A long noncoding RNA perturbs the circadian rhythm of hepatoma cells to facilitate hepatocarcinogenesis

Abstract

Clock circadian regulator (CLOCK)/brain and muscle arnt-like protein-1 (BMAL1) complex governs the regulation of circadian rhythm through triggering periodic alterations of gene expression. However, the underlying mechanism of circadian clock disruption in hepatocellular carcinoma (HCC) remains unclear. Here, we report that a long noncoding RNA (lncRNA), highly upregulated in liver cancer (HULC), contributes to the perturbations in circadian rhythm of hepatoma cells. Our observations showed that HULC was able to heighten the expression levels of CLOCK and its downstream circadian oscillators, such as period circadian clock 1 and cryptochrome circadian clock 1, in hepatoma cells. Strikingly, HULC altered the expression pattern and prolonged the periodic expression of CLOCK in hepatoma cells. Mechanistically, the complementary base pairing between HULC and the 5' untranslated region of CLOCK mRNA underlay the HULC-modulated expression of CLOCK, and the mutants in the complementary region failed to achieve the event. Moreover, immunohistochemistry staining and quantitative real-time polymerase chain reaction validated that the levels of CLOCK were elevated in HCC tissues, and the expression levels of HULC were positively associated with those of CLOCK in clinical HCC samples. In functional experiments, our data exhibited that CLOCK was implicated in the HULC-accelerated proliferation of hepatoma cells in vitro and in vivo. Taken together, our data show that an lncRNA, HULC, is responsible for the perturbations in circadian rhythm through upregulating circadian oscillator CLOCK in hepatoma cells, resulting in the promotion of hepatocarcinogenesis. Thus, our finding provides new insights into the mechanism by which lncRNA accelerates hepatocarcinogenesis through disturbing circadian rhythm of HCC.

Figure 1

HULC upregulates CLOCK and perturbs its rhythmical expression in hepatoma cells. (A and B) The protein levels of CLOCK were examined by Western blotting in L-O2 and HepG2 cells transfected with pcDNA3.1-HULC (termed as HULC), respectively. Meanwhile, the transfection efficiency of HULC was detected by qRT-PCR. (C) The protein levels of CLOCK were examined by Western blotting in HepG2.2.15 cells transfected with CLOCK siRNA. The interference efficiency of CLOCK siRNA was detected by qRT-PCR in the cells. (D) The effect of HULC on the expression of downstream target genes of CLOCK, PER1 and CRY1, was measured by qRT-PCR in HepG2 and L-O2 cells. (E and F) The effect of HULC on rhythmical expression of CLOCK was assessed by qRT-PCR in L-O2 and HepG2 cells during 24 hours, respectively. Statistically significant differences are indicated: **P < .01, ***P < .001; Student’s t test.

Figure 2

HULC interacts with the 5'UTR of CLOCK mRNA via complementary base pairing in hepatoma cells. (A) A model shows the complementary region between HULC and CLOCK 5'UTR by bioinformatics prediction, in which the generated mutant sites in (or out of) the complementary region between HULC and CLOCK 5'UTR were marked. (B and C) The protein levels of CLOCK were measured by Western blotting in L-O2 cells transfected with HULC-mut-in (or HULC-mut-out), respectively. The transfection efficiency of HULC-mut-in (or HULC-mut-out) was detected by qRT-PCR in the cells. (D and E) The experiments above were repeated in HepG2 cells. Statistically significant differences are indicated: **P < .01; Student’s t test.

Figure 3

The 5'UTR of CLOCK mRNA interacts with HULC via complementary base pairing in hepatoma cells. (A) A model shows the complementary region between HULC and CLOCK 5'UTR. Schematic diagram exhibits the generated mutant site at the CLOCK 5'UTR in complementary region binding to HULC, which is inserted in pGL3-control vector. (B) The effect of HULC on reporters of pGL3-CL-5'UTR (or pGL3-CL-5'UTR-mut) was examined by luciferase reporter gene assays in L-O2 cells. (C) The effect of HULC siRNA on reporters of pGL3-CL-5'UTR (or pGL3-CL-5'UTR-mut) was measured by luciferase reporter gene assays in HBx-elevated HULC-highly-expressing L-O2-X cells. (D and E) The experiments above were repeated in HepG2 and HepG2.2.15 cells. Statistically significant differences are indicated: **P < .01, no significance (NS); Student’s t test.

Figure 4

The expression levels of HULC are positively correlated with those of CLOCK in clinical HCC tissues. (A) The expression of CLOCK was examined by IHC staining in clinical HCC tissues and peritumor tissues using tissue microarray. Representative example of IHC observed in peritumor tissues and HCC tissues using rabbit anti-CLOCK Ab. (B) The relative mRNA levels of CLOCK were assessed by qRT-PCR in 20 pairs of clinical HCC tissues and corresponding nontumorous tissues (**P < .01; Wilcoxon signed-rank test). (C) The correlation of HULC mRNA levels with CLOCK mRNA levels was examined by qRT-PCR in 30 cases of clinical HCC tissues (**P < .01; Pearson correlation coefficient, r= 0.6532).

Figure 5

CLOCK is required for HULC-accelerated proliferation of hepatoma cells in vitro. (A) The effect of HULC-mut-in (or HULC-mut-out) on reporters of pGL3-CL-5'UTR was assessed by luciferase reporter gene assays in HepG2 cells. (B–D) The cell proliferation was measured by MTT assays, flow cytometry, and cloning formation in HepG2 cells transfected with HULC (or HULC/CLOCK siRNA) individually. (E) The effect of HULC on the neoplastic transformation in LO-2 cells was detected by soft agar assays. Statistically significant differences are indicated: *P < .05, **P < .01, NS; Student’s t test.

Figure 6

CLOCK is required for HULC-accelerated proliferation of hepatoma cells in vivo. (A and B) Photographs of dissected tumors from nude mice and their growth curve, respectively. (C) The average weight of tumors from mice. (D and E) Expression levels of HULC and CLOCK were detected by qRT-PCR and Western blotting in the tumor tissues from mice, respectively. Statistically significant differences are indicated: **P < .01, NS; Student’s t test.