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. 2019 Apr;54(4):1183-1194.
doi: 10.3892/ijo.2019.4707. Epub 2019 Feb 1.

lncRNA PLAC2 activated by H3K27 acetylation promotes cell proliferation and invasion via the activation of Wnt/β‑catenin pathway in oral squamous cell carcinoma

Fubo Chen  1 , Shengcai Qi  1 , Xu Zhang  1 , Jinjin Wu  1 , Xi Yang  2 , Raorao Wang  1
Affiliations

lncRNA PLAC2 activated by H3K27 acetylation promotes cell proliferation and invasion via the activation of Wnt/β‑catenin pathway in oral squamous cell carcinoma

Fubo Chen et al. Int J Oncol. 2019 Apr.

Abstract

As a new group of important effector molecules involved in multiple cancer types, including breast cancer, lung cancer and oral squamous cell carcinoma, long noncoding RNAs (lncRNAs) have attracted considerable attention recently. However, the underlying cause that induces the dysregulated lncRNAs in cancer remains poorly understood. In the present study, the regulatory model of the lncRNA placenta‑specific protein 2 (PLAC2) upregulation in oral squamous cell carcinoma (OSCC) was investigated and its biological functions in OSCC malignant progression was identified. A reverse transcription‑quantitative polymerase chain reaction assay identified that PLAC2 is upregulated in OSCC cell lines and primary tissue samples. Furthermore, bioinformatic analysis followed by chromatin immunoprecipitation verified an enriched histone H3 on lysine 27 (H3K27) acetylation (H3K27ac) at the promoter region of the PLAC2 gene. Knockdown of cAMP‑response element binding protein‑binding protein (CBP) significantly reduced the enrichment level of H3K27ac, and thereby induced a decreased expression of PLAC2. Functionally, overexpression of PLAC2 promotes OSCC cell proliferation, migration and invasion, whereas knockdown of PLAC2 exerted an opposite effect. Furthermore, the Wnt/β‑catenin signaling pathway was activated by PLAC2 and mediated the PLAC2‑induced malignant progress of OSCC. In conclusion, the present results indicated that lncRNA PLAC2 is transcriptionally activated by H3K27ac modification at the promoter region in OSCC, and promotes cell growth and metastasis via activating Wnt/β‑catenin signaling pathway. Therefore, PLAC2 may serve as a promising biomarker for OSCC prognosis and therapy.

Keywords: oral squamous cell carcinoma; placenta-specific protein 2; histone H3 on lysine 27 acetylation; proliferation; invasion; Wnt/β-catenin.

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Figures

Figure 1
Figure 1
PLAC2is activated by histone acetylation at the promoter region. (A) The expression level of PLAC2 in OSCC cells and normal epithelial HOK cells were measured via RT-qPCR. *P<0.05. (B) PLAC2 expression in 48 paired OSCC tissues and adjacent non-tumor tissues was assessed by RT-qPCR. *P<0.05. (C) The genome bioinformatics analysis demonstrated that the promoter of PLAC2 had a high enrichment of H3K27ac. (D) The enrichment of H3K27ac at the PLAC2 promoter was assessed via a ChIP assay in OSCC and HOK cells. *P<0.05. (E) The enrichment of H3K27ac at the PLAC2 promoter was assessed via a ChIP assay in OSCC tissues and paired non-tumor tissues. *P<0.05, compared with OSCC tissues. (F) PLAC2 expression was measured by RT-qPCR in OSCC cells treated with C646 or DMSO for 48 h. *P<0.05, compared to DMSO group. PLAC2, placenta-specific protein 2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; DMSO, dimethyl sulfoxide; H3K27ac, histone H3 on lysine 27 acetylation; OSCC, oral squamous cell carcinoma; ChIP, chromatin immunoprecipitation.
Figure 2
Figure 2
The H3K27 acetylation process is mediated by CBP. (A) CBP level was detected in OSCC and HOK cells via an immunofluorescence assay. (B) The enrichment of CBP at PLAC2 promoter was assessed in cells with a chromatin immunoprecipitation assay. *P<0.05. (C) The knockdown effect of si-CBP was evaluated with a western blot analysis assay. (D) The enrichment of H3K27ac was assessed in cells with CBP knockdown, *P<0.05, compared with CAL-27 or SCC-9 cells. (E) Reverse transcription-quantitative polymerase chain reaction was used to determine the expression level of PLAC2 in OSCC cells upon knockdown of CBP. *P<0.05 compared with the si-NC group. PLAC2, placenta-specific protein 2; CBP, cAMP-response element binding protein-binding protein; H3K27ac, histone H3 on lysine 27 acetylation; OSCC, oral squamous cell carcinoma; si, small interfering; NC, negative control.
Figure 3
Figure 3
PLAC2 promotes proliferation and invasion of OSCC cells. (A) The transfection efficiency of knockdown or overexpression of PLAC2 was determined via reverse transcription-quantitative polymerase chain reaction. ***P<0.001, compared with the control groups. (B) Cell viability was assessed with an MTT assay in OSCC cells. *P<0.05, compared with the control groups. (C) Ki-67 protein level was measured in OSCC cells with an immunofluorescence assay. (D and E) Cell migration and invasion abilities were assessed in OSCC cells using wound-healing and Matrigel assays, respectively. *P<0.05, ***P<0.001, compared with the control groups. PLAC2, placenta-specific protein 2; OSCC, oral squamous cell carcinoma; si, small interfering; NC, negative control; p-NC, negative control overexpressed plasmid; p-PLAC2, PLAC2 overexpressed plasmid.
Figure 4
Figure 4
Wnt/β-catenin signaling pathway is activated by PLAC2 in oral squamous cell carcinoma. (A) Nuclear fraction experiment and reverse transcription-quantitative polymerase chain reaction detected the abundance of PLAC2 in the nucleus and cytoplasm. GAPDH was the positive control for cytoplasm, and U1 were the positive controls for the nucleus. (B) The subcellular distribution of PLAC2 was visualized by RNA Fluorescent in situ hybridization in SCC-9 and CAL-27 cells. (C) The activity changes of indicated signaling pathways in SCC-9 cells were depicted upon overexpression of PLAC2, as indicated with a reporter activity. (D) The effect of PLAC2 on the Wnt/β-catenin signaling pathway in SCC-9 cells was assessed with a western blot analysis assay. (E) PLAC2 induced nuclear translocation of β-catenin. Immunofluorescence staining of β-catenin in SCC-9 cells treated with p-PLAC2. PLAC2, placenta-specific protein 2; MMP-7, matrix metallopeptidase-7; TCF4, transcription factor 4; p-PLAC2, PLAC2 overexpression plasmids.
Figure 5
Figure 5
PLAC2 promotes tumor growth and metastasis in vivo. (A) Representative images of the nude mice and corresponding xenograft fed for five weeks in the Lv-NC or Lv-PLAC2 cell groups. (B) Tumor volume and tumor weight in the Lv-NC and Lv-PLAC2 groups. The tumor volumes were measured every 5 days (tumor volume = length x width2/2). **P<0.01. (C) The transcript expression of PLAC2, CBP and β-catenin in implanted OSCC tissues were measured by reverse transcription-quantitative polymerase chain reaction. *P<0.05, compared with the Lv-NC group. (D) Representative images of β-catenin expression in the implanted OSCC tumors in each group as analyzed by immunohistochemistry. Scale bars: 50 µm; magnification (left, ×10; right, ×40). (E) Lung metastases were quantified using bioluminescence imaging after 5 weeks of initial implantation. Representative in vivo bioluminescent images are depicted. (F) Representative images of metastatic tumor nodules and corresponding quantitative number of metastasis. ***P<0.001. PLAC2, placenta-specific protein 2; NC, negative control; OSCC, oral squamous cell carcinoma; CBP, cAMP-response element binding protein-binding protein.
Figure 6
Figure 6
High PLAC2 is associated with poor survival of patients with OSCC. (A) The correlations of the RNA expression between PLAC2 and CBP, and PLAC2 and β-catenin in OSCC tissues were analyzed by Spearman's correlation test. (B) A receiver operating curves was used to determine the diagnostic value of PLAC2 in distinguishing patients with OSCC from healthy population. (C) Overall survival and (D) progressive-free survival of the 48 patients as represented by Kaplan-Meier curves. Expression level of PLAC2 was categorized into high and low using the median value. (E) A scheme of the proposed mechanisms, the histone H3 on lysine 27 acetylation-induced PLAC2 expression promotes tumor growth and metastasis by the activation of Wnt/β-catenin pathway. PLAC2, placenta-specific protein 2; CBP, cAMP-response element binding protein-binding protein; AUC, area under the curve; OSCC, oral squamous cell carcinoma; lnc, long non-coding.
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