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. 2020 Mar:53:102685.
doi: 10.1016/j.ebiom.2020.102685. Epub 2020 Feb 27.

BAP18 is involved in upregulation of CCND1/2 transcription to promote cell growth in oral squamous cell carcinoma

Xue Wang  1 , Chunyu Wang  2 , Guangqi Yan  3 , Yuanyuan Kang  4 , Ge Sun  2 , Shengli Wang  2 , Renlong Zou  2 , Hongmiao Sun  2 , Kai Zeng  2 , Huijuan Song  2 , Wei Liu  2 , Ning Sun  2 , Wensu Liu  2 , Yue Zhao  5
Affiliations

BAP18 is involved in upregulation of CCND1/2 transcription to promote cell growth in oral squamous cell carcinoma

Xue Wang et al. EBioMedicine. 2020 Mar.

Abstract

Background: As a reader of histone H3K4me3, BPTF associated protein of 18 kDa (BAP18) is involved in modulation of androgen receptor action in prostate cancer. However, the function of BAP18 on oral squamous cell carcinoma (OSCC) and its molecular mechanism remains to be elusive.

Methods: OSCC-derived cell lines carrying silenced BAP18 were established by Lentiviral infection. Quantitative PCR (qPCR), western blot, and ChIP assay were performed to detect gene transcription regulation and the possible mechanism. Colony formation, cell growth curve and xenograft tumor experiments were performed to examine cell growth and proliferation.

Findings: Our study demonstrated that BAP18 was highly expressed in OSCC samples compared with that in benign. BAP18 depletion obviously influenced the expression of a series of genes, including cell cycle-related genes. We thus provided the evidence to demonstrate that BAP18 depletion significantly decreases CCND1 and CCND2 (CCND1/2) transcription. In addition, BAP18 is recruited to the promoter regions of CCND1/2, thereby facilitating the recruitment of the core subunits of MLL1 complex to the same regions, to increase histone H3K4me3 levels. Furthermore, BAP18 depletion delayed G1-S phase transition and inhibited cell growth in OSCC-derived cell lines.

Interpretation: This study suggests that BAP18 is involved in modulation of CCND1/2 transcription and promotes OSCC progression. BAP18 could be a potential _target for OSCC treatment and diagnosis. FUND: This work was funded by National Natural Science Foundation of China (31871286, 81872015, 31701102, 81702800, 81902889), Foundation for Special Professor of Liaoning Province, and Supported project for young technological innovation-talents in Shenyang (No. RC170541).

Keywords: BAP18; CCND1/2; Histone methylation; Oral squamous cell carcinoma; Transcription regulation.

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Conflict of interest statement

Declaration of Competing Interest The authors declare no conflict of interests.

Figures

Fig. 1
Fig. 1
BAP18 is highly expressed in clinical OSCC samples. (a) Expression of BAP18 in unpaired fresh tissues of oral non-cancerous (NC) tissues and OSCC tissues. (b) Grayscale values of western blotting in (a), Student t-test was used for statistical significance between two groups. (c) BAP18 expression in non-cancerous (NC) tissues and OSCC samples with different clinical stages. Magnification: 4* and 20*; Scale bars: 500 μm and 100 μm. (d) Statistical significance among non-cancerous tissues, early stages tissues (I and II) and terminal stages tissues (III and IV). Average score of three independent view calculation represents the sample final score. Mann-Whitney U test were used, ***p<0.001. (e) Ki67 and BAP18 expression in OSCC samples. Magnification: 10*; Scale bars: 100 μm. (f) Scatter diagram of two proteins (BAP18 and Ki67) expression in the same sample, and the straight line was generated by Prism Graphpad7.0. (g) Disease free survival curves was generated from GEPIA showing BAP18 contribution to HNCC patients living from TCGA cohorts (http://gepia.cancer-pku.cn/index.html).
Fig. 2
Fig. 2
BAP18 significantly regulates the transcription of a series of genes in OSCC-derived cell lines. (a) The effects of BAP18 depletion on a series of genes in Cal-27 cells by real-time quantitative PCR. Three independent siRNAs against BAP18 (siBAP18s) were designed and generated for BAP18 depletion. Levels of all kinds of RNAs as indicated were normalized to that of β-Actin. Student t-test were used, ns stands for no significant, ***p<0.001, **p<0.01, *p<0.05. (b) Effect of knockdown of BAP18 on mRNA expression of CCND1, CCND2, or CCND3 in SCC9 cells. Student t-test were performed, ns stands for no significant, ***p<0.001, **p<0.01, *p<0.05. (c,d) Effects of depletion of BAP18 or ectopic expression of BAP18 on protein expression of cyclin D1 and cyclin D2 by western blotting in Cal-27 cells (c) or SCC9 cells (d) with indicated antibodies.
Fig. 3
Fig. 3
BAP18 is recruited to the promoter regions of CCND1/2. (a) Schematic representation of primers designed for CCND1 and CCND2 gene promoter-TSS regions. BBS is an abbreviation of BAP18 binding sites. (b–e) ChIP assays performed on the promoter regions of CCND1/2 with the indicated antibodies in Cal-27 cells with shCtrl or shBAP18. Student t-test was performed, ns stands for no significant, ***p<0.001, **p<0.01, *p<0.05.
Fig. 4
Fig. 4
BAP18 depletion influences the G1-S phase in OSCC-derived cell lines. (a) Cal-27 cells were infected with lentivirus against BAP18 (shBAP18) and its negative control (shCtrl). The efficiency of the shBAP18 detected by western blotting in Cal-27 cells. (b–d) The impact of BAP18 on cell cycle progression by flow cytometry assay in Cal-27 cells. (e) SCC9 cells were infected with lentivirus against BAP18 (shBAP18) and its negative control (shCtrl). The efficiency of the shBAP18 detected by western blotting in SCC9 cells. (f–h) The impact of BAP18 on cell cycle progression by flow cytometry assay in SCC9 cells. All statistics were performed by Graphpad7.0 with Student t-test, ns stands for no significant, ***p<0.001, **p<0.01, *p<0.05.
Fig. 5
Fig. 5
BAP18 promotes the cell proliferation in OSCC-derived cell lines. (a) Colony formation assays in Cal-27 cells carrying siCtrl or siBAP18s (siBAP18#1, 2, 3). Amount of 400 cells were seeded into 35 mm dishes for 14 days and stained by crystal violet. (b) Representation of histogram for colony formation experiments. Standard of account is one cell growth colony contains more than 50 cloned cells. (c) siRNAs against CCND1/CCND2 (siCCND1/2) were transfected into Cal-27 cells with overexpression plasmid of BAP18 tagged with FLAG or vector plasmids for Growth curve analysis. (d) Growth curve analysis for Cal-27 cells with overexpression plasmids of BAP18 tagged with FLAG or vector plasmids were treated with CDK4/6 inhibitor (Palbociclib, 25 nM). (e) Growth curve analysis for Cal-27 cells with depletion of BAP18 (shBAP18) or shCtrl were treated with CDK4/6 inhibitor (Palbociclib, 25 nM). The expression levels of proteins as indicated were detected by western blotting shown at the bottom of panel c, d, e. Error bars represent mean ± SD. Student t-test was used, ns stands for no significant. ***p<0.001, *p<0.05.
Fig. 6
Fig. 6
BAP18 depletion inhibits the OSCC-derived cell growth in xenograft mice. (a) Representative photos were pictured of mice xenograft tumor. shCtrl (left) and shBAP18 (right) were stably expression in Cal-27 cells and injected into male mice at 4 weeks old. (b) Xenograft tumors were shown after mice were killed. (c) The average tumor volume of shCtrl and shBAP18 were measured every six days. Bars represented standard alteration of the mean, ***p<0.001. (d) Tumor weights were measured and statistically calculated with Student t-test, ***p<0.001. (e) Expressions of BAP18 proteins in xenograft tumors were detected by IHC assays. Magnification: 10*; Scale bars: 100 μm. (f) RNA isolated from xenograft tumors and the mRNA expression of BAP18, CCND1, CCND2, ASNS, DDK1andSENP2 was shown as indicated. Student t-test was used, *p<0.05, **p<0.01. (g) Schematic representation of the function of BAP18on up-regulation of CCND1/2 transcription. BAP18 is recruited to the promoter regions of CCND1/2, thereby facilitating the recruitment of the core subunits of MLL1 complex to enhance CCND1/2 transcription, to promote the cell growth and proliferation in OSCC progression.
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