Long noncoding RNA MEG3 is downregulated in cervical cancer and affects cell proliferation and apoptosis by regulating miR-21

Recent research has found that long noncoding RNAs (lncRNAs) were involved in various human cancers. However, the role of these lncRNAs in cervical cancer remains unexplored. Therefore, we aimed to investigate the biological function of maternally expressed gene 3 (MEG3), a cancer-related lncRNA, and its underlying mechanism in cervical cancer. In this study, MEG3 expression of 108 patients’ cervical cancer tissues and adjacent normal tissues was detected by quantitative real-time PCR analysis (qRT-PCR), and the functional effect of MEG3 was determined in vitro assays. We observed that MEG3 was downregulated in cervical cancer tissues, compared to the adjacent normal tissues, and was negatively related with FIGO stages, tumor size, lymphatic metastasis, HR-HPV infection and the expression of homo sapiens microRNA-21 (miR-21). Furthermore, we focused on the function and molecular mechanism of MEG3, finding that overexpression of MEG3 reduced the level of miR-21-5p expression, causing inhibition of proliferation and increased apoptosis in cervical cancer cells. In summary, our findings indicate that MEG3 function as a tumor suppressor by regulating miR-21-5p, resulting in the inhibition of tumor growth in cervical cancer. As a result, this study improves our understanding of the function of MEG3 in cervical cancer and will help to provide new potential target sites for cervical cancer treatment.     

长链非编码RNA FOXD2-AS1在骨肉瘤细胞中的表达及功能

目的:探讨长链非编码RNA FOXD2-AS1在骨肉瘤细胞中的表达谱及其功能。方法:前期对5对组织（骨肉瘤组织和正常组织）进行转录组学测序筛选出差异表达的lncRNAs,并筛选出上调变化的具有研究意义的前五位lncRNAs。通过荧光定量PCR进一步在骨肉瘤组织及正常组织内验证lncRNA FOXD2-AS1差异表达这一趋势,利用siRNA技术干扰其表达,筛选出干扰lncRNA FOXD2-AS1效率最高的两条siRNAs,CCK-8法及流式细胞术检测敲减lncRNA后细胞增殖及凋亡是否发生变化。结果:转录组学测序筛选出上调变化明显的前5位lncRNAs,并进一步通过实时荧光定量PCR显示lncRNA FOXD2-AS1在骨肉瘤组织内高表达,CCK-8法及流式细胞术结果显示该高表达的lncRNA FOXD2-AS1促进骨肉瘤细胞增殖,且抑制骨肉瘤细胞的凋亡。结论:骨肉瘤与正常组织相比,存在明显的高表达lncRNAs,且高表达的lncRNA FOXD2-AS1促进骨肉瘤的增殖并抑制其凋亡,在骨肉瘤进展中发挥至关重要的作用。     

长链非编码RNA MAFG-AS1在骨肉瘤中的表达及功能

目的:探讨及分析骨肉瘤中差异表达长链非编码RNA（long non-coding RNA,lncRNA）的表达谱及功能。方法:本课题前期通过5对组织（骨肉瘤组织和瘤旁组织）进行高通量转录组学测序筛选出差异表达的lncRNAs,并筛选出上调变化的前5位lncRNAs,通过荧光定量PCR在骨肉瘤细胞系内检测lncRNA MAFG-AS1的差异表达,并通过小干扰RNA技术干扰其表达,CCK-8法检测敲低lncRNA后细胞增殖是否发生变化,Western-blotting检测下游蛋白变化。结果:高通量数据分词浅析显示,共有376个lncRNAs在骨肉瘤组织中差异表达,其中206个上调,170个下调,并筛选出上调变化的前5位lncRNAs。荧光定量PCR显示lncRNA MAFG-AS1在骨肉瘤细胞内高表达。CCK-8法结果显示该lncRNA MAFG-AS1具有影响骨肉瘤细胞增殖的作用,且下游蛋白RRM2表达发生变化。结论:骨肉瘤组织中存在明显的lncRNAs差异性表达。且lncRNA MAFG-AS1在骨肉瘤发生和发展中发挥中重要的作用。     

肝细胞癌组织中lncRNA H19、miR-200a表达变化与临床预后的相关性分析

目的:观察肝细胞癌（hepatocellular carcinoma,HCC）组织中lncRNA H19、miR-200a表达情况,并探讨其与HCC患者临床病理特征及预后的关系。方法:选取57例HCC患者经手术切取的HCC组织和相应癌旁组织（≥癌灶边缘5 cm）,以及60例因肝血管瘤行手术切除的周围正常肝组织。采用qRT-PCR法检测组织中lncRNA H19和miR-200a表达水平;分析其与HCC患者临床病理特征及5年总生存率（overall survival,OS）的关系。结果:HCC组织中lncRNA H19表达水平明显高于癌旁组织和正常肝组织（P＜0.05）;HCC组织中miR-200a表达水平显著低于癌旁组织和正常肝组织（P＜0.05）。HCC组织中lncRNA H19、miR-200a表达与HCC患者分化程度、TNM分期、AFP值及是否侵袭转移有关（P＜0.05）。HCC组织中lncRNA H19表达与miR-200a表达呈负相关（P＜0.05）。HCC患者lncRNA H19低表达5年OS明显高于高表达（P＜0.05）;HCC患者miR-200a高表达5年OS显著高于低表达（P＜0.05）。分化程度、TNM分期、lncRNA H19及miR-200a表达均是影响HCC患者预后的独立危险因素（P均＜0.05）。结论:HCC组织中lncRNA H19表达上调、miR-200a表达下调,二者异常表达与病情程度及5年OS关系密切,可能相互作用参与HCC发展。     

长链非编码RNA MEG3对肝癌细胞增殖及自噬能力的影响

目的:探讨长链非编码RNA MEG3对肝癌细胞增殖及自噬能力的影响。方法:实时定量PCR检测60例肝癌组织标本以及相应癌旁组织中MEG3的表达水平，并检测MEG3在肝癌细胞系中的表达情况。通过质粒转染方法构建MEG3过表达的肝癌细胞Huh7和HepG2，采用CCK-8法检测肝癌细胞增殖情况；Western blot检测自噬相关蛋白（LC3Ⅱ/Ⅰ、p62）表达变化；利用免疫荧光检测LC3变化情况。结果:MEG3在肝癌组织中表达低于癌旁组织，差异有统计学意义(P<0.05)。肝癌细胞中过表达MEG3后，其细胞增殖能力显著降低（P<0.05）；并且过表达MEG3后能减少LC3-I向LC3-Ⅱ的转化，而p62表达升高（P<0.05）；免疫荧光可见明显LC3荧光斑点减少。结论:在肝癌组织中MEG3表达低于癌旁组织；在体外细胞实验中发现，MEG3能够抑制肝癌细胞增殖和自噬水平，有望成为肝癌治疗的潜在靶点。     

小细胞肺癌组织中lncRNA MEG3的表达及其与预后关系的研究

目的 探讨长链非编码RNA MEG3（lncRNA MEG3）在小细胞肺癌（SCLC）组织中的表达及其与预后的关系。方法 收集2012年1月至2015年12月间于我院就诊的SCLC组织标本92例,采用实时荧光定量PCR（QPCR）法检测lncRNA MEG3在92例SCLC组织、40例癌旁组织及50例正常肺组织标本中的表达,分析其表达与临床病理特征和预后的关系。结果 与癌旁组织（4.082±0.86）和正常肺组织（4.209±0.82）比较,lncRNA MEG3在92例SCLC组织中的表达（2.071±0.97）明显降低,差异有统计学意义（P＜0.001）。lncRNA MEG3表达与年龄、性别无关,与分期、远处转移及生存状态有关。lncRNA MEG3低表达者（＜2.071）的中位无进展生存期为8个月,较高表达者（≥2.071）的21个月短,差异有统计学意义（P＜0.001）;高表达者的中位总生存期为32个月,较低表达组的21个月长,差异有统计学意义（P＜0.001）。Cox比例风险模型分析显示,lncRNA MEG3表达、远处转移和分期均为影响SCLC总生存期的独立因素。结论 lncRNA MEG3参与调节SCLC的发生、发展,可作为潜在的评估SCLC预后的分子标志物。     

Transcriptome profiling of lncRNA and co-expression networks in esophageal squamous cell carcinoma by RNA sequencing

Long non-coding RNAs (lncRNAs) are emerging as crucial regulators of cancer. To identify novel targets for further study in esophageal squamous cell carcinoma (ESCC), we performed a genome-wide analysis of lncRNA expression in 12 ESCC tumor and normal tissues. Publicly available RNA-seq data were downloaded from the NCBI, GEO, and Co-LncRNA databases, and lncRNA and messenger RNA (mRNA) expression profiles were analyzed. In total, 127 lncRNAs were found to be differentially expressed, with a greater than fourfold change in ESCC tumor tissues compared with normal tissues. Among these lncRNAs, 98 were upregulated and 29 downregulated. Moreover, 1469 network nodes and 1720 connection edges between 119 lncRNAs and 1350 coding genes were integrated into the lncRNA and mRNA co-expression network. Bioinformatic analysis using GO terms revealed that these dysregulated lncRNAs are associated with developmental processes, proteinaceous extracellular matrix, and protein binding activity, with ECM-receptor interaction and the PI3K-Akt signaling pathway enrichment. Lastly, qRT-PCR results verified two significantly upregulated lncRNAs and three significantly downregulated lncRNAs in 50 pairs of ESCC tissues and adjacent normal tissues. These results reveal the landscape of ESCC-associated lncRNAs and co-expression networks, providing important insight regarding the lncRNAs involved in ESCC.     

lncRNA 的生物标记作用及 MEG3在肿瘤中的研究进展

长链非编码 RNA（lncRNA）是 RNA 分子学的一个新类型,由于转录长度超过200个核苷酸缺乏蛋白编码潜能而得名。一些 lncRNA 在特殊类型的癌症中具有高度特异的表达使其成为潜在的诊断工具,另一些 lncRNA 与肿瘤生长和患者生存期不同的病理生理特点有相关性,使其成为指示预后的生物标记物。众多 lncRNA 之一的母系表达基因3（MEG3）是一种印记基因,包含12个亚型基因,编码与肿瘤发生相关的lncRNA。MEG3通过调节 p53和促血管再生等发挥作用,提示 MEG3及其亚型可作为一个新型的 lncRNA 肿瘤抑制因子。近年来,有关 lncRNA 与肿瘤的相关性已成为研究的热点,本文就 lncRNA 生物标记作用和MEG3及其亚型与肿瘤疾病的研究进展作一综述。     

lncRNA MEG3 increases the radiosensitivity of lung cancer cells by regulating miR-21

Objective To investigate the regulatory mechanism of long-chain non-coding RNA (lncRNA) MEG3 on the sensitivity of lung cancer cell line H1299 to irradiation. Methods The expression of MEG3 and miR-21-5p in lung cancer cell line H1299 was detected by qRT-PCR. Overexpression control group (transfected with pcDNA3.1), MEG3 overexpression group (transfected with pcDNA3.1-MEG3), miR-NC inhibition group (transfected anti-miR-NC), miR-21-5p inhibition group (transfected with anti-miR-21-5p), MEG3 overexpression+miR-NC overexpression group (co-transfected with pcDNA3.1-MEG3 and miR-NC), MEG3 overexpression+miR-21-5p overexpression group (co-transfected with pcDNA3.1-MEG3 and miR-21-5p mimics) were all transfected into H1299 cells by liposome method treated with 4Gy irradiation. Cell survival fraction was detected by colony formation assay. Cell apoptosis was detected by flow cytometry. The binding of MEG3 to miR-21-5p in cells was assessed by dual luciferase reporter assay. Results Compared with normal lung epithelial cells, the expression of MEG3 was significantly decreased, whereas the expression of miR-21-5p was significantly increased in the radioresistant lung cancer cells H1299. Overexpression of MEG3 or inhibition of miR-21-5p could promote the apoptosis and enhance the radiosensitivity of H1299 cells. MEG3 could targetedly regulate the expression of miR-21-5p. Overexpression of miR-21-5p could reverse the enhanced radiosensitivity of MEG3 to H1299 cells. Conclusion LncRNA MEG3 can enhance the sensitivity of lung cancer cells H1299 to irradiation. The mechanism may be related to targeting miR-21-5p.     

lncRNA MEG3通过调控miR-21表达增加肺癌细胞放射敏感性

目的 探讨长链非编码RNA (lncRNA) MEG3对肺癌细胞H1299的放射敏感性调控机制。方法 运用qRT-PCR法检测具有放射抗性的H1299细胞中MEG3、miR-21-5p的表达。将过表达对照组(转染pcDNA3.1)、过表达MEG3组(转染pcDNA3.1-MEG3)、抑制miR-NC组(转染anti-miR-NC)、抑制miR-21-5p组(转染anti-miR-21-5p)、过表达MEG3+过表达miR-NC组(转染pcDNA3.1-MEG3和miR-NC)、过表达MEG3+过表达miR-21-5p组(转染pcDNA3.1-MEG3和miR-21-5p mimics)均用脂质体法转染。克隆形成实验检测细胞存活分数,流式细胞术检测细胞凋亡,双荧光素酶报告基因检测实验检测细胞中MEG3与miR-21-5p的结合力。结果 与正常肺上皮细胞相比,H1299细胞中MEG3表达明显降低,miR-21-5p表达明显升高;过表达MEG3或抑制miR-21-5p均可促进H1299细胞凋亡,增强放射敏感性;MEG3可靶向调控miR-21-5p的表达。过表达miR-21-5p可逆转MEG3对H1299细胞放射增强作用。结论 lncRNA MEG3可增强H1299细胞放射敏感性,其机制也许可能与靶向miR-21-5p有关。     

小细胞肺癌组织中lncRNA表达谱的差异

目的：观察长链非编码RNA（ lncRNA）在未发生转移的小细胞肺癌和发生转移的小细胞肺癌组织中表达的差异。方法：利用高通量lncRNA芯片技术分别检测3例未发生转移的小细胞肺癌组织和3例发生转移的小细胞肺癌组织中lncRNA表达谱的变异,经对原始数据进行预处理达到均一化后,筛选出差异表达的lncRNA,进行聚类分析。结果：将lncRNA表达在发生转移的小细胞肺癌组织中与未发生转移的小细胞肺癌组织中的变化倍数在2倍以上并有显著差异（ P＜0.05）的lncRNA,确定为差异性表达的lncRNA。其中在3例发生远处转移的患者肺癌组织中变化均在2倍以上的共842条,占所有lncRNA的12.1％。其中,2倍以上表达上调的共440条,2倍以上表达降低的共402条;5倍以上表达升高的共31条;5倍以上表达降低的共65条。结论：发生转移的小细胞肺癌组织与未发生转移的小细胞肺癌组织比较,lncRNA表达谱发生显著变化。提示差异性表达的lncRNAs参与了小细胞肺癌侵袭和转移的恶性表型。     

Long non-coding RNA MEG3 functions as a competing endogenous RNA to regulate gastric cancer progression

Background

Long noncoding RNAs (lncRNAs) have recently emerged as important regulators in governing fundamental biological processes, and many of which are likely to have functional roles in tumorigenesis. Maternally expressed gene 3 (MEG3) gene encodes a lncRNA whose expression is lost in an expanding list of primary human tumors and tumor cell lines, however its biological role and regulatory mechanism in gastric cancer (GC) development and progression are poorly defined.

Methods

Quantitative RT-PCR analysis was used to determine whether aberrant MEG3 expression was associated with GC patients pTNM stage and pM state. Furthermore, the effect of ectopic expression of MEG3 on cell proliferation, migration, invasion and cell apoptosis was assessed by using CCK-8, wound healing, transwell invasion assays and flow cytometric analysis, respectively, in GC cell lines HGC-27 and MGC-803. Moreover, the competing endogenous RNA (ceRNA) activity of MEG3 on miR-181a was investigated via luciferase reporter assay and immunoblot analysis.

Results

MEG3 is decreased in GC patients and cell lines, and its expression was associated with metastatic GC. Furthermore, ectopic expression of MEG3 in HGC-27 and MGC-803 cells inhibited cell proliferation, migration, invasion, and promoted cell apoptosis, which might be due to MEG3 sequestering oncogenic miR-181 s in GC cells. Furthermore, MEG3 could up-regulated Bcl-2 via its competing endogenous RNA (ceRNA) activity on miR-181a.

Conclusions

These findings suggest that lncRNA MEG3, a ceRNA of miR-181 s, could regulate gastric carcinogenesis and may serve as a potential target for antineoplastic therapies.     

lncRNASNHG10靶向调控miR-532-3p促进结直肠癌SW620细胞的侵袭和迁移

目的： 探讨lncRNA SNHG10在结直肠癌组织和细胞中的表达情况及其对结直肠癌细胞侵袭和迁移的影响与可能的机制。 方法： 收集2018年1月至2019年12月在河南省人民医院行根治性结直肠癌切除术的78例患者的癌组织及对应癌旁组织标本,采用qPCR法检测结直肠癌组织、结直肠癌细胞（SW480、SW620、HT-29和LoVo）及人正常结直肠黏膜细胞FHC中lncRNA SNHG10和miR-532-3p的表达水平,并分析其与结直肠癌患者临床病理特征的关系及在组织中表达的相关性。采用双荧光素酶报告基因实验验证lncRNA SNHG10和miR-532-3p间的靶向关系。向SW620细胞中转染si-SNHG10或miR-532-3p mimic或共转染si-SNHG10+miR-532-3p inhibitor,采用Transwell实验检测其侵袭和迁移能力的改变,采用WB法检测E-cadherin,N-cadherin和vimentin蛋白表达水平变化。 结果： SNHG10 在结直肠癌组织和细胞中呈高表达（P<0.05 或 P<0.01）,其表达水平与TNM分期和远处转移有关（均P<0.05）;miR-532-3p在结直肠癌组织和细胞中呈低表达,其表达水平与TNM分期、淋巴结转移和远处转移有关（P<0.05或P<0.01）,SNHG10和miR-532-3p在结直肠癌组织中的表达呈负相关（r=-0.225, P=0.048）。双荧光素酶报告基因实验证实SNHG10靶向调节miR-532-3p的表达。下调 SNHG10 或上调 miR-532-3p 的表达后,SW620 细胞的侵袭和迁移能力显著降低（P<0.01）,E-cadherin蛋白表达水平升高（P<0.05）、N-cadherin和vimentin蛋白表达水平降低（均P<0.05）。抑制miR-532-3p表达后,敲低lncRNA SNHG10表达对结直肠癌细胞侵袭和迁移的抑制作用被逆转（均P<0.05）。 结论： lncRNA SNHG10在结直肠癌中高表达并与TNM分期和远处转移相关,lncRNASNHG10靶向调控miR-532-3p表达并通过EMT途径影响结直肠癌细胞的侵袭和转移。