MicroRNA-490-5p靶向CDK1通过ERK信号通路参与胃癌细胞周期调控

目的:探讨miR-490-5p对胃癌细胞增殖与周期的调控作用和分子机制,以期为临床治疗胃癌提供新的有效靶点.方法:收集30例胃癌患者的胃癌组织及相应的癌旁组织标本;采用Real-time PCR法检测miR-490-5p和CDK1的表达水平;分析细胞周期相关蛋白 CDK1 与 miR-490-5p 的靶向关系.MTT法和流式细胞仪检测转染后胃癌细胞生长以及细胞周期情况.结果:与癌旁组织相比,胃癌组织中miR-490-5p的表达显著下调(P<0.001);转染 miR-490-5p mimics 的细胞中miR-490-5p的表达显著上调,而miR-490-5p inhibitors中miR-490-5p显著下降(均P<0.001);CDK1 是 miR-490-5p 的靶基因;下调miR-490-5p、上调CDK1能促进胃癌细胞的增殖能力及G1/S期的转化(均P<0.001).结论:通过上调miR-490-5p 可以抑制胃癌细胞的恶性增殖,减少CDK1表达,抑制 ERK信号途径,降低了G1/S期的转化速率,从而为胃癌诊断及治疗提供新靶标.     

GADD45α在膀胱癌组织和细胞中的表达及作用机制

目的:检测GADD45α基因在膀胱癌中的表达情况及作用机制。方法:分别使用免疫组化、荧光定量PCR和Western blot实验检测GADD45α基因在膀胱癌组织中的表达情况;用慢病毒包装质粒上调膀胱癌细胞中GADD45α的表达后,流式细胞仪分析膀胱癌细胞周期比例分布情况和用Western blot实验检测细胞周期蛋白激酶水平变化情况。结果:膀胱癌组织中GADD45α的表达水平较癌旁正常组织的表达降低;上调膀胱癌细胞中GADD45α的表达后,与control细胞相比,高表达GADD45α细胞的克隆大小和数目皆明显下降（86±12.3 vs 134±13.5,P＜0.05）;升高GADD45α表达后,膀胱癌细胞G2期比例升高至56.54%,而G1期比例则由82.87%下降至40.8%,S期比例由17.13%下降至2.66%,膀胱癌细胞周期被阻滞在G2/M期,伴随细胞周期蛋白激酶cdc2/cyclinB1的表达下降。结论:GADD45α在膀胱癌中表达下降,GADD45α通过抑制cdc2/cyclinB1的表达活性以阻滞膀胱癌细胞周期在G2/M期,抑制膀胱癌细胞的增殖。     

miR-16对大肠癌细胞增殖能力的影响

目的研究miR-16在大肠癌组织中的表达及其在大肠癌细胞株HCT116和LoVo细胞增殖中的作用。方法运用Realtime PCR法检测miR-16在33对配对肠癌及癌旁正常组织中的表达。HCT116和LoVo细胞转染miR-16 抑制剂、阴性对照剂和模拟剂;CCK8法检测细胞增殖;流式细胞仪进行细胞周期检测;Western blot法检测细胞中cyclinD1、CDK6和GAPDH蛋白表达。结果（1）Realtime PCR结果显示miR-16在大肠癌组织中低表达（P=0.047）。（2）miR-16过表达可抑制HCT116和LoVo细胞增殖,诱导细胞G₀/G₁期阻滞,降低cyclinD1和CDK6蛋白表达。结论miR-16过表达可以抑制大肠癌细胞的增殖能力。     

MicroRNA-10a通过靶向作用E2F3抑制肝癌细胞的增殖

目的：探讨微小RNA-10a（microRNA-10a,miR-10a）对肝癌细胞增殖的影响及其作用机制。方法：收集广西医科大学附属肿瘤医院肿瘤科2001年10月至2005年7月144例肝癌患者手术切除的肝癌组织和癌旁组织（距癌灶组织边缘2~5 cm）标本,Real-time PCR法分析144例肝癌组织及癌旁组织中miR-10a的表达量。在肝癌细胞（QGY-7701、Huh7、PCL/PRF/5）中转染miR-10a 模拟物,Real-time PCR法检测转染后细胞miR-10a的表达水平;CCK-8法检测过表达miR-10a的肝癌细胞的增殖水平,流式细胞术检测过表达miR-10a的肝癌细胞的凋亡和细胞周期;生物信息学预测并以Western blotting检测过表达miR-10a的肝癌细胞中转录因子E2F3的表达量。结果：与癌旁组织相比,肝癌组织中的miR-10a显著低表达\[（-9.89±168） vs （-7.84±1.97）, P =0.000\]。转染miR-10a模拟物后肝癌细胞系中miR-10a的表达量是转染对照小RNA组或空白组细胞的16倍左右。过表达miR-10a可显著抑制7种肝癌细胞（QGY-7701、QGY-7703、Huh7、PCL/PRF/5、HepG2、BeL-7402、SMMC-7721）的增殖（均 P <0.05）,并引起肝癌细胞细胞周期G1/S期阻滞,但并不能诱导肝癌细胞发生凋亡。生物信息学预测显示E2F3是miR-10a可能的靶分子,Western blotting检测显示过表达miR-10a可明显抑制肝癌细胞中E2F3的表达\[（0.50±0.12) vs (0.79±0.21), P <0.05\]。结论：人肝癌组织中低表达miR-10a,转染miR-10a模拟物后多种肝癌细胞的增殖均受到明显抑制,其机制可能与miR-10a靶向作用转录因子E2F3并阻滞肝癌细胞细胞周期于G1/S期有关。     

miR-181a诱导慢性淋巴细胞白血病细胞周期阻滞及细胞凋亡的分子机制探讨

目的:探讨miR-181a在慢性淋巴细胞白血病细胞周期阻滞及细胞凋亡中的作用并初步阐明其调控机制。方法:利用荧光定量PCR检测慢性淋巴细胞白血病患者肿瘤细胞中miR-181a的表达水平;利用双荧光素酶试验验证miR-181a的靶基因MCL-1和BCL-2,利用免疫印迹试验检测过表达miR-181a对p53通路中关键蛋白MCL-1和BCL-2的影响,利用流式细胞仪检测过表达miR-181a对慢性淋巴细胞白血病细胞凋亡和周期的影响。结果:RT-qPCR结果显示,慢性淋巴细胞白血病细胞中miR-181a的表达下调;双荧光素酶试验验证发现,p53通路下游基因MCL-1和BCL-2均是miR-181a的靶基因;免疫印迹试验结果显示,过表达miR-181a抑制了MCL-1和BCL-2蛋白的表达;流式细胞仪检测结果显示,过表达miR-181a阻滞细胞周期G1/S期转换,促进细胞凋亡。结论:miR-181a 通过靶向作用p53通路中关键基因MCL-1和BCL-2,诱导细胞周期阻滞及细胞凋亡,可能在慢性淋巴细胞白血病中扮演着抑癌基因的角色。     

微小RNA-106a在膀胱癌组织中的表达及其对膀胱癌T24细胞增殖、侵袭作用的影响

目的:观察微小RNA-106a（miR-106a）在膀胱癌组织中的表达及其对膀胱癌T24细胞增殖、侵袭作用的影响。方法:提取35例膀胱癌组织及正常膀胱组织中的总RNA，实时定量RT-PCR法检测miR-106a在膀胱癌组织及正常膀胱组织中的表达量；培养膀胱癌T24细胞，分成实验组及对照组，实验组转染miR-106a mimics，对照组转染空白脂质体，噻唑蓝(MTT)法检测转染miR-106a mimics后对T24细胞增殖的影响，流式细胞仪检测T24细胞周期变化，Transwell侵袭小室检测T24细胞侵袭能力。结果:膀胱癌组织中miR-106a的表达量显著低于正常膀胱组织。膀胱癌T24细胞转染miR-106a mimics后，T24细胞增殖能力降低，细胞周期停滞于G1期，Transwell侵袭小室检测提示T24细胞侵袭能力降低。结论:miR-106a的表达量在膀胱癌组织中减少，miR-106a过表达抑制膀胱癌T24细胞的增殖及侵袭能力。     

δ-三烯生育酚通过上调MicroRNA-34a抑制宫颈癌细胞增殖

目的 探究δ-三烯生育酚对HeLa宫颈癌细胞增殖的影响以及microRNA-34a（miR-34a）是否介导这一过程。方法 用荧光定量PCR方法检测miR-34a和细胞增殖关键基因表达的变化,CCK-8检测细胞活力变化,Western blot检测细胞增殖关键基因蛋白水平的变化,流式细胞术检测细胞周期变化;并利用miR-34a模拟剂和抑制剂,探究其对细胞增殖的影响。结果 δ-三烯生育酚处理HeLa宫颈癌细胞36 h显著降低细胞活力并呈剂量依赖效应。用20 μmol/L δ-三烯生育酚处理细胞36 h显著上调miR-34a表达,显著下调细胞周期关键基因CCND1,同时S细胞比例显著下降。细胞转染miR-34a模拟剂后,CCND1表达和细胞活力都下降;而转染miR-34a抑制剂则显著减弱δ-三烯生育酚的增殖抑制作用。结论 δ-三烯生育酚通过上调miR-34a抑制宫颈癌细胞增殖。     

miR-34a稳定表达株构建及其对宫颈癌Caski细胞功能影响的观察

目的:构建人miR-34a的真核表达载体,并研究其对宫颈癌细胞株Caski细胞活性的影响。方法:以人正常组织基因组DNA为模板,用PCR法扩增得到miR-34a的前体序列,构建miR-34a真核表达载体pEGFP-C1-miR34a。同时用重组载体pEGFP-C1-miR34a转染宫颈癌细胞株Caski细胞,筛选miR-34a稳定表达细胞系作为实验组,pEGFP-C1转染组为对照组(Control),用RT-PCR及Real time RT-RCR法鉴定miR-34a在克隆细胞中的表达,并用MTT、FCM分析miR-34a的生物学特性,RT-PCR研究检测其靶基因表达改变。结果:成功构建了人miR-34a真核表达载体pEGFP-C1-miR34a,并在宫颈癌Caski细胞中上调miR-34a的表达,实验组miR-34a的表达相对于对照组升高约3.13倍;MTT提示,实验组miR-34a能够显著的抑制Caski细胞增殖,差异有统计学意义,P=0.018;实验组G0/G1期百分率为77.7%,与对照组的50%比较,差异有统计学意义,P=0.004;而实验组S期百分率为20.3%,对照组为35.4%,两组比较差异有统计学意义,P=0.027;实验组细胞miR-34a靶基因细胞周期基因CDK4、CDK6、Cyclin E2和E2F1mRNA的表达下调,与对照组比较差异有统计学意义,P=0.002。结论:成功构建人miR-34a的真核表达载体,并能显著抑制宫颈癌细胞株Caski细胞活性,为进一步研究miR-34a在宫颈癌中的功能及基因调控机制奠定了实验基础。     

斯钙素1的表达对肺癌细胞A549细胞周期及凋亡的影响

背景与目的：斯钙素1(stanniocalcin l,STC1)在多种癌组织中表达上调,且与癌组织的恶性程度相关,但STC1在肺癌细胞中的分子作用机制尚不明确。本研究旨在探讨STC1的表达对肺癌细胞A549细胞周期及凋亡的影响。方法：构建STC1基因RNA干扰的肺癌细胞株A549-STC1-siRNA和对照细胞株A549-Vector,用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)和蛋白[质]印迹法(Western blot)检测A549-Vector及A549-STC1-siRNA细胞株的细胞周期蛋白基因CyclinA、CyclinB1、CyclinD1、CyclinE、CDK2、CDK4,凋亡抑制基因Bcl-2、Bcl-xl及凋亡诱导基因Caspase-3、Bax、Bak、Bid的表达水平,用流式细胞术检测STC1基因对A549细胞周期的影响,用原位末端标记(terminal deoxynucleotidyl transferase-mediated nick-end labeling,TUNEL)检测STC1基因对A549细胞凋亡的影响。结果：与A549-Vector细胞相比,A549-STC1-siRNA的细胞周期蛋白基因CyclinA、CyclinB1、CyclinD1、CyclinE、CDK2和CDK4在转录和蛋白表达水平上均显著减少(P<0.05),G0/G1期细胞比例明显增加,S期及G2/M期细胞比例降低(P<0.05),细胞周期受阻;A549-STC1-siRNA的凋亡抑制基因Bcl-2和Bcl-xl表达下调(P<0.05),而凋亡诱导基因Caspase-3、Bax、Bak及Bid显著上调(P<0.05);TUNEL实验表明,A549-STC1-siRNA细胞的凋亡率明显增加。结论：STC1基因的低表达可阻滞肺癌细胞A549的细胞周期,抑制细胞增殖,同时促进细胞凋亡。     

miR-572 通过靶向调控 WWOX 影响肺癌细胞恶性生物学行为

目的 探讨miR-572靶向氧化还原酶的WW结构域（WW domain containing oxidoreductase,WWOX）调控肺癌细胞增殖、凋亡的分子机制。方法 选取50例2016年3月—2018年5月十堰市太和医院手术切除肺癌组织及其相应的癌旁组织。利用脂质体转染技术将miR-572 inhibitor和miR-572 mimics转染至肺癌A549和L9981细胞;采用qRT-PCR检测miR-572和WWOX的mRNA表达水平,MTT法检测细胞增殖能力,流式细胞仪检测细胞周期和细胞凋亡情况。通过生物信息学软件Targetscan分析miR-572的靶基因,荧光素酶报告基因实验检验miR-572和WWOX的靶向关系。结果 miR-572在肺癌组织和细胞中高表达（均P<0.05）。下调miR-572后,肺癌细胞A549、L9981中miR-572表达水平均降低（均P<0.05）,细胞增殖能力也降低（均P<0.05）,细胞G0/G1期比例和细胞凋亡率均升高（均P<0.05）;而上调miR-572后,肺癌A549、L9981细胞增殖能力升高（均P<0.05）,细胞G0/G1期比例和细胞凋亡率则降低（均P<0.05）。WWOX在肺癌组织和细胞中低表达（均P<0.05）,且在肺癌组织中WWOX与miR-572表达呈负相关（r=-0.669,P<0.001）。下调WWOX可逆转下调miR-572对肺癌细胞的增殖抑制、周期阻滞和凋亡促进作用。 结论 下调miR-572可抑制肺癌细胞增殖并诱导细胞凋亡,其作用机制可能与靶向负调控WWOX有关。     

MicroRNA-320c inhibits tumorous behaviors of bladder cancer by targeting Cyclin-dependent kinase 6

Background

Increasing evidence has suggested that dysregulation of microRNAs (miRNAs) could contribute to human disease including cancer. Previous miRNA microarray analysis illustrated that miR-320c is down-regulated in various cancers. However, the roles of miR-320c in human bladder cancer have not been well elucidated. Therefore, this study was performed to investigate the biological functions and molecular mechanisms of miR-320c in human bladder cancer cell lines, discussing whether it could be a therapeutic biomarker of bladder cancer in the future.

Methods

Two human bladder cancer cell lines and samples from thirteen patients with bladder cancer were analyzed for the expression of miR-320c by quantitative RT-PCR. Over-expression of miR-320c was established by transfecting mimics into T24 and UM-UC-3. Cell proliferation and cell cycle were assessed by cell viability assay, flow cytometry and colony formation assay. Cell motility ability was evaluated by transwell assay. The target gene of miR-320c was determined by luciferase assay, quantitative RT-PCR and western blot. The regulation of cell cycle and mobility by miR-320c was analyzed by western blot.

Results

We observed that miR-320c was down-regulated in human bladder cancer tissues and bladder cancer cell lines T24 and UM-UC-3. Over-expression of miR-320c could induce G1 phase arrest in UM-UC-3 and T24 cells, and subsequently inhibited cell growth. We also indentified miR-320c could impair UM-UC-3 and T24 cell motility. In addition, we identified CDK6, a cell cycle regulator, as a novel target of miR-320c. Moreover, we demonstrated miR-320c could induce bladder cancer cell cycle arrest and mobility via regulating CDK6. We also observed that inhibition of miR-320c or restoration of CDK6 in miR-320c-over-expressed bladder cancer cells partly reversed the suppressive effects of miR-320c.

Conclusions

miR-320c could inhibit the proliferation, migration and invasion of bladder cancer cells via regulating CDK6. Our study revealed that miR-320c could be a therapeutic biomarker of bladder cancer in the future.     

MiR-101 inhibits cell growth and tumorigenesis of Helicobacter pylori related gastric cancer by repression of SOCS2

Several microRNAs (miRNA) have been implicated in H. pylori related gastric cancer (GC). However, the molecular mechanism of miRNAs in gastric cancer has not been fully understood. In this study, we reported that miR-101 is significantly down-regulated in H. pylori positive tissues and cells and in tumor tissues with important functional consequences. Ectopic expression of miR-101 dramatically suppressed cell proliferation and colony formation by inducing G1-phase cell-cycle arrest. We found that miR-101 strongly reduced the expression of SOCS2 oncogene in GC cells. Similar to the restoring miR-26 expression, SOCS2 down-regulation inhibited cell growth and cell-cycle progression, whereas SOCS2 over-expression rescued the suppressive effect of miR-101. Mechanistic investigations revealed that miR-101 suppressed the expression of c-myc, CDK2, CDK4, CDK6, CCND2, CCND3, and CCNE2, while promoted tumor suppressor p14, p16, p21 and p27 expression. In clinical specimens, SOCS2 was over-expressed in tumors and H. pylori positive tissues and its mRNA levels were inversely correlated with miR-101 expression. Taken together, our results indicated that miR-101 functions as a growth-suppressive miRNA in H. pylori related GC, and that its suppressive effects are mediated mainly by repressing SOCS2 expression.     

高表达的miR-301b和miR-769-5p对非小细胞肺癌A549细胞生长的影响

目的:探讨高表达的miR-301b和miR-769-5p对非小细胞肺癌A549细胞生长的影响。方法:采用实对荧光定量聚合酶链反应检测A549细胞中miR-301b和miR-769-5p的表达;使用 qRT-PCR检测增殖相关基因miRNA表达的变化,并将实验组miR-301b和miR-769-5p表达量上调(与阴性对照组比较);采用MTT法检测并对比A549细胞增殖的改变;采用流式细胞仪检测并对比A549细胞周期以及凋亡的改变。结果:qRT-PCR结果表明,浸染了miR-301b和miR-769-5p过表达载体的A549细胞中miR-301b和miR-769-5p水平明显高于阴性对照组,差异有统计学意义（P＜0.01)。miR-301b过表达使G0/G1期的A549细胞增加,S和G2/M期的细胞减少;miR-769-5p过表达使G0/G1和G2/M期细胞增加,S期细胞减少。结论:miR-301b对靶基因FOXF2具有调控作用,miR-769-5p对靶基因ARID1A具有调控作用。miR-301b和miR-769-5p的过表达对肺癌A549细胞的细胞周期产生了明显影响,但没有影响细胞增殖和凋亡,miR-301b和miR-769-5p有可能成为肺癌分子靶向治疗调控效应靶点,可以进一步探索研究。     

miR-361-5p通过靶向CCND1逆转胃癌SGC-7901细胞奥沙利铂耐药性

目的:探讨miR-361-5p对胃癌SGC-7901细胞奥沙利铂(oxaliplatin,OXA)耐药性的影响及其作用机制.方法:采用qPCR法检测miR-361-5p在胃癌细胞MKN-45、MGC80-3、SGC-7901和OXA耐药细胞SGC-7901/OXA中的表达水平.利用脂质体转染技术分别将miR-361-5...     

miR-1297 inhibits osteosarcoma cell proliferation and growth by targeting CCND2

Cyclin D2 (CCND2) is abnormally overexpressed in many tumor types and has been associated with tumor cell proliferation. Although the important role of miR-1297 is well established, the molecular mechanism between CCND2 and miR-1297 in osteosarcoma (OS) has not been determined. In the present study, we found CCND2 was highly expressed in OS cells, and its downregulation suppressed cell proliferation, resulting in G1 phase cell cycle arrest. In contrast, miR-1297 was lowly expressed in OS compared to normal tissue. Several data platforms predicted that CCND2 was a target of miR-1297, which was validated by a dual-luciferase reporter assay that revealed miR-1297 could bind with CCND2-3’UTR. miR-1297 overexpression greatly inhibited CCND2 protein expression and exerted the same phenotypic effect as CCND2 downregulation in OS cells. Furthermore, miR-1297 inhibition could also be rescued by CCND2. Nude mice injected cells stable overexpressing miR-1297 OS cells showed lower size and tumor weight. Moreover, lower fluorescence activity recorded by in vivo imaging system and bone erosion revealed by microCT in the miR-1297 group demonstrated miR-1297 inhibited OS tumor growth via CCND2. Our findings demonstrated that miR-1297 can inhibit proliferation and tumor growth in OS by directly targeting CCND2, which indicates that miR-1297 may represent a novel therapeutic target for OS.     

MicroRNA miR-302 inhibits the tumorigenecity of human pluripotent stem cells by coordinate suppression of the CDK2 and CDK4/6 cell cycle pathways

miR-302 is the major microRNA found in human embryonic stem cells and induced pluripotent stem cells, but its function has been unclear. In mice, there is evidence that miR-302 may silence p21Cip1 (CDKN1A) to promote cell proliferation, whereas studies in human reprogrammed pluripotent stem cells suggested that elevated miR-302 expression inhibited cell cycle transit. Here, we clarify this difference, reporting that in human cells, miR-302 simultaneously suppressed both the cyclin E-CDK2 and cyclin D-CDK4/6 pathways to block>70% of the G1-S cell cycle transition. Concurrent silencing of BMI-1, a cancer stem cell marker targeted by miR-302, further promoted tumor suppressor functions of p16Ink4a and p14/p19Arf directed against CDK4/6-mediated cell proliferation. Among all G1 phase checkpoint regulators, human p21Cip1 was found not to be a valid target of miR-302. Overall, our findings indicate that miR-302 inhibits human pluripotent stem cell tumorigenicity by enhancing multiple G1 phase arrest pathways rather than by silencing p21Cip1.     

lncRNA CTD-2196E14.5通过海绵吸附miR-744-5p抑制膀胱癌细胞的增殖和侵袭

目的:探讨CTD-2196E14.5通过调控miR-744-5p表达对膀胱癌细胞增殖和侵袭的影响。方法:通过检索TCGA数据库分析膀胱癌组织及癌旁组织中CTD-2196E14.5的表达水平及其与膀胱癌患者总生存期的关系。采用荧光实时定量聚合酶链反应(fluorescence quantitative polymerase chain reaction, qPCR)检测膀胱癌细胞株MGH-U3、T24、253J、J82和正常膀胱上皮细胞SV-HUC-1中CTD-2196E14.5的表达水平。采用脂质体介导技术将pcDNA-CTD-2196E14.5质粒、pcDNA空载质粒分别转染253J细胞,即CTD-2196E14.5组和NC组。采用MTT法、流式细胞术和Transwell实验检测CTD-2196E14.5对膀胱癌253J细胞增殖、细胞周期和侵袭的影响。应用lncRNA2function软件预测和双荧光素酶报告基因实验验证CTD-2196E14.5与miR-744-5p的靶向关系。qPCR检测miR-744-5p的表达。Western blotting检测AMPK信号通路蛋白和细胞周期...     

肿瘤相关巨噬细胞对膀胱癌细胞增殖和血管生成的影响

目的:探讨肿瘤相关巨噬细胞（TAMs）对膀胱癌细胞增殖和血管生成的影响。方法:采用巨噬细胞U937的上清液培养膀胱癌细胞系T24,CCK-8法检测膀胱癌细胞的增殖能力;流式细胞术检测其对细胞周期的影响;通过实时荧光定量PCR和Western blot检测膀胱癌细胞中血管内皮生长因子（VEGF）的mRNA和蛋白表达以及AKT磷酸化水平。结果:CCK-8结果表明TAMs促进膀胱癌细胞的增殖;流式结果显示TAMs能促进细胞周期G1-S期转换;实时荧光定量PCR和Western blot 结果显示TAMs促进膀胱癌细胞内VEGF的mRNA和蛋白表达,并促进AKT磷酸化。结论:TAMs促进细胞周期G1-S期转换,促进AKT磷酸化和VEGF的表达,且TAMs有望成为膀胱癌治疗和预后判断新的靶点。