Transcriptional and H3K27ac related genome profiles in oral squamous cell carcinoma cells treated with metformin

. 2022 Mar 21;13(6):1859-1870.


doi: 10.7150/jca.63234.


eCollection 2022.

Affiliations

Item in Clipboard

Shan Liu et al.


J Cancer.


.

Abstract

Objectives: Metformin, a first-line drug that has been used for type 2 diabetes treatment, recently attracts broad attention for its therapeutic effects on diverse human cancers. However, its effect and the underlying mechanisms on oral squamous cell carcinoma (OSCC) are not well known. Materials and Methods: OSCC cells were used to evaluate the effect of metformin on cell proliferation and colony formation in vitro. Tumor formation assay in nude mice was conducted to assess the effect of metformin in vivo. Western blotting and immunohistochemistry stain were performed to investigate the effect of metformin on the expression of acetylation at lysine 27 of histone H3 (H3K27ac) and methylation at lysine 27 of histone H3 (H3K27me3) in vitro and in vivo. RNA-seq and ChIP-seq were performed to explore the genome profile to metformin treatment in OSCC cells. Results: Metformin inhibited OSCC cell proliferation and colony formation in vitro, as well as OSCC growth in vivo. Metformin increased the global H3K27ac modification in vitro. Transcriptome analysis suggested that metformin mainly downregulated pluripotency stem cell pathway, development involved pathways and upregulated cytokine and inflammatory pathways. Additionally, H3K27ac was involved in transcription, DNA repair and replication in metformin-treated OSCC cells. Conclusions: Metformin inhibits OSCC growth concomitant upregulated global level of H3K27ac in vitro. This study provides insights into the molecule and epigenome basis on application of metformin in OSCC treatment, and highlights the underlying mechanisms of reprogrammed cancer regulation and epigenetic histone modification.


Keywords:

H3K27ac; H3K27me3; Metformin; oral squamous cell carcinoma; reprogrammed cancer regulation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures


Figure 1



Figure 1

Metformin inhibited OSCC growth in vitro. (A) Cell proliferation of HaCaT, SCC25, Cal27 and HSC-2 cells induced by metformin was evaluated by CCK8 assay. Cells were treated by concentration gradient of metformin (vehicle, 2.5, 5, 10, 20, and 40 mM) for 24, 48, 72, and 96 h. (B) Proliferative ability of cells treated by metformin (vehicle, 2.5, 5 and 10 mM) was assessed by colony formation assay. Typical images (left) and quantification (right) in terms of colony numbers. Data are presented as mean ± SD from three independent experiments at least. *: p<0.05; **: p<0.01; ***: p<0.001.


Figure 2



Figure 2

Metformin inhibited OSCC tumor growth in nude mice. (A) Macroscopic appearance of the dissected tumors from mice at the end of the experiment. (B) Tumor weight histogram. (C) Tumor growth curves. (D) Ki67 staining and (E) Ki67 IHC score.


Figure 3



Figure 3

Metformin upregulated global level of H3K27ac in vitro. (A) Cells treated with metformin (vehicle, 5 and 10 mM) for 6 days were subjected to western blotting analysis. H3 served as the reference protein for H3K27ac and H3K27me3, and β-actin was used as reference protein for EZH2, EED and SUZ12. (B) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27me3. (C) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27ac. (D) IHC scores of H3K27me3 (left) and H3K27ac (right).


Figure 4



Figure 4

Transcriptome profile of OSCC cell lines treated with metformin. Volcano map (A) and gene-wise hierarchical clustering heatmap (B) of genome in response to metformin treatment in Cal27 and HSC-2 cell lines. (C and D) Enrichment pathway of downregulated (C) and upregulated (D) DEGs in Cal27. (E and F) Enrichment pathway of downregulated (E) and upregulated (F) DEGs in HSC-2.


Figure 5



Figure 5

The verification of dual effect of metformin on transcripts in OSCC cells treatment. (A) Cell cycle regulation genes. (B) Ribosome genes. (C) The oncogenes and tumor suppressors. (D) Cell metabolism genes. (E) Cytokines expression.


Figure 6



Figure 6

Genome profile modified by H3K27ac in OSCC cell lines treated with metformin. (A and B) The location of differential peaks in Cal27 (A) and HSC-2 (B). (C and D) BETA analysis predicted the function of H3K27ac on gene expression in Cal27 (C) and HSC-2 (D) treated with metformin. (E and F) BETA analysis predicted the function of H3K27me3 on gene expression in Cal27 (E) and HSC-2 (F) treated with metformin. (G) The pathway enrichment analysis based on upregulated target genes analyzed by BETA in Cal27. (H and I) The pathway enrichment analysis based on upregulated (H) and downregulated (I) target genes analyzed by BETA in HSC-2.


Figure 7



Figure 7

GSK126 showed different regulation on transcripts from metformin. (A and B) The effect of GSK126 on possible transcripts regulated by H3K27ac with metformin in Cal27 (A) and HSC-2 (B). (C) The western blot analysis of Cal27 and HSC-2 treated with metformin or (and) GSK126. (D) The cell growth of Cal27 and HSC-2 treated with metformin or (and) GSK126. Data are presented as mean ± SD from three independent experiments at least. *: p<0.05; **: p<0.01; ***: p<0.001.


Figure 8



Figure 8

The disease-free survival (A) and overall survival (B) of OSCC patients with type II diabetes treated with or without metformin in clinic.

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