HBx-upregulated lncRNA UCA1 promotes cell growth and tumorigenesis by recruiting EZH2 and repressing p27Kip1/CDK2 signaling - PubMed (original) (raw)
HBx-upregulated lncRNA UCA1 promotes cell growth and tumorigenesis by recruiting EZH2 and repressing p27Kip1/CDK2 signaling
Jiao-Jiao Hu et al. Sci Rep. 2016.
Abstract
It is well accepted that HBx plays the major role in hepatocarcinogenesis associated with hepatitis B virus (HBV) infections. However, little was known about its role in regulating long noncoding RNAs (lncRNAs), a large group of transcripts regulating a variety of biological processes including carcinogenesis in mammalian cells. Here we report that HBx upregulates UCA1 genes and downregulates p27 genes in hepatic LO2 cells. Further studies show that the upregulated UCA1 promotes cell growth by facilitating G1/S transition through CDK2 in both hepatic and hepatoma cells. Knock down of UCA1 in HBx-expressing hepatic and hepatoma cells resulted in markedly increased apoptotic cells by elevating the cleaved caspase-3 and caspase-8. More importantly, UCA1 is found to be physically associated with enhancer of zeste homolog 2 (EZH2), which suppresses p27Kip1 through histone methylation (H3K27me3) on p27Kip1 promoter. We also show that knockdown of UCA1 in hepatoma cells inhibits tumorigenesis in nude mice. In a clinic study, UCA1 is found to be frequently up-regulated in HBx positive group tissues in comparison with the HBx negative group, and exhibits an inverse correlation between UCA1 and p27Kip1 levels. Our findings demonstrate an important mechanism of hepatocarcinogenesis through the signaling of HBx-UCA1/EZH2-p27Kip1 axis, and a potential target of HCC.
Figures
Figure 1. Differential expression profile of lncRNAs in HBx transfected LO2 cells.
(a) Hierarchical clustering analysis of lncRNAs. 379 up-regulated lncRNAs (fold change ≥2, P < 0.05) and 97 down-regulated lncRNAs (fold change ≥4, P < 0.05) were differentially expressed in HBx-expressing cells. The upregulated lncRNAs are represented in red, and the downregulated lncRNAs are displayed in green. The experiments were conducted in three biological replicates. (b) The fold change of lncRNA in microarray. (c) Nine differentially expressed lncRNAs randomly selected from panel (a) were validated by real-time PCR. Two LO2-HBx stable cell lines (LO2-HBx #1 and LO2-HBx #8) and LO2-HBx mixed stable cells were used to validate lncRNA profiles. Statistically significant differences are reported (Student t-test) for three independent experiments. *P < 0.05.
Figure 2. Induction of lncRNAs UCA1 expression by HBx.
(a) The levels of HBx mRNA and UCA1 in LO2-HBx stable cells measured by conventional RT-PCR. (b) The levels of HBx mRNA and UCA1 detected in 10 liver cell lines by RT-PCR. (c,d) UCA1 levels in LO2 cells transiently transfected with an HBx-expressing plasmid pcDNA4.0-HBx at different doses. UCA1 was examined either by conventional RT-PCR (c) or real-time PCR (d). (e) The relative levels of HBx mRNA and UCA1 in Hep3B cells after transfected with different dose of an HBx-expressing plasmid. The value of control was set as 1. (f) The relative levels of HBx mRNA and UCA1 in LO2-HBx and Hep3B cells after transfected transiently with HBx-siRNA or negative control siRNA. β-actin was used as internal control in above experiments. Statistically significant differences are reported (Student t-test) for three independent experiments. *P < 0.05; **P < 0.01. (g) UCA1 levels measured by real-time PCR in 60 paired tumour and adjacent nontumor tissues. The HCC specimens are divided into two sub-groups based on their relative HBx mRNA status (HBx negative and positive group). Horizontal lines in the box plots represent the median; the boxes represent the interquartile range, and whiskers represent the 2.5th and 97.5th percentiles. Wilcoxon’s signed-rank test was used in this study, P = 0.0244. (h) The inverse correlation of levels of HBx mRNA and UCA1 in HBx positive samples. The ΔCt values (normalized to β-actin) were subjected to Pearson’s correlation analysis (R2 = 0.1276, P = 0.0237).
Figure 3. The Role of UCA1 in cells proliferation and cell cycle in vitro.
Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) assays (a to c). (a) LO2 cells were transfected with plasmids pcDNA3.1-UCA1 (a); (b,c) LO2-HBx cells (b) or Hep3B (c) cells were transfected with UCA1-specific siRNA. (d,e) colony formation assays. Representative images of colony formation of LO2 cells transfected with plasmids pcDNA3.1-UCA1 (d) and LO2-HBx stably transfected with pSUPER-EGFP-shUCA1 (e) are shown. Colonies were stained with crystal violate and counted after fourteen days. The colonies were counted and are depicted in a bar chart (right panel). All above the values indicate the mean ± s.d. for three separate experiments (*P < 0.05, independent Student t-test). (f–h) Cell cycle distribution in LO2 cells. Cell cycle distribution was examined by fluorescent activated cell sorting (FACS) assays in LO2 cells transiently transfected with either a UCA1-expressing plasmid (f,g) or an HBx-expressing plasmid (h) after stained with propidiumiodide. The bar chart represents the relative percentages of cells in each phase. The values indicate the mean ±s.d. for three separate experiments (*P < 0.05, **P < 0.01, Student t-test). (i,j) Cell cycle distribution in LO2-HBx stable cells and Hep3B cells upon UCA1 or HBx knocked down by specific siRNAs. From (h–j), Flow cytometry was 24h post-transfection. The bar chart represents the relative percentages of cells in each phase. The values indicate the mean ± s.d. for three separate experiments (*P < 0.05, **P < 0.01, Student t-test).
Figure 4. Apoptosis induced by knockdown of UCA1 in hepatic HCC cell lines.
(a) Flow cytometry analysis of cell apoptosis in LO2-HBx and Hep3B cells transfected with si-NC, si-UCA1 and si-HBx. Apoptotic cells were stained with FITC-labeled Annexin V and analysed by FACS assays. Knockdown of UCA1 and HBx resulted in significant increase of apoptotic cells when compared with control cells. (b) The apoptosis rate was calculated and depicted in a bar chart. The values indicate the mean ± s.d. for three separate experiments (Student t-test, *P < 0.05). LR, early apoptotic cells; UR, terminal apoptotic cells. (c) Western blot analysis for cleaved caspase-9, caspase-3 and caspase-8 in LO2-HBx stable cells transfected with pSUPER-EGFP-shUCA1 and control vector cells, respectively. β-actin was used as a loading control. The data are representative of three independent experiments.
Figure 5. Suppression of p27 expression by UCA1 in HCC.
(a) Protein levels of cyclin-dependent kinases (CDKs) and CDK inhibitors (CDKI) in LO2 cells with ectopically expressed UCA1 and in Hep3B cells with UCA1 knockdown. The relative protein levels were analysed by density assays and presented in a bar chart (right panel). (b,c) The UCA1 levels were examined by conventional RT-PCR (b) and p27 levels were examined by real-time PCR(c) in LO2 and QSG-7701 cells transiently transfected with UCA1 expressing or control vectors, respectively. (d) The levels of UCA1 and p27 mRNA were examined by qPCR in LO2-HBx cells stably transfected with pS-EGFP-shUCA1 and a control plasmid. (e) Knockdown of UCA1 expression upregulated p27 protein level was detected by western blot. (f) The levels of p27 mRNA were detected by real-time PCR in Hep3B cells transfected with indicated siRNA, HBx-, or UCA1-expressing plasmids, respectively. The ratio of mRNA copies of p27 to β-actin was set as 1 in the control groups in above experiments. Student t-test was conducted for statistical analysis. At least three independent experiments were conducted. *P < 0.05; **P < 0.01. (g) The relative p27 levels were assayed by real-time PCR in 31 HCC and pair-matched non-tumour tissues. (h) The Pearson’s correlation analysis showed a negative correlation of UCA1 and p27 mRNA expression (R2 = 0.1126, P = 0.015).
Figure 6. The physical association of UCA1 and EZH2 elevating p27 3MeK27H3 level in promoters.
(a) The relative mRNA levels of EZH2 and p27 after transfection of EZH2-specific siRNA or scramble siRNA into LO2-HBx cells (si-NC or si-EZH2). The EZH2 and p27 mRNA levels were examined by real-time PCR (*P < 0.05, Student t-test). (b) Upregulation of p27 protein levels by knockdown of EZH2. (c) RNA-binding protein immunoprecipitation (RIP). The lysates of LO2-HBx cells were applied to co- immunoprecipitation with antibodies against EZH2 and IgG, then HOTAR, UCA1 and β-actin were detected by real-time PCR. The values indicate the mean ± SD. Statistically significant differences are reported for three independent experiments. **P < 0.01. (d,e) ChIP assays. (d) primer sets conducted on p27 promoters. (e) Cell lysates from LO2-HBx cells without and with UCA1 knockdown were applied for immunoprecipitation using anti-EZH2, -H3K27me3, -Histone H3 and IgG antibodies. ChIP samples at the p27 promoter was quantified by real-time PCR. The ChIP results revealed that the EZH2 occupied across p27 promoters around the transcription start site (primers b, c and d) (upper), but without bind the region correspond to primer a, e, f, g and h (data not shown). Knockdown of UCA1 could not alter the H3 total levels in binding-regions (lower). The values indicate the mean ± SD. Statistically significant differences are reported for two independent experiments. *P < 0.05. (f) UCA1 was detected both in the cytosolic and nuclear compartments (n = 2).
Figure 7. Inhibition of tumour growth by knockdown of UCA1 in a xenograft mouse model.
(a) Actual tumour size after removal from mice which is injected with cells transfected by siUCA1, siHBx and control siRNA. (b) Tumour volumes were calculated after injection every week. Bars indicate S.D. The tumour growth curve shows that knockdown of UCA1 significantly inhibited tumour growth in the mice. Asterisk indicates a significant change (*P < 0.05). Data are the mean ± SD.
References
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