDH漏出率、克隆形成率和转移的细胞数均显著低于对照组(P0.05);萤光素酶报告基因实验结果显示转染miR-496 mimics细胞中的miR-496启动子活性明显增加(P0.05),分别是对照组的1.75倍、2.04倍和1.61倍。Western blot实验结果显示miR-496过表达抑制β-catenin蛋白表达,p-4E-BP1和p-LRP6的蛋白水平降低;siRNA或miR-496过表达介导的β-catenin表达下调能显著抑制MMP-7和MMP-9的表达,促进TIMP-2的表达。结论:miR-496在结肠癌细胞中低表达,在正常结肠上皮细胞中高表达;miR-496过表达抑制结肠癌细胞的生长和转移,其机制是通过抑制Wnt/β-catenin通路进一步抑制MMP-7和MMP-9表达,促进TIMP-2表达,从而抑制结肠癌细胞的恶性表型。     

circRNA_100395通过结合miR-31-5p抑制心肌细胞肥大相关基因的表达

目的:探讨环状RNA(circRNA)_100395对心肌细胞肥大表型的影响及其作用机制.方法:通过RT-qPCR检测健康器官捐献者(n=8)与心衰患者(n=14)心肌组织中circRNA_100395及其宿主基因Kelch蛋白样家族成员20(KLHL20)的表达水平.原代分离、培养乳小鼠心室肌细胞(NMVCs),分别...     

miR-183 promotes radioresistance of lung adenocarcinoma H1299 cells via epithelial-mesenchymal transition

Lung adenocarcinomas are usually sensitive to radiation therapy, but some develop resistance. Radiation resistance can lead to poor patient prognosis. Studies have shown that lung adenocarcinoma cells (H1299 cells) can develop radioresistance through epithelial-mesenchymal transition (EMT), and this process is regulated by miRNAs. However, it is unclear which miRNAs are involved in the process of EMT. In our present study, we found that miR-183 expression was increased in a radioresistant lung adenocarcinoma cell line (H1299R cells). We then explored the regulatory mechanism of miR-183 and found that it may be involved in the regulation of zinc finger E-box-binding homeobox 1 (ZEB1) expression and mediate EMT in lung adenocarcinoma cells. qPCR results showed that miR-183, ZEB1, and vimentin were highly expressed in H1299R cells, whereas no difference was observed in E-cadherin expression. Western blot results showed that ZEB1 and vimentin were highly expressed in H1299R cells, while E-cadherin expression was decreased. When miR-183 expression was inhibited in H1299R cells, radiation resistance, proliferation, and cell migration were decreased. The expression of ZEB1 and vimentin in H1299R cells was decreased, while the expression of E-cadherin was increased. Moreover, miR-183 overexpression in H1299 cells enhanced radiation resistance, proliferative capacity, and cell migration ability. The expression of ZEB1 and vimentin in H1299 cells was increased, while that of E-cadherin was decreased. In conclusion, miR-183 may promote EMT and radioresistance in H1299 cells, and targeting the miR-183-ZEB1 signaling pathway may be a promising approach for lung cancer treatment.     

miR-210通过MMP-2下调M2/M1巨噬细胞比例抑制肺癌细胞生长

目的研究miR-210通过MMP-2调控M2/M1巨噬细胞比例对肺癌细胞生长的影响及对相关机制进行初步探讨。方法将小鼠Lewis lung carcinoma (LLC)-1细胞皮下植入C57BL/6小鼠构建小鼠肺癌模型,荧光定量PCR检测miR-210表达。进一步取40只肺癌建模成功的小鼠,并随机分为4组:对照组小鼠尾静脉注射AAV9-Fugw腺相关病毒;AAV9-OE-miR-210组小鼠尾静脉注射AAV9-OE-miR-210腺相关病毒; AAV9-OE-MMP-2组小鼠尾静脉注射AAV9-OE-MMP-2腺相关病毒;AAV9-OE-miR-210+AAV9-OE-MMP-2组小鼠尾静脉注射AAV9-OE-miR-210和AAV9-OE-MMP-2腺相关病毒。所有小鼠注射腺相关病毒滴度为108TU,病毒注射时间为4周。检测4组小鼠肿瘤大小变化,WGA染色检测4组小鼠肺癌细胞大小变化。另外,取生长融合至80%的LLC1细胞,并随机分为2组:WT组转染包含野生型MMP-2 3’-UTR的构建物,Mut组转染突变型MMP-2 3’-UTR的构建物。细胞转染48 h后,荧光素酶实验检测m...     

BTG2 inhibits the proliferation and metastasis of osteosarcoma cells by suppressing the PI3K/AKT pathway

B cell translocation gene 2 (BTG2) has been reported to be a potential tumor suppressor in many types of tumors. However, the roles and molecular mechanisms of BTG2 in osteosarcoma progression are still unknown. In this study, we investigated the role of BTG2 in proliferation and metastasis of osteosarcoma and the underlying mechanism. BTG2 expression levels were measured in fresh osteosarcoma tissues and cell lines. The effects of BTG2 on cell proliferation, migration and invasion were explored by MTT, transwell assays, western blot, and in vivo tumorigenesis in nude mice. We found that BTG2 was down-regulated in human osteosarcoma tissues and cell lines. Overexpression of BTG2 inhibited the proliferation and migration/invasion of human osteosarcoma cells in vitro, it also markedly inhibited xenograft tumor growth in vivo. Furthermore, BTG2 significantly decreased the expression of phosphorylated PI3K and AKT in osteosarcoma cells. Taken together, our data indicate that BTG2 might suppress the tumor growth and metastasis via PI3K/AKT signaling pathway, implying that BTG2 may serve as a potential molecular target for the treatment of osteosarcoma.     

miR-205通过靶向调控TBX18抑制神经胶质瘤细胞的侵袭能力

目的:探讨微小RNA-205(miR-205)在神经胶质瘤组织中的表达模式及其在胶质瘤细胞侵袭中的作用及可能机制。方法:用real-time PCR检测配对神经胶质瘤组织中miR-205的表达,同时用免疫组化方法检测配对组织中TBX18蛋白的表达量;用脂质体将miR-205模拟物(miR-205 mimics)、miR-205抑制物(miR-205 inhibitor)以及相应对照(control)导入U251胶质瘤细胞后,Transwell实验检测细胞的侵袭能力变化;生物信息学分析结合萤光素酶报告基因实验观察miR-205对TBX18的靶向调控作用;采用RNA干扰技术下调U251细胞中TBX18的表达,用Transwell实验检测细胞侵袭能力变化。结果:miR-205在82.6%所检测的神经胶质瘤组织中表达下调,而TBX18在神经胶质瘤组织中表达上调,两者表达呈负相关;过表达miR-205使U251细胞的侵袭细胞数下降(P0.01),抑制miR-205表达后U251侵袭细胞数增加(P0.01);miR-205在U251胶质瘤细胞中能直接靶向抑制TBX18的表达,干扰TBX18的表达亦使U251细胞的侵袭细胞数下降(P0.01)。结论:miR-205在神经胶质瘤中表达下调,miR-205可能通过靶向调控TBX18影响胶质瘤细胞的侵袭能力,这将为完善胶质瘤侵袭的分子机制及开发新治疗策略以提高胶质瘤治疗效果提供有力的理论依据。     

miR-124-3p通过负调控Caveolin-1表达降低N2a/APPswe细胞内Tau蛋白磷酸化水平

目的探讨在阿尔茨海默病(AD)细胞模型中miR-124-3p通过调控Caveolin-1的表达对细胞内Tau蛋白磷酸化水平的影响。方法体外培养野生型N2a(N2a/WT)和N2a/APPswe细胞,荧光定量PCR及Western blot分别检测miR-124-3p、APP和Caveolin-1的表达;N2a/APPswe细胞分别转染miR-124-3p模拟物、Caveolin-1过表达载体及干扰RNA后,用荧光定量PCR及Western blot分别检测Caveolin-1、Tau和Tau-Ser404表达。结果与野生型N2a细胞比较,N2a/APPswe细胞中miR-124-3p表达降低(P0.01),APP表达升高(P0.01),Caveolin-1表达升高(P0.01)。N2a/APPswe细胞转染miR-124-3p模拟物后,Caveolin-1表达降低(P0.01),Tau-Ser404/Tau降低(P0.01)。N2a/APPswe细胞转染Caveolin-1过表达载体后,Tau-Ser404/Tau升高(P0.01)。转染干扰RNA后TauSer404/Tau降低(P0.01)。结论 miR-124-3p可能通过调节其靶基因Caveolin-1的表达而降低Tau蛋白磷酸化水平,在AD中发挥神经保护作用。     

miR-143 down-regulates TLR2 expression in hepatoma cells and inhibits hepatoma cell proliferation and invasion

Hepatoma is a tumor with high degree of malignancy. A number of oncogenes and tumor suppressor genes play certain roles in tumorigenesis and progression. Among which, miRNA, as an important class of gene regulators, play important roles in regulating tumorigenesis and development of hepatoma. So know well the unique molecular pathway is very important. Here, we showed that there is a different miR-143 expression patterns in different hepatoma tissues, and that miR-143 expressions contribute disease progress. By contrast, we down-regulated the expression of miR-143 with miR-143 mimics in HepG2 cells resulting in decreased proliferation. And the decreased proliferations of HepG2 cells were due to a G₀/G₁ arrest of cell cycle. During this progress, the increased apoptosis may be another major cause for decreased proliferation of HepG2 cells. And then, we found miR-143 down-regulation induced decreased mRNA and protein expressions of TLR2 and NF-κB. These results show that HepG2 cells depend to a greater extent on miR-143 for proliferation, and miR-143 down-regulation may induce a cell cycle arrest though TLR and NF-κB pathway. miR-143 blockade may be beneficial in therapy of Hepatoma.     

miR-503 inhibits cell proliferation and induces apoptosis in colorectal cancer cells by targeting E2F3

Objective: Colorectal cancer (CRC) is one of the major healthcare problems worldwide. A lot of miRNAs are aberrantly expressed in CRC and involved in its development and progression. The purpose of this study was to investigate the expression and function of miR-503 in CRC. Methods: miR-503 expression was detected in CRC tissues and cell lines by Quantitative real-time PCR. Cell proliferation was assessed by MTT assay. Cell apoptosis and cell cycle distribution were measured by flow cytometry. Moreover, luciferase reporter assay and western blot were performed to determine the potential target of miR-503 in CRC cells. Results: miR-503 was significantly decreased in CRC tissues and cell lines in comparison with controls. Overexpression of miR-503 in CRC cells remarkably inhibited cell proliferation and induced apoptosis. Furthermore, E2F3 was identified as a direct target of miR-503 in CRC cells and down-regulation of E2F3 had a similar effect as miR-503 overexpression on CRC cells. In addition, the expression of E2F3 was negatively correlated with miR-503 level in CRC tissues. Conclusions: miR-503 inhibits cell proliferation and induces apoptosis by directly targeting E2F3 in CRC cells, indicating its potential application in CRC diagnosis and therapy.     

miR-34a-5p抑制K562细胞红系分化

目的探索miR-34a-5p对K562细胞红系分化的影响。方法分别用miR-34a-5p模拟物和反义抑制寡核苷酸转染K562细胞,用real-time PCR法检测过表达或干扰效率,并进一步用流式细胞术和联苯胺染色法检测K562细胞向红系的分化情况;通过Western blot方法检测miR-34a-5p的靶基因。结果 miR-34a-5p在K562细胞红系分化过程中呈现表达下降趋势;在K562细胞中过表达miR-34a-5p可抑制hemin诱导的红系分化(P0.05);反之,干扰K562内源的miR-34a-5p表达会对K562红系分化产生促进作用(P0.01);另一方面,miR-34a-5p通过靶向抑制c-MYB的表达抑制细胞向红系分化。结论 miR-34a-5p通过抑制c-MYB在K562细胞早期红系分化过程中发挥促进作用。     

卵巢癌中E2F1通过影响miR-106b-5P调节RhoC的表达

目的探讨卵巢癌中E2F1、miR-106b-5P以及RhoC三者的表达的关系。方法使用Spearman相关分析方法分析卵巢癌组织中E2F1和miR-106b-5P表达水平的相关关系;OVCAR3和A2780细胞中转染或沉默E2F1,通过RT-PCR检测miR-106b-5P和RhoC的表达,转染miR-106b-5P后检测RhoC的表达。结果经分析发现卵巢癌组织中E2F1和miR-106b-5P表达水平呈负相关。卵巢癌细胞转染E2F1后miR-106b-5P表达水平下降,RhoC的表达水平上升;转染si-E2F1后结果相反;而在卵巢癌细胞中转染miR-106b-5P后发现RhoC的表达水平下降。结论 E2F1负调控miR-106b-5P表达调控RhoC的表达。     

Ginsenoside Rh2 inhibits thyroid cancer cell migration and proliferation via activation of miR-524-5p

IntroductionThyroid cancer is an important disease that threatens the health of humans. Ginsenoside Rh2 is known as an anticancer molecule; however, its function in thyroid cancer cells has not been reported. In the present study, we identified that Rh2 treatment of the thyroid cancer cell line K1 inhibited cell migration and proliferation.Material and methodsWe determined the Rh2 function in thyroid cancer cell lines. By RT-PCR, expression of miR-524-5p and related genes were determined. The cell phenotype including cell migration and proliferation were detected after serials treatment. The relevant protein level were checked by Western blot.ResultsInterestingly, we observed that miR-524-5p, a type of miRNA, had lower expression in the thyroid cancer cell lines TPC-1, K1, and NPA than in the normal thyroid cell line Nthyri3-1. Additionally, Rh2 treatment induced miR-524-5p expression. Further examination using overexpression of miR-524-5p identified that the miR-524-5p mimic inhibited cell migration and proliferation of the K1 line. Similar to Rh2-treated cells, the miR-524-5p mimic-expressing cells had increased E-cadherin and reduced vimentin levels compared to the control cells. Next, we examined the relationship between Rh2 and miR-524-5p with respect to thyroid cell migration and proliferation. Treatment with Rh2 and miR-524-5p inhibitor suppressed Rh2 action on K1 thyroid cell migration and proliferation, and the rates were similar to those in control cells, suggesting that Rh2 might induce miR-524-5p expression to inhibit thyroid cancer cell migration and proliferation.ConclusionsOur analyses identified Rh2 and miR-524-5p action on thyroid cancer cell migration and proliferation as well as the linkage between Rh2 and miR-524-5p in thyroid cancer cell development.