Li Ning-Lei, Xiao Gang, Jin Yi-Yi, Deng Yun-Yao, Liu Yu-Jiao, Yin Liang-Chun
Department of General Surgery, The Third Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong 510630, China.
Taihe Hospital of Hubei University of Medicine, Shiyan 442000, China.
Pathol Res Pract. 2022 Oct;238:154115. doi: 10.1016/j.prp.2022.154115. Epub 2022 Sep 5.
Hepatocellular carcinoma (HCC) is one of the most common cancers, and has an extremely poor prognosis. Our previous study confirmed that the microRNA miR-361-5p inhibited the proliferation, metastasis, invasiveness, and epithelial-to-mesenchymal transition (EMT) process of HCC by targeting the transcription factor Twist1. Long non-coding RNAs (lncRNAs) are key regulators of processes such as cell differentiation, inflammation, tumor formation, and immune escape. However, the underlying interactions between the lncRNA LINC00992, miR-361-5p, and Twist1 in HCC progression is still elusive. In the current study, the DIANA-lncBase database was used to identify regulatory genes upstream of miR-361-5p. Reverse transcription-quantitative PCR (RT-qPCR) was used to quantify the expression of the genes encoding LINC00992, miR-361-5p, and Twist1 in HCC cells. The cell counting kit-8 (CCK-8) was used to measure HCC cell proliferation and Transwell was used to measure HCC cell migration and invasion. The dual-luciferase reporter assay and RNA pull-down assay were performed to examine the interaction between LINC00992 and miR-361-5p. Western blotting was used to detect the levels of Twist1 protein. The result confirmed that, among three lncRNAs tested, miR-361-5p was the one most significantly affected by LINC00992. RT-qPCR revealed that LINC00992 was highly expressed in HCC tissues and cells. The follow-up results showed that the expression of LINC00992 and miR-361-5p in HCC tissues were closely correlated with the rate of metastasis or recurrence of the HCC patients. Our result showed that the expression of miR-361-5p was lower in the LINC00992 (+) group than in the LINC00992 (-) group. CCK-8 and Transwell showed that LINC00992 promoted HCC cell proliferation, migration, and invasion, whereas dual-luciferase reporter assay and RNA pull-down assay showed that LINC00992 combined with miR-361-5p to act as a miRNA decoy in HCC. RT-qPCR and Western blotting confirmed that LINC00992 upregulated the expression of the Twist1 gene in HCC cells by downregulating expression of miR-361-5p. CCK-8 and Transwell assays confirmed that LINC00992 promoted the proliferation, metastasis, and invasiveness of HCC cells by downregulating miR-361-5p levels and consequently upregulating Twist1 expression, implying that these three elements may be promising targets for HCC therapy.
肝细胞癌（HCC）是最常见的癌症之一，预后极差。我们之前的研究证实，微小RNA miR-361-5p通过靶向转录因子Twist1抑制HCC的增殖、转移、侵袭及上皮-间质转化（EMT）过程。长链非编码RNA（lncRNA）是细胞分化、炎症、肿瘤形成和免疫逃逸等过程的关键调节因子。然而，lncRNA LINC00992、miR-361-5p和Twist1在HCC进展中的潜在相互作用仍不清楚。在本研究中，使用DIANA-lncBase数据库鉴定miR-361-5p上游的调控基因。采用逆转录定量PCR（RT-qPCR）对HCC细胞中编码LINC00992、miR-361-5p和Twist1的基因表达进行定量。使用细胞计数试剂盒-8（CCK-8）检测HCC细胞增殖，使用Transwell检测HCC细胞迁移和侵袭。进行双荧光素酶报告基因检测和RNA下拉检测以研究LINC00992与miR-361-5p之间的相互作用。使用蛋白质印迹法检测Twist1蛋白水平。结果证实，在所测试的三种lncRNA中，miR-361-5p受LINC00992影响最为显著。RT-qPCR显示LINC00992在HCC组织和细胞中高表达。随访结果表明，HCC组织中LINC00992和miR-361-5p的表达与HCC患者的转移或复发率密切相关。我们的结果显示，LINC00992（+）组中miR-361-5p的表达低于LINC00992（-）组。CCK-8和Transwell检测显示LINC00992促进HCC细胞增殖、迁移和侵袭，而双荧光素酶报告基因检测和RNA下拉检测显示LINC00992与miR-361-5p结合在HCC中充当miRNA诱饵。RT-qPCR和蛋白质印迹法证实LINC00992通过下调miR-361-5p的表达上调HCC细胞中Twist1基因的表达。CCK-8和Transwell检测证实LINC00992通过下调miR-361-5p水平并因此上调Twist1表达促进HCC细胞的增殖、转移和侵袭，这意味着这三个元件可能是HCC治疗的有前景的靶点。
