circ_0044516靶向miR-1281促进食管癌细胞增殖、抑制细胞凋亡北大核心CSCD

目的:探索circ_0044516是否通过靶向miR-1281影响食管癌Eca109细胞增殖和凋亡。方法:将食管癌Eca109细胞分为si-NC组、si-circ_0044516组、miR-NC组、miR-1281组、si-circ_0044516+anti-miR-NC组和si-circ_0044516+antimiR-1281组。采用实时定量聚合酶链反应(qRT-PCR)测定食管癌组织及Eca109细胞中circ_0044516和miR-1281表达。运用CCK-8法检测Eca109细胞增殖能力,克隆形成实验测定克隆形成,Western blot测定活化的半胱氨酸蛋白酶(cleaved-caspase)3蛋白和cleaved-caspase9蛋白表达,流式细胞术测定细胞凋亡。采用荧光素酶报告实验分析circ_0044516与miR-1281的靶向结合。结果:食管癌组织中circ_0044516表达量比相匹配的癌旁组织增加2.70倍左右,miR-1281表达量较癌旁组织显著减少(P<0.05)。食管癌Eca109细胞中干扰circ_0044516表达或miR-1281过表达可使cleaved-caspase3蛋白、cleaved-caspase9蛋白表达量和凋亡率升高,细胞增殖能力降低,克隆形成数减少(P<0.05)。circ_0044516靶向调控miR-1281的表达。干扰circ_0044516表达对食管癌Eca109细胞增殖和凋亡的作用被下调miR-1281表达所逆转。结论:食管癌Eca109细胞中干扰circ_0044516表达通过靶向miR-1281抑制细胞增殖并促进其凋亡。     

miR-449a过表达抑制人神经母细胞瘤细胞SH-SY5Y增殖并促进其凋亡

目的探讨miR-449a对人神经母细胞瘤细胞系SH-SY5Y的增殖和凋亡的影响。方法用Lipofectamine TM2000将miR-449a模似物或miR-449a对照转染至SH-SY5Y细胞,分为空白、miR-449a模似物和miR-449a对照SHSY5Y细胞组;实时荧光定量PCR(q-PCR)检测各组细胞中miR-449a表达;CCK8法检测细胞增殖;流式细胞仪检测细胞凋亡和周期,Western blot检测c-Myc蛋白和Bax/Bcl-2蛋白表达。结果 miR-449a模似物瞬时转染SH-SY5Y细胞后,miR-449a的表达水平明显高于正常对照组(P0.05);SH-SY5Y细胞增殖能力受到明显抑制(P0.05);凋亡率明显增加(P0.05);c-Myc蛋白表达显著降低(P0.05);细胞促凋亡蛋白Bax表达升高;抗凋亡蛋白Bcl-2表达降低(P0.05)。结论 miR-449a可通过c-Myc影响SH-SY5Y细胞的增殖和周期,通过调节Bax/Bcl-2影响其凋亡。     

miR-181通过Prox1促进人乳腺癌细胞系MCF-7的增殖并抑制凋亡

目的观察miR-181、同源异型盒转录因子(Prox-1)对乳腺癌细胞增殖及凋亡的影响,探讨miR-181及Prox-1在乳腺癌发生发展中的作用。方法荧光定量PCR检测癌旁乳腺组织、导管内癌组织、浸润性导管癌组织中miR-181的表达;pRFP-miR-181-inhibitor质粒转染乳腺癌细胞系MCF-7,荧光定量PCR和Western blot检测Prox1 mRNA及蛋白水平;EdU检测细胞增殖;TUNEL试剂盒检测细胞凋亡。结果与癌旁乳腺组织比较,导管内癌组织、浸润性导管癌组织内miR-181 mRNA表达上调(P0.01);抑制MCF-7细胞内的miR-181的表达,Prox1在蛋白水平明显上调(P0.05);MCF-7细胞的增殖能力减弱(P0.05)而凋亡增强(P0.05)。结论 miR-181可能通过抑制Prox1促进细胞的增殖、抑制细胞的凋亡,参与了乳腺癌的发生。     

吴茱萸碱抑制人骨肉瘤HOS细胞的增殖并促进其凋亡

目的探讨吴茱萸碱对骨肉瘤HOS细胞株增殖凋亡的影响及其可能的机制。方法通过利用0、1、2、4、8μmol/L浓度吴茱萸碱处理HOS细胞24、48 h后,利用CCK-8法检测HOS细胞活性。用3μmol/L的吴茱萸碱处理HOS细0、24、48 h后,利用细胞凋亡-Hoechst染色试剂盒染色观察HOS细胞细胞核染色质的形态。用3μmol/L吴茱萸碱处理0、24、48 h后,利用流式细胞仪检测HOS细胞的凋亡率。3μmol/L吴茱萸碱处理0、24、48 h后,Westernblot检测HOS细胞内Caspase 3、Bcl-2蛋白的表达变化情况。结果 2~8μmol/L的吴茱萸碱可抑制HOS细胞的细胞活性,抑制其增殖,呈剂量-时间依赖性。8μmol/L处理48 h后细胞存活率为0.453±0.071,与对照组比较,差异有统计学意义(P<0.01)。Hoechst-33258染色观察可见凋亡细胞染色质颜色发白,呈固缩状或者碎裂状染色质,染色质着色不均匀,核形态各异。流式细胞仪检测3μmol/L吴茱萸碱处理HOS细胞0、24、48 h后的凋亡率分别为(5.32±1.62)%、(10.85±1.49)%和(12.47±0.59)%,与对照组比较,差异有统计学意义(P<0.01)。吴茱萸碱可上调HOS细胞内的Caspase 3蛋白的表达,同时下调Bcl-2蛋白的表达,与对照组相比较,差异有统计学意义(P<0.05)。结论吴茱萸碱可降低人骨肉瘤HOS细胞的细胞活性,抑制其体外增殖,诱导其发生凋亡,其机制可能与其上调Caspase 3蛋白的表达,下调Bcl-2蛋白的表达有关。     

基底样乳腺癌中microRNA-205靶向调控KLF12及对细胞侵袭能力的影响

目的探讨基底样乳腺癌(basal-like breast carcinoma,BLBC)特异性miRNAs和靶基因及生物学功能。方法提取细胞总RNA和蛋白,荧光素酶实验验证靶基因;采用qRT-PCR、Western blot和免疫组化En Vision法分析miRNA和靶基因表达;CCK8和Transwell实验检测细胞增殖和侵袭能力。结果荧光素酶实验示miR-205靶向调控KLF12(P=0.001 6)。qRT-PCR检测结果示MAD-MB-468细胞中miR-205表达下调(P=0.007),KLF12表达升高(P=0.039);转染miR-205 mimics过表达miR-205,miR-205表达显著升高(P=0.000),KLF12表达减少(P=0.038)。Western blot检测结果示miR-205过表达,MDAMB-468细胞KLF12表达显著升高(P=0.007 9)。CCK8实验示miR-205未参与细胞增殖。Transwell实验示过表达miR-205显著抑制细胞侵袭能力(P=0.001)。结论 miR-205是BLBC特异性的miRNA,通过负性靶向调控原癌基因KLF12和抑制侵袭具有抑癌基因的功能。miR-205和KLF12为BLBC的诊断和治疗提供潜在的分子标志物和新思路。     

miRNA-200b通过靶向RhoA抑制宫颈癌细胞增殖、促进细胞凋亡

目的:探讨miRNA-200b通过靶向调控RhoA对宫颈癌细胞增殖和凋亡的影响.方法:将宫颈癌HeLa细胞分为5组:空白对照组、阴性对照组、miRNA-200b mimic组、RhoA阴性对照组、RhoA过表达组,转染后48 h收集细胞.RT-PCR检测miRNA-200b表达;双荧光素酶靶标实验验证miRNA-200...     

三氧化二砷抑制大鼠血管平滑肌细胞增殖并促进细胞凋亡

目的研究三氧化二砷(As2O3)对大鼠血管平滑肌细胞(VSMCs)的抑制增殖、促进凋亡及诱导细胞周期停滞的作用。方法经组织块贴壁法原代培养的大鼠VSMCs,应用MTT法、台盼蓝拒染法3、H-胸腺嘧啶胞苷掺入法测定VSMCs的细胞活力、生长及增殖;应用流式细胞仪、DNA梯带法分析细胞周期分布及凋亡;应用Western blot法检测凋亡相关基因产物P53及细胞周期蛋白激酶抑制蛋白P21waf1/cip1。结果1~16μmol/L As2O3对VSMCs细胞活力无明显影响,但可显著抑制细胞生长及DNA合成(P<0.05),并呈时间和剂量依赖性。8μmol/L As2O3可使细胞周期S期减少(P<0.05)、G0G1期增加(P<0.05),并出现sub-G1期凋亡峰。16μmol/L As2O3对VSMCs作用不同时间后出现典型的凋亡梯带样DNA片段。8μmol/L As2O3可显著上调VSMCs的P53及P21waf1/cip1蛋白产物(P<0.05),且呈时间依赖性。结论As2O3对VSMCs具有明确的抗增殖、促凋亡、阻滞细胞周期进程的作用,并且与P53、P21waf1/cip1表达上调密切相关。     

miR-187靶向S100A4抑制胶质瘤U251细胞增殖、侵袭、迁移并诱导细胞凋亡

目的探讨miR-187对胶质瘤U251细胞增殖、侵袭、迁移和凋亡的影响及其机制。方法将体外培养的U251细胞分为Con组(未处理)、NC组(转染miR-NC)、miR-187组(转染miR-187 mimics)。采用实时定量PCR检测miR-187的表达,MTT法、Transwell小室实验和流式细胞仪分别检测细胞增殖、迁移、侵袭和凋亡,实时定量PCR和Western blot检测S100A4 mRNA和蛋白的表达,双荧光素酶报告基因实验检测miR-187和S100A4的靶向关系。结果与Con组相比,miR-187组细胞中miR-187表达水平和细胞凋亡率均明显升高,而细胞增殖活性、侵袭细胞数、迁移细胞数和S100A4 mRNA、S100A4蛋白的表达水平均明显降低(P0.05);而miR-NC组与Con组相比无显著性差异(P0.05)。双荧光素酶报告基因实验证实S100A4是miR-187的靶基因。结论 miR-187通过靶向S100A4抑制U251细胞增殖、迁移、侵袭并诱导细胞凋亡。     

miR-144靶向Bcl-2抑制儿童急性淋巴细胞白血病细胞增殖并促进凋亡的分子机制研究

目的:观察miR-144对儿童急性淋巴细胞白血病(ALL)CEM/C1细胞增殖和凋亡的影响,并探讨其作用机制。方法:运用qRT-PCR法和Western blot法检测非恶性血液病患儿脊髓组织(Normal组)、ALL患儿脊髓组织(ALL组)、CEM/C1细胞(CEM/C1组)、ALL癌细胞珠MOLT-4(MOLT-4组)和CCRF-CEM(CCRF-CEM组)中miR-144、Bcl-2mRNA和Bcl-2蛋白表达水平并比较组间差异;将不同质粒以脂质体法转染至CEM/C1细胞,分为miR-NC组(转染miR-NC)、miR-144组(转染miR-144mimics)、inhibitor NC组(转染inhibitor NC)、miR-144inhibitor组(转染miR-144inhibitor)、si-NC组(转染si-NC)、si-Bcl-2组(转染si-Bcl-2)、miR-144+Vector组(miR-144mimics和pcDNA 3.1共转染)、miR-144+Bcl-2组(miR-144mimics和pcDNA 3.1-Bcl-2共转染),Western blot检测各组细胞中Bcl-2蛋白表达;MTT法检测各组细胞增殖;流式细胞术检测各组细胞凋亡;双荧光素酶报告基因实验检测各组细胞的荧光素酶活性。结果:与Normal组相比,ALL组miR-144表达显著降低,Bcl-2mRNA和蛋白表达均显著升高(P0.01)。与CEM/C1组比较,MOLT-4组和CCRF-CEM组miR-144表达显著升高,Bcl-2mRNA和蛋白表达均显著降低(P0.01)。与miR-NC组相比,miR-144组细胞增殖率降低,凋亡率升高,Bcl-2(WT)细胞的荧光活性降低(P0.01)。与si-NC组相比,si-Bcl-2组细胞增殖率降低,凋亡率升高(P0.01)。与miR-144+Vector组相比,miR-144+Bcl-2组细胞增殖率降低,凋亡率升高(P0.01)。结论:miR-144可抑制儿童ALL癌细胞增殖,促进其凋亡,可能与其能靶向Bcl-2有关,或可为儿童ALL治疗提供新的靶点。     

乙肝病毒X蛋白抑制小鼠足细胞系MPC5增殖并促进其凋亡

目的探讨乙肝病毒X蛋白(HBx)对条件永生型小鼠足细胞系(MPC5)增殖与凋亡的影响。方法用携带HBx基因的pEX质粒转染MPC5细胞,实时荧光定量PCR(RT-qPCR)验证转染效率。实验分空白对照组(MPC5 group)、阴性对照组(MPC5-pEX-neo group)、HBx转染组(MPC5-pEX-HBx group)。MTT法检测足细胞存活率;流式细胞计量术检测细胞凋亡;Western blot检测细胞中nephrin、STAT3、JAK2、p-STAT3和p-JAK2、caspase-3蛋白表达。结果 HBx转染MPC5细胞48 h后HBx表达最高。HBx转染组中nephrin蛋白表达水平显著低于空白对照及阴性对照(均P0.01)。转染HBx基因后足细胞增殖明显受抑,同时凋亡率显著增高(均P0.01)。此外,HBx转染组STAT3、p-STAT3、JAK2和p-JAK2蛋白表达较空白对照及阴性对照组均显著增高(均P0.01), caspase-3在HBx转染组中的表达也显著增高(均P0.01)。结论 HBx可下调足细胞中nephrin蛋白表达,抑制足细胞增殖,并促进其凋亡的发生。其作用机制可能与STAT3/JAK2信号通路活化有关。     

miR-205 regulates A549 cells proliferation by targeting PTEN

miR-205 is an epithelial-specific miRNA and has been shown to orchestrate some cellular processes such as epithelial mesenchymal transition (EMT) and differentiation fate of stem cells in mammary gland. miR-205 play a part of a tumor suppressor in human cancers. However, the role of miR-205 in lung cancer is unclear. In this study, we detected the expression level of miR-205 in 46 cases clinical lung cancer specimens and adjacent normal tissues by stem-loop RT-PCR. We found that the expression of miR-205 was significantly increased in lung cancer specimens compared to adjacent normal tissues (P < 0.01). Furthermore, we observed the expressions of PTEN protein and mRNA in lung cancer tissues and adjacent normal tissues by methods of western blot and Real time PCR respectively. We found that the expressions of PTEN protein and mRNA was significantly decreased in lung cancer specimens compared to adjacent normal tissues. And then, we found there is a negative relationship between the expression of miR-205 and PTEN mRNA in lung cancer by analyzed. To validate whether PTEN was direct targets of miR-205, a dual-luciferase reporter assay was employed, the result showed that PTEN is a target gene of MiR-205. In subsequent experiments, we examined the expressions of PTEN protein and mRNA after transfection of miR-205 mimics or inhibitor into A549 cells, and A549 cell proliferation was measured by CCK-8 tests. We found that the expression of PTEN protein and mRNA in A549 cells were significantly down-regulated or up-regulated after miR-205 mimics and miR-205 inhibitors transfected into, and miR-205 could inhibits A549 cells proliferation. These results indicate that miR-205 might inhibitor the proliferation of A549 cells by regulating the expression of PTEN.     

Knockdown of NPM1 by RNA interference inhibits cells proliferation and induces apoptosis in leukemic cell line

Nucleophosmin (NPM1) is an abundant and ubiquitously expressed phosphoprotein that is known to influence solid tumors progression. However, little is known about the role of NPM1 in leukemia. Here, we knocked down the NPM1 expression by RNA interference to investigate the role of NPM1 in leukemic cells proliferation and apoptosis. The interference vector pNPM1-shRNA was constructed and transfected into the human leukemic K562 cell line. The expression levels of NPM1 mRNA and protein were detected by quantitative real-time PCR and Western blot, respectively. Cells proliferation potential in vitro was assessed by methyl thiazolyl tetrazolium (MTT) and colony formation assays. Flow cytometry was used to detect the distribution of cell cycle. Cellular apoptosis was reflected by the relative activities of caspase-3 and caspase-8. The results showed that the expression levels of NPM1 mRNA and protein in K562 cells were significantly reduced after pNPM1-shRNA transfection. The cells growth was significantly inhibited in a time-dependent manner and the number of colonies was significantly reduced in the pNPM1-shRNA transfected cells. Meanwhile, the percentage of cells in G1 phase in the K562/pNPM1-shRNA cells was significantly increased. In addition, there were higher relative activities of caspase-3/8 in the pNPM1-shRNA transfected cells. These results indicate that down-regulation of NPM1 expression inhibits leukemic cells proliferation, blocks cell cycle progression and induces cellular apoptosis. It may implicate a potential target for leukemia gene therapy.     

MiR-508-3p promotes proliferation and inhibits apoptosis of middle ear cholesteatoma cells by targeting PTEN/PI3K/AKT pathway

Cholesteatoma of the middle ear is a common disease in otolaryngology, which can lead to serious intracranial and extracranial complications. Recent studies showed that the dysregulation of microRNA may be involved in the formation of middle ear cholesteatoma. This study aimed to explore the regulatory effect of micro ribonucleic acid 508-3p (miR-508-3p) on proliferation and apoptosis of middle ear cholesteatoma cells and excavate its underlying regulatory mechanism. We found miR-508-3p expression was upregulated in tissues and cells of cholesteatoma which was inversely related to the expression of hsa_circ_0000007. Overexpression of miR-508-3p could notably facilitate cholesteatoma cell proliferation. Luciferase reporter assay showed that miR-508-3p bound the 3''-untranslated region of its downstream mRNA PTEN. Gain and loss of functions of miR-508-3p were performed to identify their roles in the biological behaviors of cholesteatoma cells, including proliferation and apoptosis. Rescue assays confirmed that PTEN could reverse the effect of miR-508-3p overexpression on cell proliferation. In a word, this study validated that the development of cholesteatoma may regulated by hsa_circ_0000007/miR-508-3p/ PTEN/ PI3K/Akt axis.     

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.     

西格列汀抑制膀胱癌细胞增殖和诱导细胞凋亡

目的观察西格列汀对膀胱癌细胞增殖和凋亡的影响。方法西格列汀处理膀胱癌细胞T24及5637后,MTS检测细胞的增殖活性;Western blot检测细胞内组织蛋白酶B以及凋亡相关蛋白的表达变化。结果西格列汀显著抑制了膀胱癌细胞的增殖,经西格列汀处理后,T24及5637细胞内组织蛋白酶B的蛋白表达水平明显下降,凋亡相关蛋白PARP的剪切体表达水平升高。结论西格列汀抑制膀胱癌细胞增殖和诱导癌细胞凋亡,同时降低细胞内组织蛋白酶B的蛋白表达。     

Knockdown of WISP1 inhibit proliferation and induce apoptosis in ALL Jurkat cells

WISP1, a Wnt-induced secreted protein, has been found to have anticancer activity. ALL is a leading cause of death. Here we investigate the WISP1 effects on ALL Jurkat cells. Cell viability was assessed by CCK-8. Cell cycle and apoptosis were detected by flow cytometry. Mitochondrial membrane potential (MMP) was monitored using TMRM. Generation of reactive oxygen species (ROS) was quantified using DCFH-DA. Western blot was used to detect the expression of cell proliferation and apoptosis related genes. The results showed that knockdown of WISP1 significantly inhibited proliferation of Jurkat cells. Parallelly, cell cycle distribution was increased at G1 phase and apoptotic rate was induced after WISP1 knockdown. Furthermore, knockdown of WISP1 induced apoptosis of Jurkat cells was also associated with loss of MMP and generation of ROS. Western blot results showed that the protein expression p-AKT, PCNA, CDK1, P-ERK, CDK2, VEGF, VEGFR2 and Bcl2 were decreased, while the expression of Bax was up-regulated. In conclusion, WISP1 plays an important role in proliferation and apoptosis of Jurkat cells in mitochondria dependent pathway, the specific mechanisms need further study.     

MicroRNA-196a overexpression promotes cell proliferation and inhibits cell apoptosis through PTEN/Akt/FOXO1 pathway

MicroRNAs (miRNAs) are endogenous, non-coding, small RNAs, which play a critical role in regulating varieties of the biological and pathologic processes. MiR-196a has been reported to take part in tumorigenic progression of osteosarcoma (OS). However, the effects of miR-196a on OS are still unclear. The objective of this study is to investigate the molecular mechanism of miR-196a in osteosarcoma cells. In the present study, the expression of miR-196a in OS cell lines was detected by real-time PCR. We found that the expression level of miR-196a was markedly up-regulated in osteosarcoma cell lines compared with normal osteoblastic cells. Then, the miR-196a mimic was transiently transfected into MG63 and U2OS cells using Lipofectamine™ 2000 reagent. Subsequently, the MTT and Brdu-ELISA results showed that up-regulation of miR-196a promoted the cell viability and proliferation. Our results also showed that miR-196a mimic accelerated cell cycle progression of MG63 and U2OS cells by down regulation of p21 and p27, and upregulation of cyclin D1. In addition, overexpression of miR-196a suppressed apoptosis of MG63 and U2OS cells due to increasing BCL2L2 and MCL-1 expressions, and then inactivating caspase-3. Eventually, the effect of miR-196a mimic on the PTEN/phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was explored by Western blot. From our results, transfection of miR-196a decreased the expression of PTEN and increased the phosphorylation of PI3K and Akt. Taken together, miR-196a should be an oncogene in osteosarcoma. The possible mechanism was that overexpression of miR-196a promoted proliferation of MG63 and U2OS cells by modulating the PTEN/PI3K/Akt signaling pathway.     

Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis

Recent studies have revealed that osthole,an active constituent isolated from the fruit of Cnidium monnieri(L.) Cusson,a traditional Chinese medicine,possesses anticancer activity.However,its effect on breast cancer cells so far has not been elucidated clearly.In the present study,we evaluated the effects of osthole on the proliferation,cell cycle and apoptosis of human breast cancer cells MDA-MB 435.We demonstrated that osthole is effective in inhibiting the proliferation of MDA-MB 435 cells,The mitochondrion-mediated apoptotic pathway was involved in apoptosis induced by osthole,as indicated by activation of caspase-9 and caspase-3 followed by PARP degradation.The mechanism underlying its effect on the induction of G1 phase arrest was due to the up-regulation of p53 and p21 and down-regulation of Cdk2 and cyclin D1 expression.Were observed taken together,these findings suggest that the anticancer efficacy of osthole is mediated via induction of cell cycle arrest and apoptosis in human breast cancer cells and osthole may be a potential chemotherapeutic agent against human breast cancer.