甘草酸通过调控miR-142/ZEB1 分子轴影响非小细胞肺癌HCC827 和A549 细胞的恶性生物学行为

目的：探讨甘草酸（GA）通过调控miR-142/锌指E 盒结合的同源盒蛋白1（ZEB1）分子轴对非小细胞肺癌（NSCLC）HCC827 和A549 细胞增殖、侵袭和迁移的影响。方法：HCC827 和A549 细胞培养和转染完成后,分成4 组：NC组（未经转染+3mmol/L GA）、miR-142 inhibitor 组（敲降miR-142+3 mmol/L GA）、pcDNA3.1-ZEB1 组（过表达ZEB1+3 mmol/L GA）和pcDNA3.1-ZEB1+miR-142 mimic 组（过表达ZEB1 及miR-142+3 mmol/L GA）。采用qPCR检测不同浓度GA处理后HCC827 和A549 细胞中miR-142 的表达水平,WB实验检测HCC827 和A549 细胞中ZEB1 蛋白的表达水平,采用MTT和Transwell 检测HCC827 和A549细胞的增殖、侵袭和迁移能力,采用双荧光素酶报告基因检测miR-142 与ZEB1 的靶向关系。结果：GA显著抑制HCC827 和A549 细胞的增殖、侵袭和迁移,且显著上调miR-142 的表达水平（P<0.05 或P<0.01）;miR-142 通过靶向结合ZEB1 的3''-UTR 区域下调ZEB1 的表达水平（P<0.05 或P<0.01）;进一步实验证实,GA通过上调miR-142 抑制ZEB1 的表达水平,进而抑制HCC827 和A549 细胞增殖、侵袭和迁移（P<0.05 或P<0.01）。结论：GA能够抑制NSCLC HCC827 和A549 细胞增殖、侵袭和迁移,其机制为GA通过上调miR-142对ZEB1 的抑制作用,从而抑制HCC827和A549 细胞的恶性生物学行为。     

Methyltransferase-like 3 promotes cervical cancer metastasis by enhancing cathepsin L mRNA stability in an N6-methyladenosine-dependent manner

N6-methyladenosine (m6A) is a highly abundant RNA modification in eukaryotic cells. Methyltransferase-like 3 (METTL3), a major protein in the m6A methyltransferase complex, plays important roles in many malignancies, but its role in cervical cancer metastasis remains uncertain. Here, we found that METTL3 was significantly upregulated in cervical cancer tissue, and its upregulation was associated with a poor prognosis in cervical cancer patients. Knockdown of METTL3 significantly reduced cervical cancer cell migration and invasion. Conversely, METTL3 overexpression markedly promoted cervical cancer cell metastasis in vitro and in vivo. Furthermore, METTL3 mediated the m6A modification of cathepsin L (CTSL) mRNA at the 5′-UTR, and the m6A reader protein insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) bound to the m6A sites and enhanced CTSL mRNA stability. Our results indicated that METTL3 enhanced CTSL mRNA stability through an m6A-IGF2BP2-dependent mechanism, thereby promoting cervical cancer cell metastasis. These findings provide insights into a novel m6A modification pattern involved in cervical cancer development.     

MicroRNA-377 Targets Zinc Finger E-box-Binding Homeobox 2 to Inhibit Cell Proliferation and Invasion of Cervical Cancer

A large number of microRNAs (miRNAs) are aberrantly expressed in cervical cancer and play crucial roles in the onset and progression of cervical cancer by acting as either an oncogene or a tumor suppressor. Therefore, investigation of the expression, biological roles, and underlying mechanisms of miRNAs in cervical cancer might provide valuable therapeutic targets in the treatment for patients with this disease. In this study, miRNA- 377 (miR-377) was downregulated in cervical cancer tissues and cell lines. Decreased miR-377 expression was strongly correlated with the International Federation of Gynecology and Obstetrics (FIGO) stage, lymph node metastasis, and distant metastasis in patients with cervical cancer. Enhanced expression of miR-377 prohibited cell proliferation and invasion in cervical cancer. Bioinformatics analysis predicted that zinc finger E-boxbinding homeobox 2 (ZEB2) was a potential target of miR-377. Subsequent experiments confirmed that ZEB2 is a direct target gene of miR-377 in cervical cancer. In addition, ZEB2 was overexpressed in cervical cancer tissues and was inversely related with miR-377 levels. Furthermore, the suppressive effects of miR-377 on cervical cancer proliferation and invasion were rescued by restored ZEB2 expression. Overall, our findings indicated that miR-377 decreases proliferation and invasion of cervical cancer cells by directly targeting ZEB2 and provides novel evidence of miR-377 as a novel therapeutic strategy for the therapy of patients with this malignancy.     

miR-30a通过靶向调控ZEB2抑制非小细胞肺癌的迁移与侵袭

目的:探讨miR-30a在非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞中的表达情况及miR-30a在肺癌细胞迁移和侵袭过程中的作用及其机制。方法:采用qRT-PCR检测miR-30a在不同NSCLC细胞株中的表达情况;采用脂质体2000转染miR-30a mimics和E盒结合锌指蛋白2（E-box binding zinc finger protein 2,ZEB2） siRNA;通过qRT-PCR检验miR-30a mimics和ZEB2 siRNA的转染效率及转染后ZEB2 mRNA的表达水平变化;Western blot检测转染miR-30a mimics和ZEB2 siRNA后ZEB2蛋白表达情况;采用双荧光素酶报告实验验证miR-30a和ZEB2的相互作用机制;划痕实验和Transwell小室侵袭实验检测上调miR-30a和干扰ZEB2对A549细胞迁移和侵袭能力的影响。结果:实验结果显示,与正常人支气管上皮细胞相比,在不同NSCLC细胞株中miR-30a的表达呈不同程度下调;NSCLC细胞转染miR-30a mimics和ZEB2 siRNA后均获得满意的转染效果:miR-30a mimics和ZEB2 siRNA明显降低了NSCLC细胞中ZEB2的mRNA和蛋白的表达水平,组间差异具有统计学意义。双荧光素酶报告实验验证miR-30a对ZEB2 3' UTR具有直接调控作用。细胞迁移实验和侵袭实验结果显示,与Blank组和NC组相比,miR-30a过表达组和ZEB2基因沉默组的A549细胞迁移和侵袭能力均明显降低,说明miR-30a mimics和ZEB2 siRNA均对A549细胞细胞迁移和侵袭有抑制作用。结论:上调miR-30a的表达水平可以负调控ZEB2转录后表达水平,通过抑制上皮-间质转化(epithelial-mesenchymal transition,EMT)的进程来抑制肺癌细胞的转移和侵袭。     

UBE2C对宫颈癌HeLa细胞增殖、迁移及侵袭能力的影响

目的:研究泛素结合酶E2C（ubiquitin-conjugating enzyme E2C,UBE2C）在宫颈癌细胞中的表达情况,并明确UBE2C对宫颈癌HeLa细胞增殖、迁移及侵袭能力的影响。方法:首先查阅GEPIA数据库,了解UBE2C在宫颈癌中的表达;其次通过qRT-PCR和Western blotting 法明确UBE2C在宫颈癌HeLa细胞及正常宫颈上皮细胞End1/E6E7中表达差异,然后分别构建si-UBE2C及si-NC转染宫颈癌HeLa细胞,检测转染后细胞中 UBE2C mRNA和蛋白表达;最后采用CCK-8 实验、细胞划痕实验及Transwell实验检测转染后细胞的增殖、迁移和侵袭能力的变化,并鉴定UBE2C下游靶基因PTEN、P-p53的表达情况。结果:UBE2C在宫颈癌中高表达,能够促进宫颈癌细胞的增殖、迁移及侵袭能力,并下调抑癌基因PTEN、P-p53的表达。结论:UBE2C能够加重宫颈癌细胞恶性表型;UBE2C可能作为宫颈癌诊断及治疗的判定指标之一。     

Targets of miR-200c mediate suppression of cell motility and anoikis resistance

Introduction  

miR-200c and other members of the miR-200 family promote epithelial identity by directly targeting ZEB1 and ZEB2, which repress E-cadherin and other genes involved in polarity. Loss of miR-200c is often observed in carcinoma cells that have undergone epithelial to mesenchymal transition (EMT). Restoration of miR-200c to such cells leads to a reduction in stem cell-like characteristics, reduced migration and invasion, and increased sensitivity to taxanes. Here we investigate the functional role of novel targets of miR-200c in the aggressive behavior of breast and endometrial cancer cells.     

miR-192-5p通过靶向ZEB2调控胰腺癌PANC-1细胞的恶性生物学行为

目的：探讨miR-192-5p靶向E盒锌指结合同源框2（ZEB2）对胰腺癌PANC-1细胞增殖、迁移、侵袭和上皮间质转化（EMT）的影响及其作用机制。方法：利用TCGA数据库数据分析miR-192-5p和ZEB2在胰腺癌组织中的表达及两者的相关性。采用qPCR法和WB法分别检测人正常胰腺上皮细胞HPNE和胰腺癌PANC-1细胞中miR-192-5p和ZEB2的表达水平。利用脂质体转染技术转染PANC-1细胞,实验分为miR-192-5p mimic组、Mimic NC组、miR-192-5p inhibitor组、Inhibitor NC组、Mimic NC+pcDNA3.1 组 、miR-192-5p mimic+pcDNA3.1 组 、miR-192-5p mimic+pcDNA3.1-ZEB2 组。CCK-8 法 、克隆形成 、划痕愈合 、Transwell实验分别检测转染PANC-1细胞的增殖、克隆形成、迁移和侵袭能力。qPCR法、WB法、双重免疫荧光实验检测PANC-1细胞中ZEB2、E-cadherin、vimentin的表达水平。通过生物信息学网站预测miR-192-5p的靶基因,并利用双荧光素酶报告基因实验验证miR-192-5p对靶基因的调控作用。结果：胰腺癌组织中ZEB2的表达显著高于正常胰腺组织（P<0.01）,且胰腺癌组织中miR-192-5p和ZEB2表达呈负相关（r=-0.419,P<0.01）。与HPNE细胞比较,PANC-1细胞中miR-192-5p低表达、ZEB2蛋白高表达（均P<0.01）。miR-192-5p过表达后,PANC-1细胞增殖、克隆形成、迁移和侵袭能力均显著降低,E-cadherin的mRNA和蛋白表达均上调、vimentin和ZEB2 mRNA和蛋白表达均下调;而抑制miR-192-5p后得到了相反的结果（P<0.05或P<0.01）。miR-192-5p靶向调控ZEB2的表达,过表达ZEB2可部分逆转上调miR-192-5p对PANC-1细胞增殖、迁移、侵袭和EMT进程的抑制作用。结论：miR-192-5p通过靶向ZEB2调控胰腺癌PANC-1细胞的恶性生物学行为。     

Knockdown of Long Noncoding RNA CAT104 Inhibits the Proliferation,Migration, and Invasion of Human Osteosarcoma Cells by Regulating MicroRNA-381

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. This study aimed to explore the effects of long noncoding RNA CAT104 and microRNA-381 (miR-381) on osteosarcoma cell proliferation, migration, invasion, and apoptosis, as well as the underlying potential mechanism. We found that CAT104 was highly expressed in osteosarcoma MG63 and OS-732 cells. Knockdown of CAT104 significantly inhibited OS-732 cell proliferation, migration, and invasion, but promoted cell apoptosis. CAT104 regulated the expression of miR-381, and miR-381 participated in the effects of CAT104 on OS-732 cells. Zinc finger E-box-binding homeobox 1 (ZEB1) was a direct target gene of miR-381, which was involved in the regulatory roles of miR-381 in OS-732 cell proliferation, migration, invasion, and apoptosis, as well as c-Jun N-terminal kinase (JNK) and Wnt/ -catenin pathways. In conclusion, our research verified that suppression of CAT104 exerted significant inhibitory effects on osteosarcoma cell proliferation, migration, and invasion by regulating the expression of miR-381 and downstream ZEB1, as well as JNK and Wnt/ -catenin pathways.     

Long non‐coding RNA urothelial cancer‐associated 1 promotes bladder cancer cell migration and invasion by way of the hsa‐miR‐145–ZEB1/2–FSCN1 pathway

Numerous studies suggest that several long non‐coding RNAs (lncRNAs) play critical roles in bladder cancer development and progression. Long non‐coding RNA urothelial cancer‐associated 1 (lncRNA‐UCA1) is highly expressed in bladder cancer tissues and cells, and it has been shown to play an important role in regulating aggressive phenotypes of bladder cancer cells. However, little is known about the molecular mechanism of lncRNA‐UCA1‐mediated bladder cancer cell migration and invasion. Here, we show that overexpression of lncRNA‐UCA1 could induce EMT and increase the migratory and invasive abilities of bladder cancer cells. Mechanistically, lncRNA‐UCA1 induced EMT of bladder cancer cells by upregulating the expression levels of zinc finger E‐box binding homeobox 1 and 2 (ZEB1 and ZEB2), and regulated bladder cancer cell migration and invasion by tumor suppressive hsa‐miR‐145 and its target gene the actin‐binding protein fascin homologue 1 (FSCN1). Furthermore, we also observed a positive correlation between lncRNA‐UCA1 and ZEB1/2 expression, and a negative correlation between lncRNA‐UCA1 and hsa‐miR‐145 expression in bladder cancer specimens. Importantly, we found that lncRNA‐UCA1 repressed hsa‐miR‐145 expression to upregulate ZEB1/2, whereas the suppression of hsa‐miR‐145 could upregulate lncRNA‐UCA1 expression in bladder cancer cells. Moreover, the binding site for hsa‐miR‐145 within exons 2 and 3 of lncRNA‐UCA1 contributed to the reciprocal negative regulation of lncRNA‐UCA1 and hsa‐miR‐145. Taken together, our results identified that lncRNA‐UCA1 enhances bladder cancer cell migration and invasion in part through the hsa‐miR‐145/ZEB1/2/FSCN1 pathway. Therefore, lncRNA‐UCA1 might act as a promising therapeutic target for the invasion and metastasis of bladder cancer.     

53BP1 suppresses epithelial–mesenchymal transition by downregulating ZEB1 through microRNA‐200b/429 in breast cancer

Epithelial–mesenchymal transition (EMT) is an important mechanism of cancer invasion and metastasis. Although p53 binding protein 1 (53BP1) has been implicated in several biological processes, its function in EMT of human cancers has not yet been reported. Here, we show that 53BP1 negatively regulated EMT by modulating ZEB1 through targeting microRNA (miR)‐200b and miR‐429. Furthermore, 53BP1 promoted ZEB1‐mediated upregulation of E‐cadherin and also inhibited the expressions of mesenchymal markers, leading to increased migration and invasion in MDA‐MB‐231 breast cancer cells. Consistently, in MCF‐7 breast cancer cells, low 53BP1 expression reduced E‐cadherin expression, resulting in increased migration and invasion. These effects were reversed by miR‐200b and miR‐429 inhibition or overexpression. Sections of tumor xenograft model showed increased ZEB1 expression and decreased E‐cadherin expression with the downregulation of 53BP1. In 18 clinical tissue samples, expression of 53BP1 was positively correlated with miR‐200b and mir‐429 and negatively correlated with ZEB1. It was also found that 53BP1 was associated with lymph node metastasis. Taken together, these results suggest that 53BP1 functioned as a tumor suppressor gene by its novel negative control of EMT through regulating the expression of miR‐200b/429 and their target gene ZEB1.     

MiR‐150 is associated with poor prognosis in esophageal squamous cell carcinoma via targeting the EMT inducer ZEB1

The association of microRNAs (miRs) with cancer progression has been established in many cancers including esophageal squamous cell carcinoma (ESCC). A public microarray database showed that the expression of miR‐150 was lower in ESCC than in normal esophageal mucosa. Here, we focused on ZEB1, epithelial‐mesenchymal‐transition (EMT)‐inducer, as a target gene of miR‐150 based on in silico predictions. The purpose of this study was to clarify the clinicopathological significance of miR‐150 in ESCC, and to investigate miR‐150′s EMT‐regulatory ability. Quantitative RT‐PCR was used to evaluate miR‐150 expression in 108 curative resected ESCC samples to determine the clinicopathological significance. Moreover, we examined the in vitro and in vivo function of miR‐150 via degradation of ZEB1. MiR‐150 expression was significantly lower in cancer tissues compared to adjacent non‐cancerous tissues (P  <  0.001). Low expression of miR‐150 in ESCC contributed to malignant potential, such as tumor depth, lymph node metastasis, lymphatic invasion, venous invasion, clinical staging, and poor prognosis (P  <  0.05). In vitro assays showed that EMT‐inducer‐ZEB1 is a new direct target of miR‐150. Moreover, miR‐150 induced MET‐like changes in TE‐8 cells through ZEB1 degradation (e.g., E‐cadherin expression, vimentin repression, epithelial morphology, and suppression of migration ability), and significantly inhibited tumorigenicity and tumor growth in a mouse xenograft model. Analysis of the regulation of ZEB1 by miR‐150 could provide new insights into preventing metastasis and also suggests novel targeted therapeutic strategies in ESCC. (Cancer Sci 2013; 104: 48–54)     

miR-26b-3p对乳腺癌细胞生物学行为的影响及作用机制研究

背景与目的：miRNA是一类长度为21~23个核苷酸的单链非编码RNA分子，其作用机制主要为靶向于mRNA的3’非翻译区（3’ untranslated region，3’UTR）从而抑制其靶基因的表达。miRNA在肿瘤的发生、发展过程中发挥着关键作用，探讨miR-26b-3p对乳腺癌生物学行为的影响及作用机制。方法：通过实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）检测miR-26b-3p在三种乳腺癌细胞系MCF-7、MDA-MB-231和MDA-MB-453中的表达，选取miR-26b-3p表达水平最低的乳腺癌细胞转染miR-26b-3p mimics后，采用细胞计数试剂盒（cell counting kit-8，CCK-8）法检测细胞的增殖，采用transwell迁移和侵袭实验检测细胞迁移和侵袭能力，通过小动物活体成像及裸鼠移植瘤模型检测miR-26b-3p对乳腺癌细胞裸鼠移植瘤生长和转移的影响，采用双荧光素酶报告基因分析检测miR-26b-3p与锌指E盒结合同源盒基因1（zinc finger E-box binding homeobox 1，ZEB1）的相互作用，采用RTFQ-PCR和蛋白质印迹法（Western blot）检测ZEB1的表达。结果：乳腺癌细胞系MDA-MB-453中miR-26b-3p表达最低，在MDA-MB-453细胞中转染miR-26b-3p mimics后，miR-26b-3p的表达水平显著升高（P<0.05），细胞的增殖能力显著降低（P<0.05），细胞的迁移（P<0.001）和侵袭能力（P<0.01）显著降低。过表达miR-26b-3p可抑制裸鼠体内乳腺癌移植瘤的生长和转移。miR-26b-3p可与ZEB1的3’UTR结合，抑制ZEB1的表达。结论：miR-26b-3p可靶向于ZEB1，抑制乳腺癌细胞的增殖、迁移和侵袭，抑制乳腺癌的生长和转移。     

lncRNA MALAT1/miR-141-3p/ZEB1 分子轴调控胃癌SGC7901 细胞的侵袭、迁移及上皮间质转化

[摘要] 目的：探究lncRNA MALAT1/miR-141-3p/ZEB1 分子轴对胃癌（GC）SGC7901 细胞侵袭、迁移及上皮间质转化（EMT）的调控作用。方法：收集2014 年4 月至2017 年5 月武汉商职医院普外科手术切除的GC组织（非坏死部分）和配对癌旁组织（距肿瘤组织>5 cm）标本38 例,同时选取正常胃上皮细胞GES1 及GC细胞系SGC7901、HGC27、BGC823、MKN45 和MKN28。qPCR实验检测MALAT1、miR-141-3p 在GC组织和细胞系中的表达水平,CCK-8 和Transwell 实验检测敲降MALAT1 对SGC7901 细胞增殖、迁移和侵袭的影响,WB 实验检测ZEB1、E-cadherin、N-cadherin 和Vimentin 的表达情况。双荧光酶素报告基因验证MALAT1、miR-141-3p 和ZEB1 的靶向关系,CCK-8 和Transwell 实验检测MALAT1/miR-141-3p/ZEB1 分子轴对SGC7901 细胞生物学行为的影响。结果：MALAT1 在GC组织和细胞系中高表达（P<0.05 或P<0.01）。敲降MALAT1 显著抑制了SGC7901 细胞增殖、迁移、侵袭及EMT（P<0.05 或P<0.01）;MALAT1 与miR-141-3p、miR-141-3p 与ZEB1 均具有直接靶向关系;进一步研究表明,同时过表达miR-141-3p 和MALAT1 或ZEB1 能够逆转miR-141-3p 对SGC7901 细胞生物学行为的抑制作用。结论：MALAT1通过靶向下调miR-141-3p 对ZEB1 的抑制作用,进而促进SGC7901 细胞侵袭、迁移及EMT。     

IGF2BP2 promotes the progression of colorectal cancer through a YAP-dependent mechanism

To explore the effect of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) on colorectal cancer (CRC) by recognizing the m6A modification of YAP mRNA thus activating ErbB2 expression. High expressions of IGF2BP2, YAP, and ErbB2 promoted the proliferation, migration and invasion of CRC cells and reduced their apoptosis. IGF2BP2 recognized the m6A on YAP mRNA and promoted the translation of mRNA. YAP regulated ErbB2 expression by promoting TEAD4 enrichment in ErbB2 promoter region. Therefore, IGF2BP2 promoted the expression of ErbB2 to enhance the proliferation, invasion and migration of CRC cells, to repress cell apoptosis, and to promote solid tumor formation in nude mice. IGF2BP2 activates the expression of ErbB2 by recognizing the m6A of YAP, thus affecting the cell cycle of CRC, inhibiting cell apoptosis, and promoting proliferation.