Decreased long intergenic noncoding RNA P7 predicts unfavorable prognosis and promotes tumor proliferation via the modulation of the STAT1-MAPK pathway in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the most common neoplasm and is a leading cause of cancer-related death. Despite advances in the diagnosis and management of HCC, its prognosis remain unfavorable. Accumulating evidence has shown that long intergenic noncoding RNAs (lincRNAs) play central roles in the development of HCC. In this study, we identified a long intergenic noncoding RNA referred to as lincRNA P7 in HCC and explored its clinical significance and biological functions in HCC. The expression level of lincRNA P7 was significantly aberrantly deceased in HCC cancer tissues and cells lines. Gain- and loss-of-function experiments revealed that overexpression of lincRNA P7 significantly inhibited the proliferation of HCC-derived cancer cells, whereas lincRNA P7 knockdown promoted cell growth. Mechanistically, lincRNA P7 blocked Erk1/2 signaling and repressed activation of the STAT1 pathway. In nude mouse models, we show that overexpression of lincRNA P7 effectively repressed HCC xenograft tumor growth in vivo. Moreover, a clinical investigation demonstrated that down-regulated lincRNA P7 expression correlated with liver cirrhosis, Hepatitis B virus (HBV) infection, clinical stage of the tumor and recurrence. A Kaplan-Meier survival analysis showed that the expression of lincRNA P7 was significantly related to overall survival (P = 0.003) and recurrence-free survival (P = 0.031). Collectively, our findings suggested that the down-regulation of lincRNA P7 predicts poor clinical outcomes for HCC patients and might be a powerful candidate prognostic biomarker and target in HCC.

Keywords: HCC; lincRNA P7; long noncoding RNA.

Figure 1. LincRNA P7 is preferentially down-regulated in HCC tissues

(A) Hierarchical clustering of the expression level of the 7 most variable lncRNAs was significantly correlated with their corresponding 25 mRNAs. LincRNA P7 is marked in red. Expression values are indicated as high (red) or low (green). (B) PATHWAY analysis of the microarray results.

Figure 2. LincRNA P7 expression levels in HCC tissues and cells

(A) Relative expression levels of lincRNA P7 in tumor tissues compared with adjacent normal tissues (n = 108). LincRNA P7 expression was evaluated by qRT-PCR and normalized to GAPDH expression. (B) Positive values for lincRNA P7 expression indicate a higher level of lncRNA expression in tumor tissue compared with nontumor tissue, while negative values indicate a lower level of lincRNA P7 expression in tumor tissue compared with adjacent tissue. (C) The level of lincRNA P7 expression was lower in HCC cell lines than in the normal liver cell line LO2 (^***p < 0.001). (D) The area under the receiver operating characteristic (ROC) curve for the prediction of HCC based on lincRNA P7 expression, which used the corresponding adjacent normal tissues as controls. (E) qRT-PCR analysis of lincRNA P7 in the subcellular fractions of SMMC 7721 and Huh7 cells. U6 and β-actin served as nuclear and cytoplasmic markers, respectively (n = 3).

Figure 3. LincRNA P7 inhibited the proliferation of HCC cells in vitro

(A) LincRNA P7 overexpression and knockdown in HCC cells lines. The expression level of lincRNA P7 was robustly enhanced or reduced when the cell lines were treated with pcDNA3.1-LincRNA P7 or with two siRNAs that target lincRNA P7, respectively. Empty vector and the siRNA control were used as the negative controls. (B) MTT assays were used to determine the cell viability of si-LincRNA P7- and pCDNA- LincRNA P7-transfected SMMC7721 and Huh7 cells. Cell number was determined every 24 h for a total of 96 h using CCK-8 assays. The results are shown as the means ± s.e.m. from three independent experiments. ^*P < 0.05, compared with the control by two-sided t-test. (C) Colony-forming assay was conducted to determine the effect of lincRNA P7 on the proliferation in SMMC7721 and Huh7 cells. (D) LincRNA P7 regulates the STAT1 and MAPK signaling pathway. Immunoblot analysis showed that the phosphorylation of STAT1 (pSTAT1) and Erk1/2 (pErk1/2) was significantly repressed in lincRNA P7-overexpressing SMMC7721 and Huh7 cells. In contrast, the phosphorylation of these proteins was rescued in si-lincRNA P7-transfected SMMC7721 and Huh7 cells.

Figure 4. Effects of lincRNA P7 down-regulation on tumor growth in a nude mouse model

(A, B) SMMC7721 cells (5 × 10⁶) that stably expressed lincRNA P7 were inoculated into nude mice, and the effect of lincRNA P7 on HCC tumor growth was examined every 3 days for 42 days (6 weeks) (n = 6). The results show that lincRNA P7 significantly increased the proliferation of HCC tumor cells compared with the control lentiviral vector in nude mice. ^***P < 0.001 (C) Tumor weight in each of the two groups is shown. The results are presented as the means ± s.d. for six mice in each group. ^*P < 0.05, compared with control cells by two-sided t-test. (D) Effect of lincRNA P7 on HCC growth was described by tumor/body weight ratio in the two groups. Data are shown as the mean ± s.d. ^**P < 0.01. (E) The expression levels of lincRNA P7 were determined by qRT-PCR in the xenograft tumors. (F) The expression levels of the phospho-Erk1/2 and phospho-STAT1 were determined by Western Blotting in the xenograft tumors.

Figure 5. Aberrant expression of lincRNA-P7 and the overall survival and recurrence-free survival rates

Kaplan-Meier analyses of the correlations between the lincRNA P7 expression level and overall survival (A) and recurrence-free survival (B) of 108 patients with HCC. The median expression level was used as the cutoff. Patients with lincRNA P7 expression values below the 50th percentile were classified as having lower lincRNA P7 levels. Patients with lincRNA P7 expression value above the 50th percentile were classified as having higher lincRNA P7 levels.