A Novel lncRNA IHS Promotes Tumor Proliferation and Metastasis in HCC by Regulating the ERK- and AKT/GSK-3β-Signaling Pathways

Abstract

Long noncoding RNAs (lncRNAs) are involved in a variety of biological processes such as tumor proliferation and metastasis. A close relationship between hepatitis B virus X protein (HBx) and SMYD3 in promoting the proliferation and metastasis of hepatocellular carcinoma (HCC) was recently reported. However, the exact oncogenic mechanism of HBx-SMYD3 remains unknown. In this study, by performing lncRNA microarray analysis, we identified a novel lncRNA that was regulated by both HBx and SMYD3, and we named it lncIHS (lncRNA intersection between HBx microarray and SMYD3 microarray). lncIHS was overexpressed in HCC and decreased the survival rate of HCC patients. Knockdown of lncIHS inhibited HCC cell migration, invasion, and proliferation, and vice versa. Further study showed that lncIHS positively regulated the expression of epithelial mesenchymal transition (EMT)-related markers c-Myc and Cyclin D1, as well as the activation of the ERK- and AKT-signaling pathways. lncIHS exerted its oncogenic effect through ERK and AKT signaling. Moreover, results from transcriptome-sequencing analysis and mass spectrometry showed that lncIHS regulated multiple genes that were the upstream molecules of the ERK- and AKT-signaling pathways. Therefore, our findings suggest a regulatory network of ERK and AKT signaling through lncIHS, which is downstream of HBx-SMYD3, and they indicate that lncIHS may be a potential target for treating HCC.

Keywords: AKT; DUSP10; DUSP5; ERK; MAP3K8; SMYD3; hepatocellular carcinoma; lncRNA-HIS; metastasis; proliferation.

Figure 1

A Novel lncRNA Is Selected by Microarray (A) lncRNAs were screened out by microarray using HBx- and SMYD3-overexpresseing HCC cell lines. (B) The expression of lncIHS in HCC with tumor thrombus was significantly more than others. (C) lncIHS was positively regulated by HBx and SMYD3. (D) HBx promoted lncIHS expression through SMYD3. Data are presented as mean ± SD for three independent experiments. *p < 0.05.

Figure 2

lncIHS Is Overexpressed in HCC and Correlates with Unfavorable Survival (A) Compared with para-tumor tissues, the expression of lncRNA was significantly increased in HCC tissues. (B and C) Kaplan-Meier analysis of overall survival (B) and disease-free survival (C) based on lncIHS expression levels in 105 cases of patients with HCC. The median level of lncIHS is used as the cutoff. (D) The expression of lncRNA was detected in a normal liver cell line and different HCC cell lines. (E) The levels of lncIHS mRNA in purified nuclear or cytoplasmic RNAs were detected using real-time PCR. U6 and β-actin served as nuclear and cytoplasmic controls, respectively. (F) lncIHS was located in the cytoplasm and nucleus, but mainly in the nucleus, as determined by FISH assays. Data are presented as mean ± SD for three independent experiments. *p < 0.05, **p < 0.01.

Figure 3

Knockdown of lncIHS Inhibits SMMC-7721 Proliferation, Migration, and Invasion In Vitro (A) lncIHS knockdown was confirmed by real-time PCR in SMMC-7721. (B) Knockdown of lncIHS suppressed the migration and invasion abilities of SMMC-7721, as determined by transwell assay. (C and D) In vitro proliferative ability was decreased in SMMC-7721 with lncIHS knockdown, as determined by MTT assay (C) and colony formation assay (D). (E) Knockdown of lncIHS inhibited cell cycle transition from G1 to S in HCC cells. Data are presented as mean ± SD for three independent experiments. *p < 0.05.

Figure 4

Knockdown of lncIHS Inhibits Tumor Growth and Lung Metastasis of HCC Cells in Nude Mice (A) SMMC-7721 cells (5 × 10⁶) with stable knockdown of lncIHS were inoculated into nude mice, and the effect of lncIHS on HCC tumor growth was examined after 6 weeks (n = 5). A photograph of the tumors is presented. (B and C) Effect of lncIHS on HCC growth was described by tumor weight (B) and tumor size (C) in the two groups. (D) Representative images of immunohistochemistry (IHC) staining of Ki67 showed that lncIHS inhibition decreased tumor proliferation in xenografted tumors. (E) The expression of lncRNA in the tumor xenografts of the sh-lncIHS group was significantly lower than in the sh-NC group. (F) Representative H&E staining of lung metastases between SMMC-7721 sh-lncIHS cells and control cells. Data are presented as mean ± SD for three independent experiments. *p < 0.05, **p < 0.01.

Figure 5

Overexpression of lncIHS Promotes HCC Cell Migration, Invasion, and Proliferation (A) Overexpression of lncIHS was confirmed by PCR. (B and C) Overexpression of lncIHS promoted the migration and invasion abilities of HCC cells, as determined by wound-healing (B) and transwell assays (C). (D) In vitro proliferative ability of HCC cells was significantly promoted by lncIHS by MTT assay. (E) lncIHS overexpression stimulated cell cycle transition from G1 to S in HCC cells. Data are presented as mean ± SD for three independent experiments. *p < 0.05, **p < 0.01.

Figure 6

lncIHS Regulates the Expression of the EMT and Cell Cycle-Associated Genes and the Activation of the ERK- and AKT/GSK-3β-Signaling Pathways (A and B) Knockdown of lncIHS reduced the expression of c-Myc, Cyclin D1, N-cadherin, and Vimentin, and it increased the level of E-cadherin in SMMC-7721 and MHCC-97H (A), and vice versa in BEL-7402 and PLC/PRF/5 (B). (C and D) Knockdown of lncIHS decreased the expression of p-MEK1/2, p-ERK1/2, p-AKT, and p-GSK-3β in SMMC-7721 and MHCC-97H (C), and vice versa in BEL-7402 and PLC/PRF/5 (D).

Figure 7

lncIHS Promotes HCC Cell Migration, Invasion, and Proliferation through the ERK- and AKT-Signaling Pathways (A) The effects of Ly294002 and U0126 on the phosphorylation of AKT, GSK-3β, MEK1/2, and ERK1/2 were validated. (B) Treatments with Ly294002 and U0126 in HCC cells reversed the lncIHS-dependent decrease of E-cadherin expression and the lncIHS-dependent increase of N-cadherin, Vimentin, c-Myc, and Cyclin D1 expressions. (C and D) Inactivation of ERK and AKT signaling significantly reduced lncIHS-enhanced cell migration, invasion, and proliferation, as determined by transwell assay (C) and MTT assay (D). Data are presented as mean ± SD for three independent experiments. *p < 0.05, **p < 0.01.

Figure 8

NGS Reveals a lncIHS-Dependent Regulatory Network of the ERK- and AKT-Signaling Pathways (A) NGS screened out several genes that were upregulated or downregulated by lncIHS. (B) The lncIHS-related regulation of genes that may be the upstream molecules of the AKT- and ERK-signaling pathways was validated by real-time PCR. (C and D) Knockdown of lncIHS decreased MAP3K8 expression and increased DUSP5 and DUSP10 expressions in SMMC-7721 and MHCC-97H (C), and vice versa in BEL-7402 and PLC/PRF/5 (D). Data are presented as mean ± SD for three independent experiments. *p < 0.05.

Figure 9

lncIHS Interacts with YBX1 in HCC Cells (A) YBX1 was a potential interactive candidate of lncIHS, as determined by RNA pull-down assay, and the enriched products were eluted and separated by SDS-PAGE and Coomassie blue staining. (B) Western blot of the proteins from antisense lncIHS and lncIHS pull-down assays. (C) The interactions of lncIHS with YBX1 were verified by an RIP assay. (D–F) The mRNA expressions of MAP3K8 (D), DUSP5 (E), and DUSP10 (F) were detected in BEL-7402-lncIHS and its control cells with or without YBX1 knockdown. (G–I) RNA stability assays were performed in BEL-7402-lncIHS and its control cells with or without YBX1 knockdown, and the degradation rates of the MAP3K8 (G), DUSP5 (H), and DUSP10 (I) mRNA were measured at the indicated time points. (J and K) YBX1 potential binding sites on the MAP3K8 gene promoter (J) and the DUSP5/DUSP10 3′ UTR (K) are shown. (L) YBX1 was mainly located in the nucleus in lncIHS-overexpressing cells. Data are presented as mean ± SD for three independent experiments. *p < 0.05, **p < 0.01.