致癌性微小RNA-106a对正常胃黏膜上皮细胞和胃癌细胞生长的影响

目的: 探讨微小RNA-106a(miR-106a)是否具有致癌特性。方法: 用脂质体把miR-106a类似物转染到正常胃黏膜上皮细胞GES-1中,然后用MTT方法检测该细胞的增殖情况。用脂质体把miR-106a抑制剂转染到胃癌细胞MGC-803和SGC-7901中,然后分别用流式细胞术和Western印迹方法检测细胞周期分布和细胞周期相关蛋白表达的变化;最后观察裸鼠胃癌移植瘤的生长情况。结果: miR-106a类似物以剂量依赖方式促进正常胃黏膜上皮细胞的增殖。miR-106a抑制剂通过抑制细胞周期素依赖的蛋白激酶(CDK)1和CDK2的表达阻滞MGC-803细胞于G₀/G₁期和G₂/M期,而通过抑制CDK1的表达阻滞SGC-7901于G₂/M期。动物实验结果显示,miR-106a抑制剂以剂量依赖方式抑制肿瘤的生长。结论: miR-106a可能参与了胃癌的发生过程。     

Hsa_circ_0020014调控胃癌细胞的增殖、迁移的作用研究

目的 探究hsa_circ_0020014在胃癌中的生物学功能及可能的调控机制.方法 利用实时荧光定量聚合酶链反应检测hsa_circ_0020014在胃生理上皮细胞及多种胃癌细胞系中的表达水平.CCK8方法及transwell实验检测hsa_circ_0020014对胃癌细胞增殖和迁移能力的作用.结果 Hsa_circ_0020014在MGC-803、HGC-27、SGC-7901、AGS等胃癌细胞系中显著高表达,且敲除hsa_circ_0020014能够明显抑制胃癌细胞的增殖、迁移能力.与GES-1相比,hsa-miR-199a/b-3p在多种胃癌细胞系中低表达,且敲除hsa_circ_0020014能上调hsa-miR-199a/b-3p的表达.结论 Hsa_circ_0020014能通过竞争性结合hsa-miR-199a/b-3p调控胃癌的增殖与迁移.     

DADS通过miR-222抑制人胃癌细胞系的增殖与侵袭

目的通过miRNA途径研究二烯丙基二硫(DADS)的抑瘤机制,以进一步阐明DADS抑制胃癌细胞增殖与转移的分子机制。方法将胃癌细胞系MGC-803细胞分为DADS处理组、miR-222模拟物组、miR-222抑制物组和阴性对照组;分别采用0、25、50、100、200和400μmol/L DADS处理MGC-803细胞;分别将miR-222模拟物、miR-222抑制物和scramble转染MGC-803细胞。qRT-PCR检测MGC-803细胞中miR-222表达;MTT法和Transwell侵袭实验检测MGC-803细胞增殖与侵袭能力;蛋白质印迹试验检测MGC-803细胞中TIMP3蛋白表达。结果 DADS可呈剂量依赖性下调MGC-803细胞中miR-222表达(P0.05);DADS能抑制MGC-803细胞的增殖与侵袭,外源高表达miR-222能促进MGC-803细胞的增殖与侵袭,而miR-222抑制物与DADS共同处理,MGC-803细胞的增殖与侵袭抑制作用最为显著(P0.05);DADS可下调MGC-803细胞中TIMP3蛋白的表达,外源高表达miR-222能上调MGC-803细胞中TIMP3蛋白的表达,而miR-222抑制物与DADS共同处理,MGC-803细胞中TIMP3蛋白的表达下调最为显著(P0.05)。结论 DADS通过下调miR-222的表达,靶向TIMP3抑制胃癌细胞的增殖与侵袭。     

shRNA抑制Nanog基因的表达对胃癌细胞SGC-7901分子生物学行为的影响

目的:利用RNA干扰技术构建并筛选出对Nanog基因有抑制作用的重组质粒pshRNA-Nanog,转染入胃癌细胞SGC-7901中,检测其对Nanog表达的影响及其对胃癌细胞SGC-7901的增殖、周期、凋亡以及迁移的影响。方法:构建出针对人Nanog基因构建出的干扰质粒pshRNA-Nanog,在脂质体的介导下将其转染胃癌细胞SGC-7901,通过荧光显微镜,RT-PCR和Western blot法检测其表达,筛选出抑制率最高的一组;将筛选出的转染效率最高的一组pshRNA-Nanog重组质粒转染胃癌细胞SGC-7901中,通过CCK-8检测其胃癌细胞SGC-7901增殖情况,流式细胞仪分析SGC-7901周期变化和凋亡情况,Transwell迁移实验验证Nanog对SGC-7901的迁移能力以及侵袭实验验证Nanog对SGC-7901侵袭能力的影响。结果:成功构建并筛选出了对胃癌细胞SGC-7901干扰效果最明显的一组重组质粒;细胞的增殖受到抑制,胃癌细胞侵袭能力及迁移能力减弱,细胞周期停滞在S期,细胞的凋亡明显增加。结论:Nanog基因与胃癌细胞的增殖能力、周期及凋亡、迁移和侵袭能力有密切的关系。     

MicroRNA-146a通过靶向TAK1诱导胃癌SGC-7901细胞凋亡

目的:观察微小RNA-146a(miR-146a)对胃癌SGC-7901细胞凋亡的影响及其可能的分子机制。方法:将miR-146a mimic(上调miR-146a表达)和miR-146a inhibitor(下调miR-146a表达)分别转染至人胃癌SGC-7901细胞中,同时设置miRNA无义序列转染为阴性对照组(NC组)。RT-q PCR检测转染后胃癌细胞miR-146a的表达水平;CCK-8法和流式细胞术检测miR-146a对SGC-7901胃癌细胞生长活力和凋亡的影响;RT-q PCR和Western blot法检测miR-146a上调或下调对转化生长因子β激活激酶1(TAK1)/核因子κB(NF-κB)通路的影响。结果:在SGC-7901细胞中,上调miR-146a表达显著促进细胞凋亡,而下调miR-146a表达则显著抑制细胞凋亡。与NC组相比,miR-146a mimics转染SGC-7901细胞后,TAK1的mRNA和蛋白表达均明显下降,差异均有统计学显著性(P0.05),而miR-146a inhibitors转染组TAK1的mRNA和蛋白质表达则显著增加(P0.05),提示miR-146a负调控TAK1表达。敲低TAK1促进SGC-7901细胞凋亡(P0.01),而TAK1过表达TAKI则抑制SGC-7901细胞凋亡(P0.01)。此外,过表达miR-146a和敲低TAK1均能显著上调NF-κB抑制蛋白α(IκBα)和下调B细胞淋巴瘤-2(Bcl-2)表达。结论:本研究结果表明miR-146a通过靶向TAK1抑制NF-κB途径,从而诱导胃癌细胞凋亡。     

MicroRNA-10b对人胃癌SGC-7901细胞增殖、迁移和侵袭能力的影响

目的探讨Micro RNA-10b(miR-10b)对人胃癌SGC-7901细胞增殖、迁移和侵袭的影响。方法应用Real-time PCR方法检测21例胃癌组织和癌旁正常组织中miR-10b的表达水平,应用Lipofectamine?LTX试剂将化学合成的miR-10b mimics和miR-10b inhibitor转染至胃癌SGC-7901细胞,采用MTT方法检测SGC-7901细胞增殖能力的变化,Transwell小室法检测SGC-7901细胞迁移和侵袭能力的变化;应用Western Blot方法检测P21蛋白表达水平。结果与正常组织相比,miR-10b在胃癌组织中的表达水平显著增高。与对照组相比,miR-10b表达沉默能够显著抑制人胃癌SGC-7901细胞的增殖、迁移和侵袭能力,显著增加P21的蛋白表达水平。miR-10b过表达的作用与之相反。结论 MiR-10b沉默抑制胃癌SGC-7901细胞的增殖、迁移和侵袭可能与上调P21蛋白表达相关。     

PRRX2通过调控Wnt/β-catenin信号通路促进胃癌细胞活力和迁移

目的:研究配对相关同源框2(paired-related homeobox 2,PRRX2)基因对胃癌细胞活力和迁移能力的影响,并分析其调控Wnt/β-catenin信号通路的机制。方法:通过生物信息学分析在线数据库中胃癌和正常胃组织的PRRX2表达水平及PRRX2在胃癌组织中的表达与胃癌患者总生存率的相关性;将PRRX2小干扰RNA(si RNA)和过表达质粒分别转染到胃癌细胞MGC-803和SGC-7901中,敲低和过表达PRRX2基因;MTT法和Transwell实验检测胃癌细胞的活力和迁移能力;Western blot和TOPflash/FOPflash双萤光素酶报告基因实验检测Wnt/β-catenin信号通路的活化情况;免疫共沉淀实验检测PRRX2与β-catenin蛋白的相互作用。结果:敲低PRRX2能够减弱胃癌细胞MGC-803的活力和迁移能力(P0.05)。过表达PRRX2能够增强胃癌细胞SGC-7901的活力和迁移能力(P0.05),增加β-catenin、c-Myc和cyclin D1的蛋白的表达水平(P0.05),增强TOPflash/FOPflash双萤光素酶报告基因活性(P0.05)。PRRX2与β-catenin蛋白存在相互作用。结论:PRRX2可促进胃癌细胞的活力和迁移,其机制可能与Wnt/β-catenin信号通路有关。     

MiR-141对胃癌细胞SGC-7901增殖、侵袭能力的影响

目的观察miR-141在胃癌组织中的表达变化,及miR-141对胃癌细胞株SGC-7901增殖、侵袭能力的影响。方法收集116例胃癌患者的胃癌组织及癌旁正常胃组织,采用real-time PCR方法检测miR-141的表达水平,将化学合成的miR-141拟物和miR-141抑制剂转染至胃癌SGC-7901细胞,分别于培养12、24、48、72 h后采用MTT法检测细胞增殖能力,培养24 h后Transwell小室检测细胞侵袭能力,采用Western blot法检测细胞中的MMP-2、MMP-9蛋白的表达。结果胃癌组织中miR-141相对表达量显著低于癌旁组织(P0.01)。转染miR-141模拟物24、48、72 h后,SGC-7901细胞增殖能力显著降低,转染miR-141抑制剂后SGC-7901细胞增殖能力显著升高(P0.01)。转染miR-141模拟物24 h后,穿膜细胞数、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)蛋白相对表达量显著降低,转染miR-141抑制剂后穿膜细胞数、MMP-2、MMP-9蛋白相对表达量显著升高(P0.01)。结论胃癌组织中miR-141低表达可能与胃癌的发生发展有关,miR-141抑制胃癌细胞增殖、侵袭的作用机制可能与下调MMP-2、MMP-9蛋白表达有关。     

MAD2 RNA干扰载体的构建及其对胃癌细胞生长的影响

目的：构建MAD2（有丝分裂阻滞缺陷蛋白2）特异性RNA干扰真核表达载体,体外观察抑制MAD2基因表达对胃癌细胞生长和细胞周期的影响.方法：构建MAD2 siRNA真核表达载体,脂质体法将pSilencer3.1空载体和pSilencer3.1/MAD2-siRNA1和pSilencer3.1/MAD2-siRNA2真核表达载体分别导入人胃癌细胞系SGC7901.稳定转染后Western blot和RT-PCR检测胃癌细胞内MAD2蛋白及mRNA水平的表达情况,挑选出抑制效果最好的单个克隆.MTF法检测各实验组细胞生长情况,流式细胞术检测纺锤体抑制剂长春新碱作用后,胃癌细胞MAD2-siRNA转染组和空载体组细胞周期的分布.结果：成功构建MAD2 siRNA真核表达载体,转染胃癌细胞SGC7901可使其MAD2蛋白及mRNA表达显著下调.MAD2表达降低的胃癌细胞生长速度明显增快（P＜0.01）,经长春新碱作用后不能被阻滞于有丝分裂期.结论：小干扰RNA技术可以有效地特异性抑制胃癌细胞纺锤体检查点关键蛋白MAD2的表达.MAD2的抑制可使胃癌细胞SGC7901生长加速,增殖能力增强,同时减弱纺锤体抑制剂长春新碱阻滞细胞周期的作用.     

miR-216a-5p通过抑制HMGB1降低胃癌细胞系MGC-803的增殖和迁移

目的探讨miR-216a-5p对胃癌细胞增殖和迁移的影响及其分子机制。方法用Western blot检测胃癌和癌旁组织中高迁移率族蛋白1(HMGB1)的表达,RT-qPCR检测miR-216a-5p的表达。用人工合成的miR-216a-5p模拟物和其抑制物转染胃癌细胞系MGC-803。MTT检测细胞增殖,划痕法检测细胞迁移。双荧光素酶报告实验和Western blot检测miR-216a-5p和HMGB1之间的靶向关系。结果与癌旁组织相比,HMGB1在胃癌组织中高表达,miR-216a-5p在胃癌组织中低表达(P0.05)。miR-216a-5p模拟物上调胃癌细胞miR-216a-5p的表达水平,抑制细胞的增殖和迁移;miR-216a-5p抑制物下调miR-216a-5p的表达水平,促进胃癌细胞的增殖和迁移(P0.05)。miR-216a-5p可作用于HMGB1的3′UTR并抑制其表达。结论 miR-216a-5p通过靶向抑制HMGB1的表达降低胃癌细胞系MGC-803的增殖和迁移。     

MicroRNA-301b-3p accelerates the growth of gastric cancer cells by targeting zinc finger and BTB domain containing 4

MicroRNAs (miRNAs) have been found to be aberrantly expressed and exert essential roles in the tumorigenesis and progression of gastric cancer (GC). miR-301b-3p has been recognized as a cancer-related miRNA in lung cancer, bladder cancer and hepatocellular carcinoma. However, the function of miR-301b-3p in GC progression and its underlying mechanism have not been studied yet. In this study, we found that miR-301b-3p expression was up-regulated in GC tissues compared to adjacent noncancerous tissues. Furthermore, the elevated levels of miR-301b-3p were detected in GC cell lines (SGC-7901, AGS, MKN-45 and MGC-803) as compared with GES-1 cells. Interestingly, GC tissues from patients with tumor size ≥ 5 cm and advanced tumor stages showed obvious higher levels of miR-301b-3p compared to matched controls. Functionally, miR-301b-3p knockdown prominently inhibited cell proliferation, and induced cell cycle arrest at G1 phase and apoptosis in MGC-803 cells. Meanwhile, ectopic expression of miR-301b-3p conversely regulated these biological behaviors of MKN-45 cells. Next, we found that miR-301b-3p knockdown increased, whereas miR-301b-3p overexpression reduced the expression of zinc finger and BTB domain containing 4 (ZBTB4) in GC cells. Accordingly, luciferase reporter assay identified ZBTB4 as a direct target of miR-301b-3p. ZBTB4 overexpression markedly restrained the growth of MGC-803 cells. More importantly, ZBTB4 silencing partially reversed miR-301b-3p knockdown-induced tumor suppressive effects on MGC-803 cells. In conclusion, we firstly revealed that miR-301-3p was highly expressed in GC and contributed to tumor progression via attenuating ZBTB4, which might provide a novel molecular-targeted strategy for GC treatment.     

miR-199a-3p targets ETNK1 to promote invasion and migration in gastric cancer cells and is associated with poor prognosis

PurposeTo investigate the prognostic significance of miR-199a-3p and its role in invasion and metastasis in gastric cancer.MethodsmiR-199a-3p expression in 436 formalin-fixed and 39 frozen gastric cancer tissues was investigated by in situ hybridization and RT-PCR, respectively. The role of miR-199a-3p in the migration and invasion of gastric cancer cells was determined in overexpression and inhibitor studies using transwell assays and the SGC-7901, BGC-823 and MGC-803 gastric cancer cells lines. The effect of miR-199a-3p expression on ethanolamine kinase 1 (ETNK1) levels was determined by western botting.ResultsmiR-199a-3p was significantly up-regulated in AGS, SGC-7901, BGC-823 and MGC-803 gastric cancer cells, when compared with GES-1 non-malignant gastric epithelial cells. In situ hybridization studies revealed that human non-tumor gastric mucosa samples were negative for miR-199a-3p expression, while 162 of 436 (37.16%) cases of gastric cancer demonstrated positive expression. miR-199a-3p overexpression was associated with tumor size, Lauren classification, depth of invasion, lymph node and distant metastasis, TNM stage and prognosis. In patients with I, II and III stage tumors, high miR-199a-3p expression was associated with a significantly lower 5-year survival rate. miR-199a-3p overexpression was associated with increased cell migration and invasion. ETNK1 expression was inhibited following miR-199a-3p overexpression in BGC-823 and SGC-7901 cells, and elevated following miR-199a-3p suppression in MGC-803 cells.ConclusionmiR-199a-3p is highly expressed in gastric cancer, and correlates with invasion, metastasis and prognosis. miR-199a-3p regulates the invasion and migration of gastric cancer cells by targeting ETNK1. Consequently, miR-199a-3p may serve as a prognostic indicator in gastric cancer.     

Heparanase promotes human gastric cancer cells migration and invasion by increasing Src and p38 phosphorylation expression

Gastric cancer is one of the most common cancers and it remains difficult to cure, primarily because most cancer stem like cells possess higher capability of invasion and metastasis. Heparanase acts as a master regulator of the aggressive tumor phenotype in part by enhancing expression of proteins and activating signaling molecules. There were less associated with heparanase of molecular biology mechanism in human gastric cancer. We first evaluated the endogenous expression of heparanase in human gastric cancer cell lines and found Heparanase expression higher in SGC-7901 than MGC-803. Using the technology of RNAi in SGC-7901 cells down regulated heparanase gene, and reduced SGC-7901 cells migration and invasion. On the other hand, recombinant heparanase protein added in MGC-803 cells enhanced MGC-803 cell migration and invasion. The elevated cell migration and invasion were impaired by treatment of Src inhibitor pp2 or p38 inhibitor SB 203580. We further found that Stable knockdown of heparanase in SGC-7901 cells decreased phosphorylation of Src and p38. The phosphorylation of p38 was inhibited in response to pp2 treatment while the addition of SB 203580 to SGC-7901 cells did not change phosphorylation of Src. These data suggest that heparanase facilitates invasion and migration of human gastric cancer cells probably through elevating phosphorylation of Src and p38.     

miR-140在人胃癌组织中的表达及对SGC-7901胃癌细胞功能的影响

 目的: 研究microRNA-140(miR-140)在人胃癌和正常胃组织中的表达水平,以及调控miR-140表达后对SGC-7901胃癌细胞功能的影响。方法: 采用实时荧光定量PCR检测miR-140在人胃癌和正常胃组织中的表达水平;将miR-140 mimics(miR-140上调表达)和miR-140 inhibitors(miR-140下调表达)分别通过脂质体转染至人胃癌SGC-7901细胞中,同时设置未转染对照组(control组)和miRNA无义序列转染对照组(NC组)。实时荧光定量PCR检测转染后各组细胞中miR-140的表达变化;MTT方法检测各组细胞的生长活力和顺铂(DDP)作用下的生长抑制率;流式细胞术检测各组的细胞周期和凋亡率;Transwell实验检测各组细胞侵袭能力;Western blot检测各组细胞中组蛋白脱乙酰酶4(HDAC4)蛋白表达。结果: miR-140在人胃癌组织中表达水平显著低于正常胃组织(P<0.05)。与control和NC组相比,miR-140 mimics组中SGC-7901细胞活力和侵袭能力下降,细胞周期被阻滞,DDP作用下细胞生长抑制率和凋亡率上升,且HDAC4蛋白表达下调,差异均有统计学意义(P<0.05);而miR-140 inhibitors组中SGC-7901细胞活力和侵袭能力上升,细胞周期被促进,DDP作用下细胞生长抑制率和凋亡率下降,且HDAC4蛋白表达上调,差异均有统计学意义(P<0.05)。结论: miR-140在胃癌组织中低表达,可作为抑癌因子调控胃癌细胞活力、细胞周期变化、凋亡、侵袭并通过下调HDAC4发挥作用。miR-140可能作为胃癌诊断和治疗的新靶点。     

MicroRNA-125b在胃癌组织中的高表达并促进胃癌细胞系HGC-27和MGC-803的增殖

 目的 探讨microRNA-125b (miR-125b)基因在胃癌患者组织中的表达改变情况,及其对胃癌细胞系增殖和凋亡的影响。方法 使用real-time PCR方法检测miR-125b在40例临床诊断为胃癌患者的癌组织与癌旁对照组织中的表达情况。随后使用miR-125b mimic转染胃癌细胞系HGC-27和MGC-803,确认过表达成功后,分别使用CCK-8试剂盒和流式细胞仪检测过表达miR-125b对细胞增殖和凋亡的影响。结果 证实在胃癌患者组织中miR-125b的表达水平显著高于癌旁对照组（P<0.01）。在胃癌细胞系HGC-27和MGC-803中过表达miR-125b后,细胞增殖明显增加：转染72h,HGC-27（scramble组：1.632±0.09,mimic组：2.473±0.08）,MGC-803（scramble组：1.603±0.05,mimic组：2.554±0.07））,同时细胞凋亡也受到抑制。结论 miR-125b可能作为癌基因在胃癌中发挥作用,并对细胞增殖和凋亡具有显著影响。     

miR-152-3p 靶向TCF4 对人胃癌SGC-7901 细胞增殖和凋亡的调控作用

目的:探究miR-152-3p 对人胃癌SGC鄄7901 细胞增殖和凋亡的作用及机制。方法:用miR鄄152 类似物(miR-152 mimic) 转染细胞,RT鄄PCR 检测miR-152 及转录因子4(TCF4) 的表达;荧光素酶报告实验证明miR1523p 与TCF4 的靶 向关系;miR-152 mimic 和TCF pcDNA 重组质粒(pc鄄TCF4) 分别或同时转染细胞,Western blot 检测TCF4 的表达,CCK8 检测细 胞增殖情况;流式检测细胞凋亡情况。结果:miR152 mimic 能显著升高SGC鄄7901 细胞miR-1523p 的表达水平,降低TCF4 的 mRNA 水平;同时,miR152 mimic 还能显著降低TCF4 野生型荧光素酶活性;此外,miR-152 mimic 能显著降低SGC-7901 细胞 的增殖倍数,升高细胞凋亡率;pc-TCF4 能显著减弱miR-152 mimic 抑制SGC7901 细胞增殖及诱导细胞凋亡的作用。结论: miR-1523p 能抑制人胃癌SGC7901 细胞增殖并诱导细胞凋亡,作用机制与靶向调控TCF4 表达有关。     

MicroRNA-9通过NRP1抑制胃癌SGC-7901细胞上皮-间充质转化功能

目的:研究微小RNA-9(microRNA-9,miR-9)对胃癌SGC-7901细胞上皮-间充质转化(EMT)功能的影响及其相关机制。方法:SGC-7901胃癌细胞株分别转染miR-9 mimics和阴性对照序列(negative control mimic,NCM),作为miR-9组和NCM组,并设立未转染对照(control)组,采用RT-qPCR法检测各组细胞miR-9的含量,Transwell实验检测3组细胞迁移能力和侵袭能力,Western blot法检测3组细胞的N-cadherin、E-cadherin、α-catenin和神经纤毛蛋白1(NRP1)表达水平。采用Western blot法检测NRP1过表达对miR-9抑制EMT的拮抗作用。双萤光素酶实验检测miR-9与NRP1的关系。结果:miR-9组的miR-9表达水平明显上调,为control组的538倍(P0.05)。miR-9组的迁移细胞数量明显低于control组(P0.05)。miR-9组的侵袭细胞数量明显低于control组(P0.05)。miR-9组细胞的N-cadherin和NRP1蛋白表达量明显降低,E-cadherin及α-catenin蛋白表达量明显升高。而NRP1及miR-9均过表达组胃癌细胞中N-cadherin蛋白表达量明显升高,E-cadherin及α-catenin蛋白表达量明显降低。双萤光素酶检验结果显示NRP1为miR-9的下游靶基因(P0.05)。结论:miR-9可能通过降低下游靶基因NRP1水平影响EMT相关蛋白表达,抑制胃癌SGC-7901细胞的EMT功能。