Low expression of miR-199 in hepatocellular carcinoma contributes to tumor cell hyper-proliferation by negatively suppressing XBP1

Abstract

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide, and microRNAs (miRs) are considered to serve important functions in the pathogenesis of HCC by regulating the expression of specific target genes. The present study was conducted to investigate the role of miR-199 and its putative target X-box binding protein 1 (XBP1) in HCC, as well as of the downstream gene cyclin D. The expression levels of miR-199, XBP1 and cyclin D were detected in clinical HCC specimens. The effect of miR-199 on the regulation of HCC cell proliferation and its underlying mechanism were examined in Hep3B2.1-7 cells, through expression assays and measurement of cell proliferation (via Cell Counting Kit-8, and 5-ethynyl-2'-deoxyuridine and DAPI double-staining assays) coupled with gain- and lose- of function experiments. The expression of XBP1 and cyclin D was significantly increased in HCC tissues when compared with adjacent non-HCC tissues, while the expression of miR-199 was decreased. Exogenous miR-199 significantly suppressed the expression of XBP1 and cyclin D in Hep3B2.1-7 cells. However, the expression of XBP1 and cyclin D significantly increased on treatment with miR-199 inhibitor. Consistently, Hep3B2.1-7 cells co-transfected with a wild type reporter plasmid [XBP1-3'untranslated region (UTR)-WT] and exogenous miR-199 exhibited lower relative luciferase enzyme activity than cells co-transfected with negative control miRNA and XBP1-3'UTR-WT, while cells co-transfected with mutated plasmid (XBP1-3'UTR-MU) and miR-199 exhibited no change. It was further observed that knockdown of XBP1 by small interfering RNA significantly decreased the expression of cyclin D in Hep3B2.1-7 cells. Additionally, exogenous miR-199 decreased the proliferation of Hep3B2.1-7 cells, which was contrary to the effect of miR-199 inhibitor. In conclusion, it was demonstrated that miR-199 negatively regulated the expression of XBP1 by directly binding to its 3'UTR and that XBP1 impacted cyclin D expression, which was associated with the cell cycle regulation in Hep3B2.1-7 cells. These findings suggested that a miR-199/XBP1/cyclin D axis may serve an important role in the pathogenesis of HCC.

Keywords: X-box binding protein 1; cell proliferation; cyclin D; hepatocellular carcinoma; liver cancer; microRNA-199; microRNAs.

Figure 1.

miR-199, XBP1 and cyclin D are involved in HCC. The data show the liver tissue expression levels of (A) miR-199, (B) XPB1 mRNA, (C) XBP1 protein, (D) cyclin D mRNA and (E) cyclin D protein, and (F) the results of bioinformatic analysis. All values were expressed as means ± standard error of the mean (n=15). miR and mRNA levels were expressed relative to U6 and β-actin expression, respectively. *P<0.05 vs. control. miR, microRNA; XBP1, X-box binding protein 1; control, normal lung tissue; HCC, hepatocellular carcinoma; UTR, untranslated region.

Figure 2.

miR-199 directly suppresses the expression of XBP1 by binding with the 3′UTR. (A) miR-199 level; (B) XPB1 mRNA level; (C) XBP1 protein level; (D) schematic diagram of luciferase reporter plasmid construction; (E) relative luciferase activity. All values were expressed as means ± standard error of the mean. *P<0.05 vs. control; **P<0.01 and ***P<0.001 vs. control; ^#P<0.05 vs. NC miRNA + XBP1-3′UTR-WT group. miR, microRNA; XBP1, X-box binding protein 1; NC, negative control; UTR, untranslated region; WT, wild type; MU, mutant.

Figure 3.

Effect of the inhibition or knockdown of XBP1 on the expression of cyclin D. (A) Cyclin D mRNA level; (B) cyclin D protein level; (C) XBP1 mRNA level; (D) XBP1 protein level; (E) cyclin D mRNA level; (F) cyclin D protein level. All values were expressed as means ± standard error of the mean. *P<0.05 vs. control. MiR, microRNA; XBP1, X-box binding protein 1; siRNA, small interfering RNA; NC, negative control.

Figure 4.

Effect of exogenous miR-199 mimic or inhibitor on the proliferation of Hep3B2.1–7 cells. (A) Representative images of EdU and DAPI double staining (×200); (B) Cell Counting Kit-8 assay results. All values were expressed as means ± standard error of the mean. *P<0.05 and **P<0.01 vs. control. miR: microRNA; EdU, 5-ethynyl-2′-deoxyuridine; NC, negative control.