miRNA-21对结直肠癌耐药细胞ATM/CHK2/CDC25A信号通路的影响

目的 探讨miRNA-21在结直肠癌奥沙利铂(L-OHP)耐药细胞株HCT116/L-OHP中对毛细血管扩张性共济失调突变基因(ATM)/细胞周期检测点激酶(CHK)2/细胞分裂周期素(CDC)25A信号通路的影响。方法 应用细胞转染技术干扰HCT116/L-OHP细胞中miRNA-21的表达，采用实时荧光定量PCR(RT-qPCR)检测HCT116/L-OHP中ATM、CHK2、CDC25A和细胞周期蛋白依赖性激酶(CDK)2 mRNA表达，Western印迹检测该细胞株中miRNA-21表达变化后ATM、CHK2、CDC25A和CDK2蛋白表达。结果 转染miRNA-21 mimic使miRNA-21表达升高时，ATM、CHK2、CDC25A和CDK2 mRNA和蛋白的表达均随之升高，且与未转染组和阴性转染组相比较差异均具有统计学意义(P<0.05);转染miRNA-21 inhibitor使miRNA-21表达降低时，ATM、CHK2、CDC25A和CDK2 mRNA和蛋白的表达均随之降低，且与未转染组和阴性转染组相比较差异均具有统计学意义(P<0.05)。结论 在结...     

miR-21靶向调控TET1促进结直肠癌HCT15和HT29细胞增殖、侵袭及迁移

目的探究miR-21对结直肠癌细胞生物学行为的影响以及潜在调控机制。方法利用实时荧光定量PCR(qRT-PCR)检测结直肠癌组织中miR-21和TET1的表达水平并分析其相关性;利用生物信息学网站预测miR-21潜在靶基因TET1,利用双荧光素酶实验进行验证;利用细胞转染实验对两种结直肠癌细胞株进行miR-21 mimics、miR-21 inhibitor、si-TET1及相关对照的细胞转染,采用CCK-8分析、Transwell实验及细胞划痕实验检测转染后细胞增殖、侵袭及迁移能力的变化;利用Western blotting实验检测转染后细胞中TET1蛋白的表达。结果 qRT-PCR结果显示,结直肠癌组织中miR-21呈高表达水平,TET1 mRNA呈低表达水平,且两者表达水平呈负相关。靶基因预测及验证实验结果显示TET1是miR-21的靶基因,miR-21表达引起结直肠癌细胞中TET1表达下调。细胞转染实验结果显示,miR-21促进细胞增殖、侵袭及迁移。Western blotting实验结果显示,转染miR-21 mimics抑制TET1蛋白表达,转染miR-21 inhibitor促进TET1蛋白表达。结论 miR-21通过靶向调控TET1促进结直肠癌细胞的增殖、侵袭及迁移。     

miR-613靶向KLF4对结肠癌增殖及迁移的影响及其分子机制

[目的]确定miR-613在结肠癌组织和结肠癌细胞系中的表达以及miR-613靶向KrüPPel样因子4(KLF4)对人结肠癌细胞增殖、迁移的影响。[方法]采用实时荧光定量PCR(qRT-PCR)检测35例结肠癌组织及其癌旁组织中miR-613和KLF4的表达水平,并进行相关性分析。并采用qRT-PCR检测miR-613在结肠癌细胞系(SW480,SW620,HCT116,DLD1,LOVO,NCM460,RKO)中的表达。在HCT116和SW480中分别转染miR-613模拟物和抑制剂。CCK8及EDU实验用于检测转染前后细胞增殖能力的改变。Transwell实验检测细胞迁移能力的改变。使用双荧光素酶报告基因测量miR-613与KLF4的相互作用关系。功能回复实验用于确定miR-613靶向KLF4对结肠癌细胞增殖、迁移的影响。[结果]miR-613 mRNA在结肠癌组织中表达明显高于其癌旁组织(P0.05);KLF4在结肠癌组织中表达明显低于其癌旁组织(P0.05);miR-613与KLF4表达呈负相关(R~2=0.74,P0.001)。在结肠癌细胞系中,HCT116中miR-613的表达水平最低,SW480中miR-613的表达水平最高。在HCT116中,miR-613的表达上调后,细胞增殖活性显著于阴性对照组(P0.05);细胞迁移能力明显升高(P0.05);KLF4蛋白表达水平明显降低(P0.05)。SW480细胞中,抑制miR-613表达后,增殖活性明显低于阴性对照组(P0.05);细胞迁移能力明显降低(P0.05);KLF4蛋白表达水平明显降低(P0.05)。在HCT116细胞中,miR-613模拟物与KLF4 3'UTR野生型质粒共转染,与阴性对照组比较,荧光素酶活性显著增加,差异有统计学意义(P0.05)。miR-613抑制剂和si-KLF4共转染至结肠癌细胞后,si-KLF4可挽救由于miR-613低表达导致的细胞增殖、迁移能力的降低。[结论]miR-613在结肠癌组织中高表达;miR-613通过靶向KLF4基因调控结肠癌细胞的增殖和迁移。     

miR-483-3p对结直肠癌DLC1基因表达的影响

背景:抑癌基因表达抑制在肿瘤发生、发展过程中起重要作用。一些microRNAs可通过调节抑癌基因的表达影响肿瘤发生。目的:探讨miR-483-3p对结直肠癌肝癌缺失基因1(DLC1)表达的靶向调节作用。方法:纳入2012年10月~2013年4月南京鼓楼医院收治的结直肠癌患者16例,采用蛋白质印迹法检测癌组织及其相应癌旁非癌组织的DLC1表达,qRT-PCR检测miR-483-3p表达。构建含DLC1 3’非翻译区(3’UTR)的双荧光素酶报告基因质粒,在人结肠癌细胞株HCT116中验证miR-483-3p对DLC1表达的调节作用。以miR-483-3p mimic转染HEK293T细胞,采用蛋白质印迹法检测DLC1表达;以miR-483-3p mimic转染HCT116细胞,采用CCK-8实验检测细胞增殖。结果:结直肠癌组织的DLC1表达水平显著低于癌旁非癌组织,miR-483-3p表达水平显著高于癌旁非癌组织(P0.05)。miR-483-3p mimic可靶向结合DLC1的3’UTR而抑制其表达。转染miR-483-3p mimic的HCT116细胞增殖能力显著增强(P0.05)。结论:DLC1是miR-483-3p的靶基因,miR-483-3p可在转录后水平抑制DLC1表达,参与促进结直肠癌发生。     

七氟醚通过上调miR-34a抑制结直肠癌细胞的迁移和侵袭的机制研究

目的研究七氟醚对人结直肠癌HCT116细胞迁移和侵袭能力的影响,并探讨其分子机制.方法以细胞计数法(CCK-8)检测七氟醚对HCT116细胞增殖能力的影响;采用实时荧光定量PCR(qRT-PCR)检测细胞中miR-34a表达水平, Transwell实验检测细胞迁移和侵袭能力,蛋白免疫吸附法(Western blot)检测细胞中基质金属蛋白酶-2(matrix metalloprotease,MMP-2)和基质金属蛋白酶-9(MMP-9)蛋白表达水平.结果通过CCK-8实验筛选出七氟醚的作用浓度为4%.以4%七氟醚处理HCT116细胞4h后miR-34a表达水平升高了79%.转染miR-34a inhibitor后,细胞中miR-34a的表达水平下降了81%.七氟醚干预后迁移细胞数由(97.75±16.10)降低到(36.60±8.58),侵袭细胞数由(64.22±6.25)降低到(26.48±3.10),MMP-2蛋白表达水平由(0.68±0.11)下调到(0.24±0.04), MMP-9蛋白表达水平由(0.48±0.07)下调到(0.13±0.02);而抑制miR-34a的表达后,迁移细胞数由(40.15±9.02)提高到(88.65±12.74),侵袭细胞数由(26.12±3.02)提高到(59.24±4.68),MMP-2蛋白表达水平由(0.22±0.03)上调到(0.61±0.09),M M P-9蛋白表达水平由(0.14±0.03)上调到(0.43±0.06).结论七氟醚通过上调miR-34a的表达抑制人结直肠癌HCT116细胞的迁移和侵袭,可能与下调MMP-2和MMP-9蛋白表达有关.     

微小RNA-144对人结肠癌细胞株增殖、侵袭迁移影响及其与同源盒基因A10结合力

目的 观察微小RNA-144(miR-144)对人结肠癌细胞株增殖、迁移、侵袭的影响及其与同源盒基因A10(homeobox gene A10,HOXA10)的结合力。方法 (1)人结肠癌细胞株HCT116、SW480、CL-11和永生化正常人结肠上皮细胞株FHC中miR-144及HOXA10 mRNA检测：采用实时荧光定量PCR法检测HCT116、SW480、CL-11及FHC中miR-144与HOXA10 mRNA，观察人结肠癌细胞和人结肠上皮细胞miR-144、HOXA10 mRNA表达变化，选择miR-144相对表达量最低HCT116细胞为后续研究对象。(2)miR-144对人结肠癌细胞株增殖、侵袭及迁移的影响观察：取对数生长期HCT116细胞分为3组，1组细胞顺序转染miR-144模拟物（miR-144 mimics）和HOXA10过表达质粒pcDNA3.1-HOXA10,2组细胞转染miR-144 mimics,3组细胞转染空白质粒。转染24 h时测算各组细胞增殖活性、观察各组细胞的侵袭及迁移情况、检测各组细胞miR-144及HOXA10蛋白。(3)HCT116细胞中miR...     

miR-199对结直肠癌细胞增殖、侵袭的影响及机制

目的 探讨微小RNA-199(miR-199)对结直肠癌细胞增殖、侵袭的影响及其作用机制。方法 常规培养人正常结肠上皮细胞系（HCoEpiC细胞）、结直肠癌细胞系（LOVE、SW620、SW480、HT29、DLD-1细胞），用实时定量PCR法检测细胞中miR-199表达。将SW620细胞随机分为miR-199过表达组、阴性对照组及miR-199+转铁蛋白受体1(TFR1)共表达组（miR-199mimics+TFR1组），分别将miR-199mimics、mimic-NC、miR-199mimics+TFR1过表达质粒转染至细胞内；另取部分未转染细胞作为空白对照组。实时荧光定量PCR法检测TFR1 mRNA表达，MTT实验检测细胞增殖能力，Transwell侵袭实验检测细胞侵袭能力，Western blotting法检测TFR1蛋白表达，生物学软件TargetScan及双荧光素酶报告基因实验分析miR-199的靶基因。结果 人结直肠癌细胞系中miR-199相对表达量均低于人结肠上皮细胞系（P均<0.05），且SW620细胞中miR-199相对表达量最低，故选SW620细胞为实验...     

MicroRNA-21(miR-21)在大肠癌细胞中的表达及其靶基因

目的探讨微RNA(miRNA)-2l在大肠癌(CRC)细胞中的表达,抑制miRNA-21(miR-21)表达对大肠癌细胞中磷酸醋酶张力蛋白同源物(PTEN)、PI3K、蛋白激酶B(Akt)、基质金属蛋白酶-9(MMP-9)、mTOR表达的影响及其可能调控的靶基因。方法通过反义寡核苷酸(ASO)技术抑制大肠癌细胞株HCT116中miR-21的表达并加以验证。通过荧光定量PCR(FQ-PCR)技术及Western印迹法检测HCT116细胞中PTEN、PI3K、Akt、MMP-9、mTOR表达的改变。利用生物信息学分析预测miRNA-21可能的靶基因,荧光素酶双报告基因系统检测PTEN活性。结果 miR-21在HCT116中表达最高,在SW480中表达最低。miRNA-21在细胞株HCT116中的表达得到有效抑制,并且PTEN蛋白的表达明显上调,PI3K、Akt、MMP-9、mTOR表达下调。筛选并确认了PTEN为miR-21的调控靶基因之一。结论抑制miR-21的表达可促进HCT116细胞PTEN表达升高,PI3K、Akt、MMP-9、mTOR表达下调,PTEN可作为miR-21的靶基因参与调控过程。miRNA-21在CRC中表达失控,通过抑制PTEN表达促进肿瘤细胞侵袭转移。     

LncRNA SOX21-AS1靶向调控miR-202-5p影响结直肠癌细胞生物学行为的实验研究

背景:长链非编码RNA(lncRNA)可通过调控miRNA的表达来参与肿瘤发生、发展过程,但lncRNA在结直肠癌发展和转移中的分子机制尚未阐明。目的:探讨lncRNA SOX21-AS1通过调控微小RNA-202-5p(miR-202-5p)的表达影响结直肠癌细胞生物学过程的分子机制。方法:以SOX21-AS1小分子干扰RNA(si-SOX21-AS1)、miR-202-5p模拟物及其抑制剂转染结直肠癌HCT116、SW480细胞,采用实时荧光定量聚合酶链反应(qRT-PCR)法检测结直肠癌细胞株SOX21-AS1、miR-202-5p mRNA表达,MTT法检测细胞增殖情况,流式细胞术检测细胞凋亡情况,Transwell实验检测细胞迁移和侵袭情况,双萤光素酶报告实验检测SOX21-AS1与miR-202-5p的靶向调控关系,蛋白质印迹法检测细胞周期蛋白1(cyclin D1)、MMP-2、cleaved caspase-3蛋白表达。结果:结直肠癌细胞中SOX21-AS1 mRNA表达显著高于人正常结肠黏膜上皮细胞(P 0. 05),miR-202-5p mRNA表达显著降低(P 0. 05)。沉默SOX21-AS1或上调miR-202-5p表达可显著抑制HCT116、SW480细胞增殖、迁移和侵袭,细胞凋亡率显著增加(P 0. 05),并可抑制cyclin D1、MMP-2蛋白表达(P 0. 05),促进cleaved caspase-3蛋白表达(P 0. 05)。SOX21-AS1可靶向结合miR-202-5p,并可负向调控miR-202-5p的表达和活性;抑制miR-202-5p表达可逆转沉默SOX21-AS1对结直肠癌细胞增殖、凋亡、迁移和侵袭的影响。结论:沉默SOX21-AS1表达可通过上调miR-202-5p的表达从而抑制结直肠癌细胞增殖、迁移和侵袭,并可诱导细胞凋亡。     

干预膜联蛋白A4表达对结直肠癌细胞增殖、侵袭和转移能力的影响

目的探讨干预膜联蛋白(ANX) A4表达对结直肠癌HCT116细胞增殖、黏附、剥脱和侵袭的影响及机制。方法采用RT-PCR和Western印迹检测结直肠癌HCT116细胞和正常结肠上皮NCM460细胞中ANXA4 m RNA和蛋白表达情况。将HCT116细胞分为Control组(未转染)、NC组(转染阴性对照)和ANXA4组(转染si RNA-ANXA4),脂质体lipofectamineTM2000转染HCT116细胞,RT-PCR检测各组细胞中ANXA4 m RNA表达,四甲基偶氮唑盐(MTT)法检测HCT116细胞增殖能力,黏附实验、剥脱实验和侵袭实验分别检测HCT116细胞的黏附能力、剥脱能力和侵袭能力,Western印迹检测ANXA4、埃兹蛋白(Ezrin)、骨桥蛋白(OPN)和细胞表面黏附分子(CD44v6)蛋白的表达。结果 HCT116细胞中ANXA4 m RNA和蛋白表达水平均显著低于NCM460细胞;与Control组相比,干扰ANXA4表达后,HCT116细胞的增殖能力受到抑制,同时黏附能力、剥脱能力和侵袭能力明显降低,差异有统计学意义(P0. 05); ANXA4组细胞中ANXA4 m RNA表达水平及ANXA4、Ezrin、OPN和CD44v6蛋白表达水平较Control组均显著减弱,差异有统计学意义(P0. 05);而NC组与Control组各指标差异无统计学意义(P0. 05)。结论干预ANXA4表达能够抑制结直肠癌HCT116细胞的增殖、黏附、剥脱和侵袭能力,其分子机制可能与下调Ezrin、OPN和CD44v6蛋白表达有关。     

MicroRNA-21 promotes phosphatase gene and protein kinase B/phosphatidylinositol 3-kinase expression in colorectal cancer

AIM: To explore the regulatory mechanism of the target gene of microRNA-21 (miR-21), phosphatase gene (PTEN), and its downstream proteins, protein kinase B (AKT) and phosphatidylinositol 3-kinase (PI3K), in colorectal cancer (CRC) cells.METHODS: Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression levels of miR-21 and PTEN in HCT116, HT29, Colo32 and SW480 CRC cell lines. Also, the expression levels of PTEN mRNA and its downstream proteins AKT and PI3K in HCT116 cells after downregulating miR-21 were investigated.RESULTS: Comparing the miR-21 expression in CRC cells, the expression levels of miR-21 were highest in HCT116 cells, and the expression levels of miR-21 were lowest in SW480 cells. In comparing miR-21 and PTEN expression in CRC cells, we found that the protein expression levels of miR-21 and PTEN were inversely correlated (P < 0.05); when miR-21 expression was reduced, mRNA expression levels of PTEN did not significantly change (P > 0.05), but the expression levels of its protein significantly increased (P < 0.05). In comparing the levels of PTEN protein and downstream AKT and PI3K in HCT116 cells after downregulation of miR-21 expression, the levels of AKT and PI3K protein expression significantly decreased (P < 0.05).CONCLUSION: PTEN is one of the direct target genes of miR-21. Thus, phosphatase gene and its downstream AKT and PI3K expression levels can be regulated by regulating the expression levels of miR-21, which in turn regulates the development of CRC.     

miR-574-3p在结直肠癌中的表达和作用机制研究

目的 检测并分析结直肠癌组织和细胞系中miR-574-3p的表达情况及其对结直肠癌细胞发生、发展的影响.方法 收集武汉市第三医院11对手术切除结直肠癌患者的癌组织和对应癌旁组织,3种结直肠癌细胞系和1种正常结直肠上皮细胞,通过实时荧光定量聚合酶链反应(qRT-PCR)检测临床样本及结直肠癌细胞系中miR-574-3p的...     

MiR-25-3p attenuates the proliferation of tongue squamous cell carcinoma cell line Tca8113

ObjectiveTo investigate the effects of miR-25-3p on the occurrence, development and proliferation of tongue squamous cell carcinoma cells.MethodsTo establish tongue squamous cell carcinoma cell line Tca8113 that stably and highly express miR-25-3p using recombinant retroviral vector-mediated gene transfer method. The proliferation of transfected Tca8113 was detected by thiazolyl blue tetrazolium bromide (MTT) and cell colony formation assays. cyclinD1, p21^cip1 and p27^kip1 mRNA expressions in the transfected Tca-8113 were detected by quantitative PCR. cyclinD1, p21, p27^kip1, AKT, p-AKT, FOXO1 and p-FOXO1 expressions in the transfected Tca8113 were detected by western blot analysis. In addition, miR-25-3p expression in the tongue squamous cell carcinoma cell line and tissue specimen was also detected by quantitative PCR.ResultsQuantitative PCR showed that miR-25-3p expression in the tongue squamous cell carcinoma cell lines and tissue specimen was significantly lower than that in the adjacent tissue. MTT and cell colony formation assays showed that after miR-25-3p overexpression, the proliferation of transfected Tca8113 was obviously attenuated. Western blot analysis and quantitative PCR showed that after miR-25-3p overexpression, p21^cip1 and p27^kip1 expressions were upregulated, while cyclinD1, AKT, FOXO1 expressions were downregulated, and AKT and FOXO1 phosphorylation was inactivated in the transfected Tca8113 cells.ConclusionsMiR-25-3p inhibited the proliferation of tongue squamous cell carcinoma cells and regulated cell cycle-related protein expression, playing an important role in the occurrence and development of squamous cell carcinoma of the tongue.     

应用实时荧光定量PCR检测结直肠癌与癌旁组织中miRNA-221的差异表达状况

目的分析结直肠癌与毗邻癌旁组织中miRNA-221差异表达状况。方法常规抽提30例结直肠癌及对照癌旁组织中总RNA,应用实时荧光定量PCR方法检测标本中miRNA-221的表达状况。结果与癌旁组织相比,结直肠癌组织中miRNA-221表达明显上调（P〈0．01）。结论实时荧光定量PCR是一种较精确的miRNA定量检测方法;miRNA-221可能通过转录后基因沉默机制促进结直肠癌的发生发展。     

Long Noncoding RNA GAPLINC Promotes Cells Migration and Invasion in Colorectal Cancer Cell by Regulating miR-34a/c-MET Signal Pathway

Background

Gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC) has been detected in colorectal cancer (CRC) cells and reportedly performs many functions related to tumor proliferation and metastasis. Aim The present study aimed to comprehensively explore the biological functions of GAPLINC and their underlying mechanism in CRC cell.

Methods

The human cancer LncRNA PCR array was used to detect the differentially expressed long noncoding RNAs in human CRC samples. Real-time PCR, dual-luciferase assay, RNA pull-down assay, Transwell assay, and western blot analysis were performed to explore the molecular mechanism underlying GAPLINC functions related to migration and invasion of a human CRC cell line (HCT116).

Results

Compared to the non-cancerous tissues, GAPLINC expression was obviously increased in CRC tissues. In HCT116, silencing of GAPLINC weakened cell migration and invasion, while overexpression of GAPLINC significantly promoted cell migration and invasion. Through dual-luciferase, RNA pull-down, and Transwell assays, we verified that miR-34a was the downstream molecule of GAPLINC and that miR-34a negatively regulated the migration and invasion of HCT116 cell. Furthermore, we found that GAPLINC positively regulated the miR-34a target gene c-MET in CRC tissues.

Conclusions

Our findings revealed that GAPLINC was up-regulated in CRC tissues and was involved in the migration and invasion of CRC cells by regulating miR-34a/c-MET signaling pathway.

     

MiR-129-5p通过靶定胸苷酸合成酶调节结肠癌细胞对5-氟尿嘧啶的敏感性

目的 研究miR-129-5p通过靶定胸苷酸合成酶（TYMS）调控结肠癌细胞对5-氟尿嘧啶（5-Fu）敏感性的分子机制.方法 构建结肠癌耐药细胞株LoVo/5-Fu,并用实时荧光定量聚合酶链反应（PCR）检测miR-129-5p的表达水平.流式细胞术和MTT毒性实验检测过表达miR-129-5p对LoVo/5-Fu凋亡和半数致死剂量（IC50）的影响.构建TYMS 3＇UTR区荧光素酶报告载体,验证miR-129-5p对TYMS的靶向调控作用.Western Blot检测转染miR-129-5p后LoVo/5-Fu细胞中TYMS的蛋白表达.在LoVo/5-Fu内转染miR-129-5P siRNA,抑制其表达,检测LoVo/5-Fu细胞的IC50和凋亡.结果 过表达miR-129-5p后,LoVo/5-Fu细胞用5-Fu处理后凋亡增加,IC50降低.荧光素酶活性检测表明miR-129-5p能够抑制TYMS的荧光素酶活性.在LoVo/5-Fu细胞中miR-129-5p与TYMS的表达呈负相关.敲减TYMS后,LoVo/5-Fu细胞用5-Fu处理后凋亡增加,IC50降低.结论 MiR-129-5p能够通过靶定TYMS而增加结肠癌细胞对5-Fu的敏感性.     

Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells

Accumulating evidence suggests a role for microRNAs in human carcinogenesis as novel types of tumor suppressors or oncogenes. However, their precise biological role remains largely elusive. In the present study, we aimed to identify microRNA species involved in the regulation of cell proliferation. Using quantitative RT-PCR analysis, we demonstrated that miR-34a was highly up-regulated in a human colon cancer cell line, HCT 116, treated with a DNA-damaging agent, adriamycin. Transient introduction of miR-34a into two human colon cancer cell lines, HCT 116 and RKO, caused complete suppression of cell proliferation and induced senescence-like phenotypes. Moreover, miR-34a also suppressed in vivo growth of HCT 116 and RKO cells in tumors in mice when complexed and administered with atelocollagen for drug delivery. Gene-expression microarray and immunoblot analyses revealed down-regulation of the E2F pathway by miR-34a introduction. Up-regulation of the p53 pathway was also observed. Furthermore, 9 of 25 human colon cancers (36%) showed decreased expression of miR-34a compared with counterpart normal tissues. Our results provide evidence that miR-34a functions as a potent suppressor of cell proliferation through modulation of the E2F signaling pathway. Abrogation of miR-34a function could contribute to aberrant cell proliferation, leading to colon cancer development.     

CircRNA_0084927 promotes colorectal cancer progression by regulating miRNA-20b-3p/glutathione S-transferase mu 5 axis

BACKGROUNDColorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer-related death worldwide. The 5-year survival rate of patients with early-stage CRC could reach 90%, but it is very low in patients with advanced-stage CRC. Recent studies have shown that circular RNAs play important roles in regulating the migration and invasion of CRC cells.AIMTo elucidate the role of circRNA_0084927 (circ_0084927) in the migration and invasion of CRC cells and its underlying mechanism.METHODSClinical tissue samples and cells were collected, and the expression of circ_0084927 was detected by quantitative polymerase chain reaction (qPCR). The diagnostic performance of circ_0084927 was assessed by receiver operating characteristic curve analysis. The role of circ_0084927 in CRC cell proliferation, migration, and invasion was determined using cell counting kit-8 assay, wound healing assay, and transwell assay, respectively. The regulatory relationship among circ_0084927, miRNA-20b-3p (miR-20b-3p), and glutathione S-transferase mu 5 (GSTM5) was identified using databases, luciferase reporter assay, qPCR, and Western blot analysis. AKT-mTOR signaling was also verified after circ_0084927 knockdown or miR-20b-3p mimic treatment.RESULTSThe expression of circ_0084927 was significantly increased in CRC tissues and cells, and it was higher in advanced-stage CRC compared with early-stage CRC. The area under the curve (AUC) of circ_0084927 was 0.806 [95% confidence interval (CI): 0.683-0.896]. In addition, the AUC was 0.874 (95%CI: 0.738-0.956) in patients with advanced-stage CRC and 0.713 (95%CI: 0.555-0.840) in those with early-stage CRC. Knockdown of circ_0084927 inhibited the migration and invasion of HCT116 cells. Moreover, circ_0084927 was found to act as a sponge of miR-20b-3p. MiR-20b-3p activation reduced the circ_0084927 level, whereas miR-20b-3p inhibition increased the circ_0084927 level. But the effect was not found after circ_0084927 mutation. In addition, miR-20b-3p expression in CRC patients was also reduced and negatively correlated with circ_0084927 expression. The function of circ_0084927 in HCT116 cells with circ_0084927 knockdown was rescued by miR-20b-3p. Moreover, GSTM5 expression was significantly decreased after overexpressing miR-20b-3p or inhibiting circ_0084927, but its expression was rescued when circ_0084927 and miR-20b-3p were both inhibited. Finally, AKT-mTOR signaling was markedly regulated by circ_0084927 and miR-20b-3p.CONCLUSIONThe expression of circ_0084927 is significantly increased in CRC and higher in advanced-stage CRC than in early-stage CRC. Moreover, circ_0084927 potentially regulates CRC cell migration and invasion via the miR-20b-3p/GSTM5/ AKT/mTOR pathway.     

抑制细胞外信号调节激酶转导结直肠癌细胞组蛋白磷酸化和基因表达的影响

目的 观察人结直肠癌细胞中细胞外信号调节激酶-丝裂原激活蛋白激酶(ERK-MAPK)信号转导途径与组蛋白磷酸化水平及其相关蛋白表达的关系,及其对细胞增殖和细胞周期的影响.方法 培养结直肠癌细胞系SW1116、HCT116,以终浓度分别为0、10、20及40μmol/L的ERK-MAPK阻断剂U0126干预细胞.细胞计数试剂盒(cell counting kit,CCK-8)检测细胞活力影响,以流式细胞术(flow cytometry,FCM)研究细胞周期变化,Western免疫印迹法分析组蛋白H3激酶核糖体S6丝氨酸-苏氨酸激酶2(ribosomal S6 kinase 2,RSK-2)、丝裂原和应激-激活的激酶1和2(mitogen-and stress-activated kinase 1 and 2,MSK1和MSK2)、组蛋白H3(Ser10)磷酸化水平及c-Fos蛋白表达变化情况.结果 CCK-8法检测发现ERK-MAPK阻断剂能以剂量和时间依赖方式明显抑制2种结直肠癌细胞的增殖,增殖率可下降至对照组的47%;并能明显增加G0/G1期细胞百分比(P<0.01),而S期细胞百分比明显减少(P<0.01).Western印迹结果显示U0126干预后,H3激酶MSK1和RSK2表达明显降低,分别为对照HCT116的28%和40%,而MSK2蛋白水平在2个细胞系中均无明显变化.相应地,组蛋白H3磷酸化(Ser10)水平亦降低,相关蛋白c-Fos的表达亦明显下降.结论 抑制ERK-MAPK信号转导可能通过抑制组蛋白H3激酶MSK1、RSK2活性而降低组蛋白H3磷酸化水平,进而减少c-Fos蛋白表达,从而抑制人结直肠癌细胞的增殖和生长.     

MicroRNA-15b 下调Bcl-2促进耐药肺癌细胞凋亡

目的 探讨微小RNA-15b （microRNA-15b,miR-15b）对人肺癌耐药细胞A549/DDP凋亡的影响.方法 采用体外转染法将MicroRNA-15b瞬时转染到A549/DDP细胞后,应用Real-time PCR检测A549/DDP细胞中MicroRNA-15b的表达情况;流式细胞仪（FCM）检测细胞凋亡变化;并用Western印迹法（Western blot）检测细胞中Bcl-2表达.结果 转染后miR-15b组的miR-15b表达水平显著增加（P〈0.05）;miR-15b组细胞凋亡率为：32.4%±5.1%,与Mock组（5.73%±1.2%）和miR-15b-Cont（6.24%±2.4%）比较,差异具有统计学意义（P〈0.05）;miR-15b组的Bcl-2表达量较对照组明显增加.结论 miR-15b可能通过下调Bcl-2的表达从而诱导A549/DDP细胞的凋亡,这可能为肺癌耐药的治疗提供新靶点.