miR-204 inhibits invasion and epithelial-mesenchymal transition by targeting FOXM1 in esophageal cancer

MicroRNAs (miRNAs), endogenous noncoding small RNAs, have been reported to play crucial roles in epithelial-mesenchymal transition (EMT) in cancers. Deregulation of microRNA-204 (miR-204) has been documented in many cancers, but its role in the development of esophageal cancer (EC) has not been studied. Here, we reported the role of miR-204 in invasion and EMT in EC. We identified an inverse correlation between miR-204 expression level and the invasion and EMT phenotype of EC cells, and up-regulation of miR-204 inhibited invasion and EMT phenotype of EC cells. Furthermore, we showed that forkhead box protein M1 (FOXM1) was a direct target gene of miR-204, and miR-204 regulated invasion and EMT in EC by acting directly on the 3’UTR of FOXM1 mRNA and suppressing its protein expression. We also explored the anti-tumor effect of miR-204, and found that overexpression of miR-204 suppressed the growth of esophageal tumors in vivo. These findings suggest that miR-204 might be a suppressor of invasion and EMT in EC, which offers a novel potential therapeutic target for EC.     

miR-106a overexpression and pRB downregulation in sporadic colorectal cancer

Rb1 plays an important role in cell cycle progression and therefore may be involved in malignant transformation of colonic cells. The aim of our research was to define the potential role of Rb1 as a prognostic biomarker in tumorigenesis of sporadic colorectal cancer, and to examine the role of miR-106a in Rb1 regulation as it functionally binds to 3′UTR of transcribed mRNA. We examined LOH and promoter methylation status. Real-time PCR was used for Rb1 mRNA and miR-106a, and immunohistochemistry for protein expression analysis. All the results obtained from patients' samples were correlated with the clinicopathological parameters in order to determine its influence on the sporadic colorectal carcinogenesis. LOH showed no correlation with mRNA and pRb expression. 51.5% of tumor samples were scored negative for pRb staining. Despite this finding, we detected overexpression of Rb1 mRNA in tumor samples in comparison to the adjacent normal tissue (p = 0.023). mRNA overexpression was consistent with Rb1 promoter methylation analysis results, which showed no methylation in the investigated samples. Expression analysis of miR-106a in the patients samples showed its overexpression in colorectal cancer (p < 10^− 4). Negative pRb score was expected according to the definition of tumor suppressor genes and their proposed role in the malignant transformation of the cells. The observed discrepancy between mRNA and protein expression can be explained by a regulatory mechanism that inhibits translation, such as microRNA silencing. Our results suggest that miR-106a might have a regulatory role for Rb1 in sporadic colorectal cancer.     

MiR-873-5p inhibits cell migration,invasion and epithelial-mesenchymal transition in colorectal cancer via targeting ZEB1

Recent studies have demonstrated that dysregulation of mircoRNAs (miRNAs) greatly affected biological processes of human cancers, including colorectal cancer. As a member of miRNAs family, miR-873-5p has been proved to be a tumor suppressor in some human cancers. Here, we aim to investigate the effects of miR-873-5p on the migration, invasion and epithelial-mesenchymal transition (EMT) of colorectal cancer cells. The low expression of miR-873-5p in colorectal cancer cells was identified by conducting qRT-PCR analysis. Gain of function assays were designed and conducted to demonstrate the specific function of miR-873-5p overexpression in colorectal cancer progression. Transwell assay and western blot assay were conducted and revealed that miR-873-5p inhibited cell migration, invasion and EMT formation. To find the downstream molecular mechanism of miR-873-5p, mechanism assays were designed and performed to find the downstream target of miR-873-5p. ZEB1 (Zinc finger E-box-binding homeobox 1) was certified to be the target of miR-873-5p through bioinformatics analysis, luciferase activity assay and pull-down assay. Finally, rescue assays were carried out to demonstrate the effects of miR-873-5p-ZEB1 axis on the migration, invasion and EMT process of colorectal cancer cells. In conclusion, we confirmed that miR-873-5p suppressed cell migration, invasion and EMT in colorectal cancer via targeting ZEB1.     

miR-145-5p suppresses proliferation,metastasis and EMT of colorectal cancer by targeting CDCA3

MicroRNAs play a critical role in regulating the carcinogenesis of colorectal cancer (CRC). Even though its role is unclear in CRC, miR-145-5p has been reported to have anti-oncogene properties in several tumors. Our research examined the function of miR-145-5p in CRC and the potential underlying mechanism. From the bioinformatics and qRT-PCR analysis, miR-145-5p levels were lower in CRC samples and cell lines. LoVo and SW480 cells were treated with miR-145-5p mimics and inhibitor, respectively. Cell cycle, CCK-8 and EdU assays revealed that overexpression of miR-145-5p suppressed cell viability and G1/S phase transition. Conversely, miR-145-5p inhibitor promoted cell growth and cell cycle transition. Elevated miR-145-5p expression also suppressed the migration, invasion and EMT of CRC cells, while miR-145-5p reduction had a reverse effect. CDCA3 was identified as a downstream effector of miR-145-5p and had a negative correlation with the miR-145-5p expression in CRC. In addition, co-transfection of miR-145-5p inhibitor and si-CDCA3 showed that CDCA3 in SW480 cells could reverse the effect caused by miR-145-5p. In conclusion, our findings demonstrated that miR-145-5p could act as a tumor suppressor in CRC by targeting CDCA3, and serve as a diagnostic and therapeutic biomarker of CRC.     

联合靶向阻断miR-29b/92b/106b抑制胃癌细胞生长及迁移

目的:本研究旨在寻找胃癌转移相关微小RNA(microRNA,miRNA,miR),揭示其对胃癌细胞生物学功能的影响,并探讨联合阻断候选miRNA在胃癌治疗中的临床应用价值。方法:收集发生淋巴结转移及无转移患者的胃癌标本各3例,采用miRNA表达芯片筛选转移相关的候选miRNA;将锁核酸修饰的候选miRNA的反义寡核苷酸转染BGC-823胃癌细胞株,运用CCK-8法、流式细胞术、划痕实验及Transwell迁移实验分析候选miRNA抑制前后胃癌细胞生物学功能的变化;构建多西环素诱导多重靶向候选miRNA的裸鼠移植瘤模型,观察联合靶向阻断候选miRNA后,肿瘤细胞体内生长情况。结果:miRNA表达芯片发现miR-29b、miR-92b及miR-106b在发生淋巴结转移患者的胃癌组织最高,并将它们确定为胃癌转移相关候选miRNA;分别在BGC-823细胞中抑制上述miRNA,可导致细胞活力减弱,凋亡诱导增加,迁移能力显著下降(P 0. 05);同时,体内联合阻断miR-29b/92b/106b,裸鼠移植瘤的形成较对照组显著减少。结论:miR-29b、miR-92b及miR-106b与胃癌细胞的迁移有关,联合阻断上述miRNA可明显抑制胃癌细胞体内外生长。这3个miRNA可能作为潜在的治疗靶点。     

In situ hybridization analysis of the expression of miR-106b in colonic cancer

Background: MicroRNA-106b (miR-106b) is thought to be an oncogenic microRNA that promotes tumor growth and metastasis. The potential predictive value of miR-106b was studied in colonic cancer patients. Methods: The expression of miR-106b was examined in 180 colonic cancer cases using in situ hybridization (ISH) technique and was evaluated semi-quantitatively by examining the staining index. The Correlation of miR-106b expression and clinic-pathological features was analyzed by Spearman Rank Correlation. Wilcoxon signed rank test was used for assessing the expression difference of miRNA-106b between colonic cancerous and para-cancerous ones, and their effects on patient survival were analyzed by a log-rank test and the Kaplan-Meier method. Results: MiR-106b was higher expressed in para-cancerous tissues, compared with colonic cancerous ones (P < 0.001). A positive correlation of miR-106b levels between colonic and para-cancerous tissues was also observed (CC = 0.523, P < 0.001). Furthermore, the expression of miR-106b was not significantly correlated with clinic-pathological parameters, including gender, age, histological grade, tumor size, pT stage, pN stage, pM stage and pTNM stage of the patients. Histological grade was positively correlated with pT stage (P = 0.011), pN stage (P = 0.036) and pTNM stage (P = 0.009). Patients expressing high levels of miR-106b both in colonic cancer tissues and para-cancerous ones have a relatively longer survival time but the difference is not statistically significant (P = 0.16). Conclusions: The expression difference of miR-106b levels between colonic tissues and para-cancerous tissues is statistically significant, but the miR-106b levels were not quite correlated with clinic-pathological characteristics and overall survival times of patients with colonic cancer. Lower levels of miR-106b may be connected with neoplastic effects due to interference with TGF-β signaling, providing evidence that down-regulation of miR-106b might also play an important role in the progression of the disease. The study results are consistent with the literature and support the notion that miR-106b is an oncogenic microRNA.     

The potential value of miR-1 and miR-374b as biomarkers for colorectal cancer

The mortality of colorectal cancer (CRC) is growing due to the unsatisfactory specificity and sensitivity of the existing screening methods. Previous studies have focused on the role of miRNAs as CRC biomarkers. However, few studies have examined the miRNA profiles at each stage. The objective of this study was to identify miRNAs that distinguish CRC patients from normal people to prevent the misdiagnosis of patients with certain stages of CRC. We performed miRNA profiling of 1547 human miRNAs by qRT-PCR in CRC patients with stage II and stage III disease. The statistical analyses showed that there were 96 miRNAs that were significantly dysregulated in CRC relative to normal tissues (P<0.05). There were 28 dysregulated miRNAs associated with separate or combined stages II and III disease. There were 25 downregulated miRNAs, including the following: miR-1, -145, -145*, -137, -363, -143, -4770, -490-5p, -9, -144*, -99a, -99b, -23b, -143*, -100, -768-3p, -24-1*, -125a-5p, -30e*, -574-3p, -126, let-7b, miR-1979, -374b, and -140-3p. We found an upregulation of miR-203, 182, and 96. Our results demonstrated that the expression of miR-1 and miR-374b was significantly decreased in each stage and may function as a biomarker of CRC. Furthermore, 20 miRNAs were dysregulated both in stage II disease without lymph node or distant metastasis and in stage II-III tumors but not in stage III tumors. Only miR-4794 was involved exclusively with stage II tumors, and there were 19 miRNAs that were dysregulated only in stage III disease with lymph node metastasis and in stage II-III disease. There were only 6 miRNAs that were uniquely dysregulated in stage III. Our results indicate that miRNA expression may be valuable in the clinic. However, large prospective studies are required to confirm the role of miRNAs. This study provides a new model for analyzing novel CRC biomarkers by considering more clinical factors.     

miR-106a promotes growth and metastasis of non-small cell lung cancer by targeting PTEN

MicroRNAs are a class of small non-coding RNAs that play essential roles in cancer development and progression. Recent studies suggested that abnormal expression of miRNAs occurs frequently in non-small cell lung cancer (NSCLC) tissues compared to adjacent normal tissues. In this study, we investigated the expression and the biological roles of miR-106a in non-small cell lung cancer. Our results showed that miR-106a was up-regulated in NSCLC tissues and cell lines. Inhibition of miR-106a in NSCLC cells substantially inhibited cell proliferation, migration, and invasion. Phosphatase and tensin homolog (PTEN) was identified as a direct target of miR-106a, and over-expression of miR-106a suppressed PTEN by direct binding to its 3’-untranslated region (3’-UTR). Furthermore, the presence of miR-106a was inversely correlated with PTEN in NSCLC tissues. Overall, this study suggested that miR-106a inhibited the growth and metastasis of NSCLC cells by decreasing PTEN expression. These data provide novel insights with potential therapeutic applications for the treatment of NSCLC.     

同期放化疗诱导人结直肠癌细胞发生上皮-间质转化

目的:探讨放化疗抵抗的结直肠癌细胞发生上皮-间质转化(epithelial-mesenchymal transition,EMT)的意义.方法:采用5-FU化疗同期进行放疗对人结直肠癌野生型细胞(HCTll6)进行干预,诱导放化疗共同抵抗的细胞株(HCT1 16CRR)并采用克隆形成实验进行放化疗抵抗性的鉴定.高倍显微镜下观察细胞形态学变化.采用Real-time PCR和Western印迹,检测上皮表型标志物E-cadherin,间质表型标志物N-cadherin、波形蛋白(vimentin)、核转录因子(Snail)mRNA及其蛋白的表达.结果:放化疗抵抗的结直肠癌细胞发生与EMT相符的形态学改变,细胞呈纺锤体状,极性消失,并出现伪足;Real-time PCR和Western印迹结果显示E-cadherin mRNA及蛋白表达下调;N-cadherin,vimentin,Snail mRNA及蛋白表达上调,差异有统计学意义(P＜0.05).结论:放化疗抵抗后的人结直肠癌细胞发生EMT,其与结直肠癌的治疗抵抗相关.     

miR-125b通过下调HAX-1的表达提高CD133~+ SW480细胞对顺铂的敏感性

目的:探讨miR-125b在CD133~+结直肠癌细胞中发挥的作用并研究其是否和顺铂的体外治疗有关。方法:用RT-qPCR方法检测miR-125b在常规SW480肿瘤细胞及CD133~+ SW480肿瘤细胞中的表达水平。流式细胞术检测miR-125b和顺铂对SW480细胞系中CD133~+ 细胞比例的影响。MTT法检测miR-125b对顺铂杀伤CD133~+ SW480细胞能力的影响。利用生物信息学及Western blot方法验证miR-125b是否调节CD133~+ SW480细胞中HAX-1的表达。运用JC-1染色、Annexin V染色及Western blot方法研究miR-125b影响顺铂疗效的信号通路的效应。结果:CD133~+ SW480细胞中的miR-125b表达水平显著低于正常结直肠上皮细胞系FHC和常规SW480肿瘤细胞。顺铂体外单独治疗能提高SW480细胞系中CD133~+细胞的比例,然而联用miR-125b模拟物后CD133~+ SW480细胞的比例显著下降。MTT实验结果表明miR-125b可显著增强顺铂对CD133~+ SW480细胞的杀伤活性。Western blot实验表明miR-125b的靶基因可能为HAX-1。miR-125b联合顺铂可引起CD133~+ SW480细胞线粒体膜电位的丧失并诱导线粒体内细胞色素C的释放,进而引起细胞凋亡。转染HAX-1表达载体后miR-125b联合顺铂对CD133~+ SW480细胞的凋亡诱导效应显著降低。结论:miR-125b通过下调HAX-1的表达提高CD133~+结直肠癌细胞对顺铂的敏感性。     

miR-125靶向SMAD4抑制脂多糖诱导的人结肠上皮细胞向肿瘤细胞转化

目的 观察脂多糖(LPS)对正常结肠上皮细胞向肿瘤细胞转化的影响,探索miR-125和SMAD4在细胞转化过程中的作用和机制.方法 以人正常结肠上皮细胞系HCoEpiCs为研究对象,用CCK-8法探索LPS的最佳刺激浓度;用LPS连续刺激HCoEpiCs细胞40代并检测细胞周期相关基因cyclinD1、癌基因c-myc...     

MicroRNA-145 suppresses cell migration and invasion by targeting paxillin in human colorectal cancer cells

A number of cancers show increased expression of paxillin which plays a central role in tumor progression, including colorectal cancer. However, the mechanisms causing paxillin upregulation remains unclear. In our study, bioinformatics analyses suggested that paxillin is predicted to be a direct target of miR-145. We firstly identified paxillin as a new target of miR-145 and demonstrated that miR-145 inhibits paxillin expression by binding to the paxillin mRNA 3’UTR. Therefore, we assume overexpression of paxillin induced by suppression of miR-145 may promote cell migration and invasion. We detected the expression of paxillin and miR-145 in human colorectal cancer tissues by real-time quantitative PCR. Higher expression of paxillin and lower expression of miR-145 was observed in colorectal cancer tissues than corresponding paracancerous tissue. Moreover, the expression of paxillin was negatively correlated with miR-145 expression. A dual-luciferase reporter assay was used to confirm that paxillin was a direct target of miR-145. In CRC cell lines, overexpression of miR-145 could downregulate paxillin protein expression levels, and ectopic overexpression of miR-145 mimics or inhibitor could inhibit or promote cell migration, invasion, proliferation and clone formation in vitro. Taken together, these data suggested that miR-145 plays a pivotal role in colon cancer through inhibiting cell proliferation migration and invasion, and miR-145 may serve as a tumor suppressor by targeting paxillin gene.     

miR-106b表达下调介导疟疾红内期疫苗免疫保护性下降

目的探讨miR-106b在疟疾红内期感染后控制疫苗免疫保护性随时间逐渐降低中的作用。方法 C57BL/6小鼠通过尾静脉接种约氏疟原虫P.yoelii 17XNL感染的红细胞,接种小鼠给予氯喹控制红内期感染的发生。接种完成后不同时间点(30 d、90 d、180 d)首先检测小鼠免疫保护性,ELISA检测小鼠血清抗红内期疟原虫抗体滴度,q RT-PCR检测血清miR-106b表达情况。然后,通过流式细胞技术检测脾脏B细胞TACI(transmembrane activator and CAML interactor)表达变化。最后通过体外培养B细胞,加入miR-106b mimic后检测B细胞TACI表达变化。结果免疫后30 d小鼠可产生100%的免疫保护,免疫后90 d小鼠在i RBC攻击后第3天均出现感染,而免疫后180 d小鼠在i RBC攻击后第2天均出现感染。同时,免疫后180 d与30 d相比抗体滴度明显下降(P<0.01),并且小鼠血清中miR-106b的表达也呈现明显的下降趋势。流式细胞分析发现免疫后180 d小鼠脾脏记忆性B细胞(memory B cells,MBCs)表面TACI表达比免疫30 d及90 d小鼠均出现明显下降(P<0.01)。体外培养B细胞加入miR-106b mimic后TACI表达增高。结论疟原虫红内期感染后控制疫苗保护性降低与血清中miR-106b及脾脏MBCs表面TACI表达降低有关,提高miR-106b的浓度可增加MBCs表面TACI的表达。     

miR-489通过调控TWIST1的表达抑制结肠癌干细胞表型和上皮间质转换

目的探讨miR-489在结肠癌干细胞表型和上皮间质转换中的作用和调控机制。方法 RT-q PCR检测多个人结肠癌细胞系(HT29、SW480、SW620和HCT116)和正常肠道上皮细胞HIEC中miR-489表达;采用基因转染技术提高结肠癌细胞中miR-489表达,Western blot检测过表达miR-489对结肠癌干细胞标志物CD133、CD44、Ep CAM、ALDH1和上皮细胞标志物E-cadherin、间质细胞标志物vimentin和N-cadherin的表达;RT-q PCR和Western blot检测TWIST1 mRNA和蛋白表达水平。结果 4个结肠癌细胞系中的miR-489相对表达量显著低于正常肠道上皮细胞HIEC中的表达(P<0.05)。在HT29和HCT116细胞中过表达miR-489,4个结肠癌干细胞标志物表达降低(P<0.05);上皮细胞标志物表达增加(P<0.05);间质细胞标志物表达减少(P<0.05)。TWIST1是miR-489潜在靶基因,在2个细胞系中过表达miR-489,TWIST1 mRNA和蛋白表达水平均显著降低(P<0.05)。结论 miR-489在结肠癌细胞中低表达,过表达miR-489可能通过调控TWIST1抑制结肠癌干细胞表型和上皮间质转换。     

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。     

MiR-223 inhibited cell metastasis of human cervical cancer by modulating epithelial-mesenchymal transition

Accumulating evidence have emerged important roles for microRNAs (miRNAs) participating in epithelial-mesenchymal transition (EMT) process and are associated with metastasis in cervical cancer. We hypothesized that miR-223 played an important role in cell metastasis of cervical cancer. Here, we found miR-223 was downregulated in human cervical cancer cell lines and clinical tumor tissues. Result of wound healing and cell migration assays revealed that miR-223 inhibited cell migration, whereas miR-223-in showed the opposite effect. In terms of mechanism, miR-223 influenced the expression of the EMT-associated proteins by upregulating the epithelial markers E-cadherin and α-cadherin and downregulating the mesenchymal marker vimentin. In conclusion, miR-223 inhibited cell metastasis of human cervical cancer by modulating epithelial-mesenchymal transition.