Department of Gastroenterology, Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Department of Interventional Oncology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
J Exp Clin Cancer Res. 2019 Apr 10;38(1):153. doi: 10.1186/s13046-019-1128-9.
Hepatocellular carcinoma (HCC) is the major histological type of liver cancer with high morbidity and mortality worldwide. Long noncoding RNAs (lncRNA) has been proved to be associated with various cancer types, while its regulation in HCC is largely unknown.
To figure out the specific role of lncRNA EPB41L4A-AS2 in HCC. Fluorescence in situ hybridization (FISH) was first used to determine the cellular sublocalization of EPB41L4A-AS2 to determine its primary mode of action. QRT-PCR, Western blot and hematoxylin-eosin staining were then used to measure the expression of genes in cells and tissues. Cell proliferation and invasion assays were performed to determine the effects of EPB41L4A-AS2, miR-301a-5p and FOXL1 on the malignant phenotype of tumor cells. With luciferase reporter assay, the direct interaction between target genes were further confirmed for research on molecular mechanism. Finally, the mice hepatocarcinoma model was also established to disclose the tumor suppressor effects of EPB41L4A-AS2 in vivo.
Here, we have identified a novel lncRNA EPB41L4A-AS2, which is significantly downregulated both in HCC cells and tissues, and plays a negative regulatory role in HCC proliferation and invasion. Mechanistically, cytoplasmic lncRNA EPB41L4A-AS2 functions as an efficient miR-301a-5p sponge, thereby release the expression inhibition of forkhead box L1 (FOXL1). Indeed, lncRNA EPB41L4A-AS2 inhibits proliferation and migration by upregulating FOXL1 expression and FOXL1 was confirmed as a direct target of miR-301a-5p. MiR-301a-5p shows an inverse correlation with EPB41L4A-AS2 expression and was verified as a direct target of EPB41L4A-AS2 as well. Correspondingly, FOXL1 and miR-301a-5p show opposite biological effects in cell proliferation and migration. Moreover, miR-301a-5p overexpression rescued the EPB41L4A-AS2 upregulation induced depression in proliferation, migration and invasion of HCC cells, as well as promotion effect on FOXL1 expression. Also, in vivo experiments proved that EPB41L4A-AS2 suppress tumor growth and extrahepatic metastasis (lung) via the miR-301a-5p-FOXL1 axis.
Taken together, this research revealed a concrete mechanism of lncRNA EPB41L4A-AS2 in HCC, which may serve as a potential biomarkers and novel therapeutic targets for further clinical application.
肝细胞癌（HCC）是全球发病率和死亡率较高的主要肝癌组织学类型。长链非编码 RNA（lncRNA）已被证明与多种癌症类型有关，但其在 HCC 中的调控作用尚不清楚。
为了确定 lncRNA EPB41L4A-AS2 在 HCC 中的具体作用。首先使用荧光原位杂交（FISH）来确定 EPB41L4A-AS2 的细胞亚定位，以确定其主要作用方式。然后使用 qRT-PCR、Western blot 和苏木精-伊红染色来测量细胞和组织中基因的表达。进行细胞增殖和侵袭实验以确定 EPB41L4A-AS2、miR-301a-5p 和 FOXL1 对肿瘤细胞恶性表型的影响。通过荧光素酶报告基因实验进一步证实了靶基因之间的直接相互作用，以研究分子机制。最后，还建立了小鼠肝癌模型以在体内揭示 EPB41L4A-AS2 的肿瘤抑制作用。
在这里，我们鉴定了一种新型 lncRNA EPB41L4A-AS2，其在 HCC 细胞和组织中均显著下调，并在 HCC 增殖和侵袭中发挥负调控作用。机制上，细胞质 lncRNA EPB41L4A-AS2 作为有效的 miR-301a-5p 海绵，从而释放叉头盒 L1（FOXL1）的表达抑制。事实上，lncRNA EPB41L4A-AS2 通过上调 FOXL1 表达来抑制增殖和迁移，FOXL1 被证实是 miR-301a-5p 的直接靶标。miR-301a-5p 与 EPB41L4A-AS2 的表达呈负相关，并且也被证实是 EPB41L4A-AS2 的直接靶标。相应地，FOXL1 和 miR-301a-5p 在细胞增殖和迁移中表现出相反的生物学效应。此外，miR-301a-5p 的过表达挽救了 EPB41L4A-AS2 上调引起的 HCC 细胞增殖、迁移和侵袭的抑制以及对 FOXL1 表达的促进作用。此外，体内实验证明，通过 miR-301a-5p-FOXL1 轴，EPB41L4A-AS2 可抑制肿瘤生长和肝外转移（肺）。
综上所述，这项研究揭示了 lncRNA EPB41L4A-AS2 在 HCC 中的具体作用机制，可为进一步的临床应用提供潜在的生物标志物和新的治疗靶点。
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