The β-catenin/TCF-4-LINC01278-miR-1258-Smad2/3 axis promotes hepatocellular carcinoma metastasis

Abstract

Hepatocellular carcinoma (HCC) metastasis is largely responsible for HCC-associated recurrence and mortality. We aimed to identify metastasis-related long non-coding RNAs (lncRNAs) to understand the molecular mechanism of HCC metastasis. We first identified that miR-1258 was downregulated in HCC tissues both in The Cancer Genome Atlas (TCGA) and Sun Yat-sen University Cancer Center (SYSUCC) dataset. MiR-1258 expression negatively correlated with recurrence-free survival and overall survival of HCC patients. MiR-1258 overexpression inhibited migration and invasion of HCC cells both in vitro and in vivo, whereas miR-1258 downregulation promoted cell metastasis. Luciferase assays verified direct binding of miR-1258 to Smad2 and Smad3, thereby attenuating TGF-β/Smad signaling. We further established that lncRNA LINC01278 was a negative regulator of miR-1258. In vivo and in vitro assays demonstrated that LINC01278-mediated HCC metastasis was dependent on miR-1258 expression. Furthermore, miR-1258 downregulation in turn increased LINC01278 expression. We also observed that TCF-4 could bind to the LINC01278 promoter site. In addition, LINC01278 downregulation decreased migration and invasion of HCC cells induced by β-catenin and TGF-β1 both in vitro and in vivo. We uncovered a novel mechanism for β-catenin/TCF-4-LINC01278-miR-1258-Smad2/3 feedback loop activation in HCC metastasis, and the study indicated that LINC01278 could serve as a therapeutic target for HCC metastasis.

Fig. 1. miR-1258 suppresses HCC cell metastasis in vitro and in vivo and its expression associated with E-HCC recurrence and patient’s survival.

a The expression of miR-1258 in 20 paired E-HCC tissues and adjacent normal tissues. b Correlation of recurrence-free survival (RFS) and overall survival (OS) and miR-1258 expression by Kaplan–Meier analysis in TCGA datasets. c Correlation of RFS and OS and miR-1258 expression by Kaplan–Meier analysis in SYSUCC datasets. d The expression of miR-1258 in immortalized liver cells (THLE-3) and four HCC cell lines (SMMC-7721, Huh-7, HepG2, and Hep3B). e The wound-healing assay of HCC cells. f The invasion assay of HCC cells. g The numbers of lung metastasis. Experiments performed in triplications. miR-1258, HCC cells were transfected by miR-1258 expression vector. Anti-miR-1258, HCC cells were transfected by miR-1258 antisense plasmid. *P < 0.05.

Fig. 2. MiR-1258 target Smad2/3 in HCC cells.

a The predicted target sequence of miR-1258 in 3′UTR of Smad2 (Smad2-3′UTR) and Smad3 (Smad3-3′UTR) and mutant containing three altered nucleotides in the seed sequence of miR-1258 (miR-1258-mut). b Luciferase assay of pGL3-Smad2-3′UTR and pGL3-Smad3-3′UTR in the presence of miR-1258 mimics, miR-1258-mutant, and anti-miR-1258. c The Smad2/3 mRNA levels after transfected by miR-1258 mimics, miR-1258-mutant, and anti-miR-1258. d The protein levels of P-Smad2, Smad2, P-Smad3, Smad3, PAI-1, E-cadherin, and vimentin after transfected by miR-1258 mimics and anti-miR-1258 in HCC cell. e The correlation between Smad2/3 gene expression and miR-1258 level in TCGA dataset. f The correlation between Smad2/3 gene expression and miR-1258 level in SYSUCC dataset. g, h The wound-healing assay and transwell assay of HCC cells transfected with miR-1258, miR-1258+Smad2, miR-1258+Smad3. Experiments performed in triplications. miR-1258, HCC cells were transfected by miR-1258 expression vector. Anti-miR-1258, HCC cells were transfected by miR-1258 antisense plasmid. miR-1258+Smad2, co-overexpressed miR-1258, and Samd2 in HCC cells. miR-1258+Smad3, co-overexpressed miR-1258, and Samd3 in HCC cells. *P < 0.05.

Fig. 3. MiR-1258 was regulated by LINC01278.

a The correlation between LINC01278 gene expression and miR-1258 level in TCGA dataset. b The correlation between LINC01278 gene expression and miR-1258 level in SYSUCC dataset. c The expression of miR-1258 in HCC cells transfected by LINC01278 and shLINC01278. d LINC01278 is abundant in cytoplasm of SMMC-7721 and Hep3B cells. U2 and actin were used as positive control. e Cytoplasm enrichment of LINC01278 in HCC patients’ tissues. f The predicted binding sites between miR-1258 and LINC01278. The muted site (LINC01278-mut) was used for luciferase reporter assay. The relative luciferase activities were detected in 293 T cells transfected by LINC01278-WT and LINC01278-mut. g RNA-IP was used to identify the inhibition of miR-1258 by LINC01278. The expression levels of LINC01278 and miR-1258 were detected using qRT-PCR. LINC01278, ectopic LINC01278 expression in HCC cells. shLINC01278, HCC cells were transfected by shRNA target LINC01278. *P < 0.05.

Fig. 4. LINC01278 increases HCC metastasis in vivo and in vitro.

a The expression of LINC01278 in paired HCC tissues and adjacent normal tissues in TCGA datasets (n = 49) and SYSUCC dataset (n = 20). b Correlation of recurrence-free survival (RFS) and overall survival (OS) and LINC01278 expression by Kaplan–Meier analysis in SYSUCC dataset. c The wound-healing assay in HCC cells. d The invasion assay in HCC cells. e The Smad2/3 mRNA expression of HCC cells transfected by LINC01278, shLINC01278, LINC01278+miR-1258, and shLINC01278+anti-miRNA-1258. f The protein expression of Smad2 and Smad3 in HCC cells transfected by LINC01278, shLINC01278, LINC01278+miR-1258, and shLINC01278+anti-miRNA-1258. g The protein levels of P-Smad2, Smad2, P-Smad3, Smad3, PAI-1, E-cadherin, and vimentin in metastasis tumor tissues. LINC01278, ectopic LINC01278 expression in HCC cells. h The lung metastasis of HCC cells transfected by LINC01278 and shLINC01278. shLINC01278, HCC cells were transfected by shRNA target LINC01278. miR-1258, HCC cells were transfected by miR-1258 expression vector. Anti-miR-1258, HCC cells were transfected by miR-1258 antisense plasmid. *P < 0.05.

Fig. 5. LINC01278 was regulated by TCF-4.

a The expression of LINC01278 in HCC cells transfected by miR-1258 and anti-miR-1258. b The TCF-4/LEF-1 binding site in the LINC01278’s promoter sequence was identified. The mutant sequence was designed for the luciferase reporter assay. c The relative luciferase activity was detected in 293 T cells co-transfected by binding-WT/binding-mut and TCF-4. d ChIP assay was used to detect the TCF-4 to the TRE (TCF responsive element: TTCAAAG) regions in LINC01278 promoter. e The relative expression of LINC01278 in HCC cells transfected by β-catenin and siTCF-4. f The relative expression of LINC01278 in HCC cells treated with TGF-β1 (0.5 ng/ml) and siTCF-4. g, h The wound-healing assay and invasion assay in SMMC-7721 cells. i The lung metastasis of SMMC-7721 cells treated with TGF-β1 (0.1 µg/each time, three times a week) and shLINC01278. j Schematic illustration of “β-catenin/TCF-4-LINC01278-miR-1258-Smad2/3” axis. miR-1258, HCC cells were transfected by miR-1258 expression vector. Anti-miR-1258, HCC cells were transfected by miR-1258 antisense plasmid. TCF-4, HCC cells were transfected by TCF-4 expression vector. β-catenin, HCC cells were transfected by β-catenin expression vector. siTCF-4, HCC cells were transfected by siRNA target TCF-4. shLINC01278, HCC cells were transfected by shRNA target LINC01278. *P < 0.05.