FOXO3-induced lncRNA LOC554202 contributes to hepatocellular carcinoma progression via the miR-485-5p/BSG axis

Abstract

Long non-coding RNAs (LncRNAs) have played very important roles in the malignancy behaviors of hepatocellular carcinoma (HCC). LncRNA LOC554202 (LOC554202) was a newly identified tumor-related lncRNA. However, its expression and function in HCC remained unknown. In this study, we firstly reported that LOC554202 expression was distinctly upregulated in HCC specimens and cell lines. Clinical assays indicated that increased LOC554202 expression had a diagnostic value for HCC patients and was positively associated with advanced stages and poor clinical prognosis. Additionally, forkhead box O3(FOXO3) could bind directly to the LOC554202 promoter region and activate its transcription. Functionally, we observed that knockdown of LOC554202 suppressed the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) progress of HCC cells, and promoted apoptosis. Mechanistically, LOC554202 competitively bound to miR-485-5p and prevented the suppressive effects of miR-485-5p on its target gene basigin (BSG), which finally led to HCC metastasis, EMT, and docetaxel chemoresistance. Our data demonstrated that FOXO3-induced LOC554202 contributed to HCC progression by upregulating BSG via competitively binding to miR-485-5p, which suggested that the regulation of the FOXO3/LOC554202/miR-485-5p/BSG axis may have beneficial effects in the treatment of HCC.

Fig. 1. The distinct upregulation of LOC554202 in HCC patients and its clinical significance.

A RT-PCR for the expression of LOC554202 in HCC specimens and matched non-tumor specimens. B The expression of LOC554202 in advanced grades using RT-PCR. C ROC curves of tissue LOC554202 expression for differentiating HCC tissue from normal tissue. D ROC curves of tissue LOC554202 expression for differentiating advanced HCC tissue from early HCC tissue. E Relative expression of LOC554202 in five HCC cell lines and human normal liver cell (LO2). F, G The levels of LOC554202 were determined in LM3/ADM and Huh7/ADM cells by RT-PCR. H, I Kaplan–Meier curves of the overall survival and disease-free survival of 137 HCC patients. *p < 0.05.

Fig. 2. LOC554202 is activated by FOXO3 in HCC.

A FOXO3-binding site prediction in the LOC554202 promoter region using JASPAR. B RT-PCR for the levels of FOXO3 in our cohort. C ROC curves of mRNA FOXO3 expression for differentiating advanced HCC tissue from early HCC tissue. D Relative expression of FOXO3 in five HCC cell lines and LO2. E The expression of FOXO3 in Huh and LM3 cells transfected with si-FOXO3 or pcDNA3.1-FOXO3. F RT-qPCR analysis of LOC554202 expression in Huh and LM3 cells after FOXO3 downregulation or upregulation. G ChIP-qPCR analysis of FOXO3 occupancy in the LOC554202 promoter in Huh and LM3 cells. H Construction of the luciferase reporter vector pGL3F, pGL3P1, and pGL3P2. I, J Relative luciferase activities as analyzed in Huh and LM3 cells after various transfection. *p < 0.05.

Fig. 3. The effect of LOC554202 on HCC cell proliferation and apoptosis.

A Oligonucleotides targeting LOC554202 (si-LOC554202-#1 and si-LOC554202-#2) and negative controls (si-NC) were transfected into Huh and LM3 cells. B CCK-8 assays of Huh and LM3 cells after transfection. C Huh and LM3 cells were treated with ADM (0, 4, 8, 12, 16 μM) for 48 h. The inhibition rate increased with the increase of concentration of ADM. D Knockdown of LOC554202 suppressed colony-formation as demonstrated by colony-formation assays in HCC cells. E Cell proliferation was evaluated using EdU incorporation assays. F Flow cytometry assays were applied to detect cell apoptosis in si-LOC554202-#1 and si-LOC554202-#2 transfected Huh and LM3 cells. G The activities of caspase 3/9 were determined. *p < 0.05, **p < 0.01.

Fig. 4. Knockdown of LOC554202 suppressed the migration and invasion of HCC cells.

A Wound-healing assay was used to detect cell migration in si-LOC554202-#1 and si-LOC554202-#2 transfected Huh and LM3 cells. B The invasion abilities of tumor cells were assessed using transwell assays. C The influences of LOC554202 knockdown on EMT-related factors in Huh and LM3 cells were respectively analyzed using western blot. *p < 0.05, **p < 0.01.

Fig. 5. LOC554202 competitively sponged miR-485-5p.

A Relative LOC554202 expression levels in nuclear and cytosolic fractions of Huh and LM3 cells. B The schematic diagram presents the complementary binding sites within LOC554202 and miR-485-5p. C Venn analysis of the potential targeting genes of miR-485-5p in miRDB, Starbase, and TargetScan. D, E KEGG pathways for the above potential targeting genes. F The dysregulated expression of miR-485-5p in HCC tissues in TCGA data sets. G The distinct upregulation of miR-485-5p expression in HCC specimens from our cohort. H ROC assays for the diagnostic value of miR-485-5p expression in distinguishing HCC specimens. I The expression of miR-485-5p in HCC cell lines and LO2. J After overexpression of miR-485-5p, HCC cell proliferation was determined by CKK-8 assays. K After overexpression of miR-485-5p, the invasion ability of HCC cells was determined by transwell assays. L RNA-pulldown for the exploration of the association between LOC554202 and miR-485-5p. M Overexpression of miR-485-5p decreased the Luciferase activity of wild-type MSI1 3′ UTR construct in both Huh and LM3 cells. N Knockdown of LOC554202 suppressed the expression of miR-485-5p. O Overexpression of miR-485-5p inhibited the levels of LOC554202, which was determined by RT-PCR. *p < 0.05, **p < 0.01.

Fig. 6. BSG was a target gene for miR-485-5p.

A The top 50 upregulated lncRNAs in HCC tissues were shown in a heat map using TCGA data sets. B Volcano plots show differentially expressed lncRNAs in HCC tissues by analyzing TCGA data sets. C The predicted binding sites of miR-485-5p within BSG were shown. D Pan-cancer analysis of BSG expression using TGCA data sets. E The expression pattern of BSG in HCC tissues with different clinical grades and with or without metastasis by analyzing TGCA data sets. F The distinct upregulation of mRNA BSG expression in HCC and its diagnostic significance in our cohort. G Higher levels of mRNA BSG in HCC specimens and its diagnostic value were observed. H Relative expression of mRNA BSG in five HCC cell lines and LO2. I The prognostic value of high BSG expression was determined in HCC patients, which was analyzed using GEPIA. J Survival analysis of 137 HCC patients by Kaplan–Meier methods. K Relative luciferase activity was analyzed in Huh and LM3 cells. L, M Overexpression of miR-485-5p suppressed the expression of BSG at mRNA and protein levels. *p < 0.05, **p < 0.01.

Fig. 7. Deficiency of miR-485-5p attenuates the regulatory effect of LOC554202 knockdown on the progression of HCC cells.

A The expression levels of BSC in LM3 and Huh7 cells after knockdown of LOC554202 and/or inhibition of miR-485-5p. The CCK-8 assays (B), colony-formation assays (C), Edu assays (D), Cell invasion (E), and migration (F) assays following knockdown of LOC554202 and/or inhibition of miR-485-5p. *p < 0.05, **p < 0.01.

Fig. 8. Hypothetical model for LOC554202 function in HCC.

LOC554202 served as a sponge of miR-485-5p to increase BSG expression, thus further promoting the progression of HCC.