microRNA-671-5p在肝细胞癌中的表达及其负向调控丝切蛋白2与上皮细胞-间质转化的关系
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石磊, Email: 1215448013@qq.com

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陕西省科学技术研究发展计划基金资助项目(S2015YFSF0507)。


Expression of microRNA-671-5p in hepatocellular carcinoma and the relationship between its negative regulating cofilin 2 and epithelial-mesenchymal transition 
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    摘要:

    背景与目的:近年研究发现,microRNA-671-5p(miR-671-5p)参与了多种恶性肿瘤的发生发展,同时与多种病毒介导的肝损伤相关,但其与肝细胞癌(HCC)之间的关系目前仍未见报道。本研究的目的为观察miR-671-5p在HCC中的表达情况,分析其功能及其与HCC生物学行为及临床病理特征的联系,并初步探讨作用机制。
    方法:用qRT-PCR检测80例HCC组织及癌旁组织样本、不同HCC细胞系(Hep3B、MHCC-97H、HepG2、SMMC-7721)及人正常肝细胞(L02)中miR-671-5p的表达,且在同时分析TCGA数据库中miR-671-5p在HCC组织与癌旁组织的表达差异。分析miR-671-5p表达量与临床病理因素的关系;用miR-671-5p抑制物敲低MHCC-97H细胞系中miR-671-5p的表后,分别采用CCK-8实验及Transwell实验分别检测HCC细胞转染miR-671-5p抑制物后增殖、侵袭及迁移能力的变化。利用TargetScan及Starbase网站预测miR-671-5p的靶基因,并通过Western blot、双荧光素酶实验及TCGA数据库分析验证。用Western blot观察降低miR-671-5p表达对HCC细胞中miR-671-5p靶基因及上皮细胞-间质转化(EMT)相关蛋白(E-cadherin、N-cadherin、vimentin)表达的影响,以及在此基础上同时敲低靶基因的表达后,以上蛋白表达的变化。
    结果:miR-671-5p的表达在HCC组织中明显高于其癌旁组织,在各种HCC细胞系中均明显高于正常肝细胞,且随着样本肿瘤分期与HCC细胞的侵袭力的增加而升高(均P<0.05);TCGA数据库分析也显示,miR-671-5p在HCC组织中的表达量明显高于癌旁组织(P<0.05)。miR-671-5p的表达水平与AFP水平、肿瘤数目、静脉侵犯、Edmondson-Steiner分级及TNM分期明显有关(均P<0.05)。转染miR-671-5p抑制物后,MHCC-97H细胞的增殖、侵袭及迁移能力均明显降低(均P<0.05)。生物信息学分析及双荧光素酶实验均显示丝切蛋白2(CFL2)是miR-671-5p潜在靶基因,TCGA数据库分析也显示miR-671-5p与CFL2的表达呈负相关(均P<0.05)。降低MHCC-97H细胞中miR-671-5p的表达后,CFL2蛋白的表达水平升高,同时EMT相关蛋白表达明显降低(均P<0.05),但同时干扰CFL2的表达后,以上变化均有明显程度的逆转(均P<0.05)。
    结论:miR-671-5p在HCC中表达上调,且与HCC的不良临床病理特征密切相关。miR-671-5p可促进HCC细胞的增殖、侵袭、迁移,其机制可能与抑制CFL2的表达而促进EMT发生有关。

    Abstract:

    Background and Aims: Recent investigations demonstrated that microRNA-671-5p (miR-671-5p) participates in the occurrence and development of several types of cancer, and is associated with the liver injury induced by various types of virus. However, the role of miR-671-5p in hepatocellular carcinoma (HCC) has not yet been reported to date. This study was conducted to observe the miR-671-5p expression in HCC, analyze its function and its association with the biological behaviors and clinicopathologic features of HCC, and preliminarily investigate the possible mechanism. 
    Methods: The miR-671-5p expressions in 80 paired specimens of HCC and tumor adjacent tissue as well as different HCC cell lines (Hep3B, MHCC-97H, HepG2 and SMMC-7721) and normal human hepatic cell line (L02) were determined by qRT-PCR method, and meanwhile, the expression difference in miR-671-5p between HCC tissue and tumor adjacent tissue were analyzed in TCGA database. The relations of miR-671-5p expression level with the clinicopathologic factors were analyzed. In MHCC-97H cells after knockdown of miR-671-5p expression by transfection with miR-671-5p inhibitors, the changes in proliferative and invasion/migration abilities were examined by CCK-8 assay and Transwell assay, respectively. The target genes of miR-671-5p were predicted by using the TargetScan and Starbase online websites, and then verified by Western blot, dual luciferase assay and TCGA database analysis, respectively. The influences of miR-671-5p knockdown on the expressions of target gene of miR-671-5p and the proteins (E-cadherin, N-cadherin and vimentin) associated with epithelial-mesenchymal transition (EMT) in MHCC-97H cells, as well as the changes in expressions of above proteins upon the same condition with the synchronous knockdown of the target gene were detected by Western blot.
    Results: The miR-671-5p expressions in HCC tissue and all studied HCC cell lines were significantly higher than that in tumor adjacent tissue or the normal hepatic cell line, and was increased with the elevation of the tumor stage of the sample and invasion ability of the HCC cells (all P<0.05); the TCGA database analysis also showed that the miR-671-5p expression level in HCC tissue was significantly higher than that in tumor adjacent tissue (P<0.05). The miR-671-5p expression level was significantly related to AFP level, tumor number, venous invasion, Edmondson-Steiner grade and TNM stage (all P<0.05). In MHCC-97H cells after transfected with miR-671-5p inhibitors, the proliferative, invasion and migration abilities were all significantly reduced (all P<0.05). Bio-informatics analysis and dual luciferase assay suggested that cofilin 2 (CFL2) was the potential target gene of miR-671-5p, and the TCGA database analysis also showed that there was a negative correlation between miR-671-5p expression and CFL2 expression (P<0.05). In MHCC-97H cells with down-regulated miR-671-5p expression, the CFL2 expression was significantly increased and the expressions of EMT-associated proteins were significantly decreased (all P<0.05), but all these changes were reversed to significant extents by synchronous interference of the CFL2 expression (all P<0.05).
    Conclusion: The miR-671-5p expression is up-regulated in HCC, which is closely associated with the unfavorable clinicopathologic features of HCC. MiR-671-5p can promote the proliferation, invasion and migration of HCC cells, and the mechanism may be probably related to its down-regulating CFL2 expression and thereby promote EMT process.

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李亚昭, 姚博文, 石磊. microRNA-671-5p在肝细胞癌中的表达及其负向调控丝切蛋白2与上皮细胞-间质转化的关系[J].中国普通外科杂志,2020,29(1):43-52.
DOI:10.7659/j. issn.1005-6947.2020.01.006

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  • 收稿日期:2019-08-02
  • 最后修改日期:2019-12-20
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  • 在线发布日期: 2020-01-25