lncRNA ST8SIA6-AS1 facilitates proliferation and invasion in liver cancer by regulating miR-142-3p

Abstract

Long non-coding RNA ST8 α-N-acetyl-neuraminide α-2,8-sialyltransferase 6 antisense 1 (ST8SIA6-AS1) has been identified as a novel oncogene in breast cancer. However, its involvement in liver cancer has remained elusive. In the present study, the expression of ST8SIA6-AS1 and microRNA (miR)-142-3p in liver cancer tissues and cell lines was detected by reverse transcription-quantitative PCR. Tumor cell proliferation, migration and invasion assays were performed to determine the biological functions of ST8SIA6-AS1. The targeting interaction between ST8SIA6-AS1 and miR-142-3p predicted by bioinformatics was verified by a luciferase reporter assay and a biotin pulldown assay. The results indicated that ST8SIA6-AS1 was highly expressed in liver cancer tissues and cell lines, and the high expression of ST8SIA6-AS1 in liver cancer tissues was associated with poor prognosis. Knockdown of ST8SIA6-AS1 inhibited the proliferation, metastasis and invasion of liver cancer cells. Mechanistic investigation revealed that ST8SIA6-AS1 sequesters miR-142-3p and negatively regulates miR-142-3p expression in liver cancer cells. Further investigation indicated that the tumor-inhibitory effect of ST8SIA6-AS1 silencing was reversed by miR-142-3p depletion. In conclusion, ST8SIA6-AS1 was indicated to exert an oncogenic function in liver cancer by competitively sponging miR-142-3p.

Keywords: ST8SIA6-AS1; hepatocellular carcinoma; invasion; miR-142-3p; proliferation.

Figure 1

Upregulated ST8SIA6-AS1 correlates with poor prognosis in liver cancer. (A) Expression of ST8SIA6-AS1 explored in a cohort from The Cancer Genome Atlas-LIHC containing data of 369 liver cancer tumor tissues and 160 normal liver samples, indicating that ST8SIA6-AS1 was increased in tumor tissues. ^*P<0.05. (B) Compared with that in adjacent normal tissues, ST8SIA6-AS1 expression was elevated in liver cancer tissues. (C) Compared with that in normal liver HL-7702 cells, ST8SIA6-AS1 expression was elevated in liver cancer cell lines. (D) Patients with high ST8SIA6-AS1 expression (n=20) had a lower survival rate than those with low expression of ST8SIA6-AS1 (n=31) as demonstrated by Kaplan-Meier curves. Values are presented as the mean ± SD and are representative of three individual experiments. ^**P<0.01 as indicated or vs. HL-7702. ST8SIA6-AS1, ST8 α-N-acetyl-neuraminide α-2,8-sialyltransferase 6 antisense 1; LIHC, liver hepatocellular carcinoma; HCC, hepatocellular carcinoma; T, tumor; N, normal.

Figure 2

Knockdown of ST8SIA6-AS1 inhibits liver cancer cell proliferation, metastasis and invasion. (A) The efficiency of ST8SIA6-AS1 knockdown was assessed in HepG2 and Hep3B cells. (B and C) Cell Counting Kit-8 assay was used to analyze the viability of (B) HepG2 and (C) Hep3B cells after transfection. (D) Colony formation assay. Quantified numbers of colonies (left panel) and representative images of colonies (right panel) (magnification, x100). (E) Effects of ST8SIA6-AS1 knockdown on the migration ability of HepG2 and Hep3B cells. Quantified numbers of migrated cells (left panel) and representative images of the Transwell migration assay for HepG2 and Hep3B cells (right panel) (magnification, x400). (F) Effects of ST8SIA6-AS1 knockdown on the invasion ability of HepG2 and Hep3B cells. The quantified numbers of invaded cells (left panel) and representative images of the Transwell invasion assay for HepG2 and Hep3B cells (right panel) are provided (magnification, x400). Values are presented as the mean ± SD and are representative of three individual experiments. ^*P<0.05, ^**P<0.01 as indicated or vs. sh-NC. ST8SIA6-AS1, ST8 α-N-acetyl-neuraminide α-2,8-sialyltransferase 6 antisense 1; NC, negative control; sh, short hairpin RNA.

Figure 3

ST8SIA6-AS1 sponges miR-142-3p in liver cancer. (A) The binding sites of ST8SIA6-AS1 and miR-142-3p were identified via bioinformatics prediction. (B and C) The luciferase reporter assay confirmed the direct binding interaction between miR-142-3p and ST8SIA6-AS1 with the WT binding sites in (B) HepG2 and (C) Hep3B cells. (D) Fold enrichment of ST8SIA6-AS1 expression after RNA pull-down experiment with HepG2 and Hep3B cell extracts in different groups. (E) The expression of miR-142-3p in HepG2 and Hep3B cells with ST8SIA6-AS1 knockdown. (F) Relative expression of miR-142-3p in human liver cancer tissues. (G) The correlation between ST8SIA6-AS1 and miR-142-3p in liver cancer patients was examined using Spearman's correlation analysis. (H) Relative expression of miR-142-3p in human liver cancer cell lines. Values are presented as the mean ± SD and are representative of three individual experiments. ^**P<0.01, ^***P<0.001 as indicated or vs. HL-7702. ns, no significance. ST8SIA6-AS1, ST8 α-N-acetyl-neuraminide α-2,8-sialyltransferase 6 antisense 1; miR, microRNA; WT, wild-type; MUT, mutant; hsa, Homo sapiens; NC, negative control; HCC, hepatocellular carcinoma; Bio, biotin.

Figure 4

ST8SIA6-AS1 functions as an oncogene in liver cancer by sponging miR-142-3p. (A) The efficiency of miR-142-3p knockdown was assessed in HepG2 and Hep3B cells. Cell Counting Kit-8 assay was used to analyze cell viability in (B) HepG2 and (C) Hep3B cells with ST8SIA6-AS1 and miR-142-3p knockdown. ^**P<0.01 as indicated or vs. sh-NC; ^#P<0.05, ^##P<0.01 vs. sh-ST8SIA6-AS1. (D) Colony formation assays. Quantified numbers of colonies (left panels) and representative images of colonies (right panels) (magnification, x100). (E) Effect of ST8SIA6-AS1 and miR-142-3p knockdown on the migration ability of HepG2 and Hep3B cells. Quantified numbers of migrated cells (left panel) and representative images of the Transwell migration assay for HepG2 and Hep3B cells (right panel) (magnification, x400). (F) Effect of ST8SIA6-AS1 and miR-142-3p knockdown on the invasion ability of HepG2 and Hep3B cells. Quantified numbers of invaded (left panel) and representative images of the Transwell invasion assay for HepG2 and Hep3B cells (right panel) are provided (magnification, x400). Values are presented as the mean ± SD and are representative of three individual experiments. ^**P<0.01, ^***P<0.001. ST8SIA6-AS1, ST8 α-N-acetyl-neuraminide α-2,8-sialyltransferase 6 antisense 1; miR, microRNA; hsa, Homo sapiens; NC, negative control; sh, short hairpin RNA.