EffectofmiRNA-210-3pinInvas,LiWanming,YuMin,,KeyLaboratoryofCellBiology,MinistryofPublicHealth，KeyLaboratoryofMedicalCellBiology,MinistryofEducation,ChinaMedicalUniversity,Liaoning,China

AbstractObjectiveTo investigate the effect of miR-210-3p in colorectal cancer (CRC) cells migration and invasion and to analyze the enriched signaling pathways of target genes by bioinformatic expression of miR-210-3p in colorectal cancer was analyzed by dbDEMC and OncomiR database. Real-time quantitative PCR was used to detecte the expression of miR-210-3p in five types of CRC cells (CL187,HCT-8,SW480, HCT-116,LoVo). miRNA mimics or inhibitor was transfected in CRC cells to up or down regulate the expression of miR-210-3p. Transwell assays were used to evaluated migration and invasion of CRC cells after transfection. Target gengs were pridected by miRDB, , starBase and miRPathDB. Gene ontology analysis and signal pathway enrichment were performed by and OncomiR database showed that miR-210-3p was highly expressed in colorectal cancer, which was related to tumor metastasis and prognosis. Real-time quantitative PCR tested expression of miR-210-3p in highly invasiive cell lines(HCT-116 and LoVo) was significantly higher than that in moderate invasive cell lines SW480. And low invasive cell lines(CL187, HCT-8) revealed the lowest expression. Overexpression of miR-210-3p significantly enhanced SW480 cell migration and invasion, while LoVo cell migration and invasion was decreased after knockdown miR-210-3p. A total of 3035 target genes were predicted and the functions were mainly enriched in biological processes such as cell migration, adhesion, protein metabolism. The enriched pathways include cancer pathway, HIF signaling pathway and Rap1 signaling pathway. RGMA is significantly down regulated in clinical CRC tissues and may be the target gene of can improve the migration and invasion abilities of colorectal cancer cells, which indicates that it might be a new biological target for colorectal cancer treatment.

KeywordsmiR-210-3p; Colorectal cancer; Invasion; Bioinformatic analysis; Target gene

大肠癌是消化系统常见的一种恶性肿瘤，其发生率和病死率居高不下。引发大肠癌患者死亡的主要原因是早期诊断不及时，确诊时约30%的患者已发生转移[1]。因此寻找肿瘤转移的分子标志物对了解大肠癌转移机制和提高患者预后有重要意义。microRNA(miRNA)是一类长约22个核苷酸的内源性非编码RNA，与靶mRNA 3′UTR结合，促进其降解或抑制其翻译，从而对基因表达进行负调控[2]。大量研究表明，miRNA在多种肿瘤中表达异常，参与肿瘤的发生、发展及转移[3～5]。

miR-210-3p位于人11号染色体短臂，多项研究表明其在多种肿瘤中发挥着重要的作用。研究报道，在三阴性乳腺癌、非小细胞肺癌、胰腺癌等恶性肿瘤中，miR-210-3p的表达水平与患者的临床病理特征相关，可以作为预后及诊断的生物学标志物[6～8]。在肺腺癌中，miR-210-3p通过抑制赖氨酰氧化酶样4(LOXL4)促进癌细胞增殖和迁移，发挥癌基因作用；而在膀胱癌中，miR-210-3p则发挥抑制肿瘤生长和转移的抑癌作用[9,10]。但在大肠癌中miR-210-3p的作用仍不清楚，本研究旨在探讨miR-210-3p对大肠癌细胞迁移和侵袭能力的影响，预测相关靶基因及其富集的GO功能和信号转导通路，为深入开展miR-210-3p对大肠癌的转移机制的研究提供理论依据。

材料与方法

1.细胞系及主要试剂：选取不同转移潜能的大肠癌细胞系，高转移细胞系HCT-116和LoVo，中低转移细胞系SW480，低转移细胞系CL187、HCT-8，以上细胞系购自中国科学院上海细胞库。miR-210-3p模拟物，miR-210-3p抑制剂，miRNA NC和Transfection Kit均购自广州锐博生物科技有限公司；miRNA Isolation Kit购自美国Invitrogen公司；miRNA反转录引物、反转录试剂盒、检测试剂盒(All-in-one miRNA qRT-PCR Detection Kit)购自广州复能公司；胎牛血清、RPMI1640和DEME培养基购自以色列BI公司。

2.细胞培养及转染： HCT-8、SW480、HCT-116和LoVo细胞培养于含10%胎牛血清的RPMI 1640培养基，CL187细胞培养于含10%胎牛血清的DMEM培养基，置于37℃、5% CO2培养箱中培养。将对数生长期的细胞接种于培养皿中，培养至细胞汇合度达到30%～50%。将miR-210-3p 模拟物、miR-210-3p抑制剂、miRNA-NC按照转染试剂盒说明书提供的方法转染至SW480细胞中，继续培养用于后续实验。

3.miRNA的提取及实时荧光定量PCR实验：在无RNA酶条件下严格按照miRNA提取试剂盒说明书提取细胞miRNA。将miRNA反转录合成cDNA后，进行实时荧光定量PCR扩增。以U6为内参，采用2-ΔΔCt法计算miRNA表达。

迁移、侵袭实验：实验分为空白对照组、阴性对照组、miR-210-3p 模拟物组、miR-210-3p 抑制剂组。用无血清培养基制备细胞悬液，调整细胞浓度至2.0×105个/毫升，接种至无基质胶包被或包被Matrigel基质胶的Transwell小室上层，下室加入600μl含10%胎牛血清的培养基，培养48h后，95%乙醇固定30min，4%台盼蓝染色30min，于显微镜下计迁出细胞数。随机选择5个视野计算平均值。

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