Histone methyltransferase SUV39H2 regulates LSD1-dependent CDH1 expression and promotes epithelial mesenchymal transition of osteosarcoma

Abstract

Objective: Osteosarcoma (OS) is a malignant tumor characterized by the direct production of bone or osteoid tissues by proliferating tumor cells. Suppressor of variegation 3-9 homolog 2 (SUV39H2) is implicated in the occurrence of OS. Therefore, we designed this study to investigate effects of SUV39H2 in OS meditated by the lysine specific demethylase-1/E-cadherin (LSD1/CDH1) axis.

Methods: Clinical OS tissues and paracancerous tissues were collected for analysis of SUV39H2, LSD1 and CDH1 expression, and Kaplan-Meier survival analysis was applied to test the relationship between SUV39H2 expression and overall survival. Loss- and gain-of-function assays were conducted to determine the roles of SUV39H2, LSD1 and CDH1 in OS epithelial mesenchymal transition (EMT) and migration in OS cells, with quantitation of relevant proteins by immunofluorescence. We confirmed the effects of modulating the SUV39H2/CDH1 axis in a mouse OS tumor model.

Results: SUV39H2 and LSD1 were highly expressed, while CDH1 was downregulated in OS tissues and cells. SUV39H2 expression correlated inversely with overall survival of patients with OS. SUV39H2 positively regulated LSD1 expression, while LSD1 negatively regulated CDH1 expression. SUV39H2 or LSD1 overexpression, or CDH1 silencing promoted migration and EMT, as indicated by reduced E-cadherin and dramatically upregulated Vimentin and N-cadherin of OS cells. SUV39H2 expedited the progression of OS, which was reversed by CDH1 repression in the setting of OS in vitro and in vivo.

Conclusions: Collectively, our results demonstrate highly expressed SUV39H2 in OS elevates the expression of LSD1 to downregulate CDH1 expression, thereby aggravating OS, providing a potential therapeutic target for treatment of OS.

Keywords: E-cadherin gene; Histone methyltransferases; Lysine specific demethylase-1; Osteosarcoma; Suppressor of variegation 3–9 homolog 2.

Fig. 1

The expression of SUV39H2 is upregulated in patients with OS in association with reduced survival rate. a SUV39H2 expression in OS and normal samples from the TCGA database detected through UALCAN website. b Kaplan–Meier survival curves showing the effect of SUV39H2 expression on OS patient survival from the TCGA database. c SUV39H2 mRNA expression in OS tissues and paracancerous tissues tested using RT-qPCR. d SUV39H2 protein expression normalized to GAPDH in OS tissues and paracancerous tissues tested using Western blot analysis. *p < 0.05 vs. paracancerous tissues. e SUV39H2 expression in hFOB1.19, U2OS, HOS and MG63 cells assessed by RT-qPCR. *p < 0.05 vs. hFOB1.19 cells. f The relationship between SUV39H2 expression and the overall survival rate of patients with OS (n = 58) analyzed using Kaplan-Meier survival analysis. Measurement data were expressed as mean ± standard deviation derived from at least 3 independent experiments. Paired t-test was used for comparison between the data of carcinoma and its paracancerous tissues. One-way ANOVA was applied for comparisons among data of hFOB1.19, U2OS, HOS and MG63 cells. Kaplan-Meier graphical analysis was used to calculate the survival rate of patients (n = 58)

Fig. 2

SUV39H2 contributes to accelerated EMT and migration of OS cells. MG63 cells were transfected with sh-NC, sh1-SUV39H2 and sh2-SUV39H2. a Transfection efficiency of SUV39H2 evaluated using RT-qPCR. b The positive rates of N-cadherin, E-cadherin and Vimentin detected using Immunofluorescence. c The protein expression of SUV39H2, E-cadherin and Vimentin normalized to GAPDH in MG63 cells detected by Western blot analysis. d Cell migration and invasion tested by the means of Transwell assay. e Cell apoptosis detected by flow cytometry. *p < 0.05 vs. sh-NC-transfected MG63 cells. Measurement data are expressed as mean ± standard deviation derived from at least 3 independent experiments. One-way ANOVA was conducted for comparison among multiple groups, followed by Tukey’s post hoc test

Fig. 3

SUV39H2 promotes the expression of LSD1, thereby accelerating EMT and migration of OS cells. MG63 cells were transfected with oe-NC, oe-SUV39H2, oe-SUV39H2 + sh-NC, oe-SUV39H2 + sh1-LSD1 or oe-SUV39H2 + sh2-LSD1. a Venn diagram with each circle respectively displaying the OS-related genes, genes that interacted with SUV39H2 through GeneCard and genes that were positively associated with SUV39H2 in OS by UALCAN. The middle part refers to the intersection. b Expression of LSD1 in OS samples of TCGA database. c The co-expression relationship between SUV39H2 and LSD1 in OS samples obtained using Chipbasev2.0 website. d LSD1 mRNA expression in OS tissues and paracancerous tissues detected using RT-qPCR. e LSD1 protein expression normalized to GAPDH in OS tissues and paracancerous tissues detected using Western blot analysis. *p < 0.05 vs. paracancerous tissues, n = 58. f The LSD1 mRNA expression in MG63 cells after overexpressing SUV39H2 detected using RT-qPCR. g H3K4m3 enriched in LSD1 promoter region through UCSC website. h H3K4m3 enrichment in LSD1 promoter region evaluated using ChIP assay. i The transfection efficiency of LSD1 in MG63 cells determined by RT-qPCR. j EMT-related protein expression in MG63 cells detected using immunofluorescence. k The protein expression of E-cadherin, N-cadherin, Vimentin, LSD1 and SUV39H2 normalized to GAPDH in each group detected using Western blot analysis. l The cell migration capacity evaluated using Transwell assay. m The cell apoptosis detected using flow cytometry. *p < 0.05 vs. oe-NC transfected MG63 cells and #p < 0.05 vs. MG63 cells co-transfected with oe-SUV39H2 + sh-NC. Measurement data were expressed as mean ± standard deviation derived from at least 3 independent experiments. Paired t-test was used for the data of OS tissues and paracancerous tissues, and unpaired t-test was used for the other two groups. The data comparison between multiple groups was performed using one-way ANOVA

Fig. 4

LSD1 promotes EMT and migration of OS cells by inhibiting the expression of CDH1. a The interactive relationship of OS-related genes analyzed using STRING. The x-axis indicates the number of interactions shared by, and the y-axis indicates the gene name. b Heatmap displaying genes in OS samples of TCGA database predicted using UALCAN. The x-axis represents the sample and y-axis represents the name of the gene. c Expression of CDH1 in OS samples of TCGA database from UALCAN. d The co-expression relationship between LSD1 and CDH1 identified using Chipbasev2.0. e Expression of CDH1 in OS tissues and paracancerous tissues from patients with OS examined using RT-qPCR. f Expression of CDH1 normalized to GAPDH in OS tissues and paracancerous tissues from patients with OS examined using Western blot analysis. *p < 0.05 vs.. paracancerous tissues, n = 58. g CDH1 expression after overexpressing LSD1 measured using RT-qPCR. h High enrichment of H3K4m3 in CDH1 promoter region found through the UCSC website. i The enrichment of H3K4m3 in CDH1 promoter region assessed using ChIP assay. j EMT-related protein expression tested using immunofluorescence. k N-cadherin, E-cadherin and Vimentin protein expression normalized to GAPDH measured using Western blot analysis. l Cell migration capacity detected using Transwell assay. m Cell apoptosis detected by flow cytometry. *p < 0.05 vs. oe-NC transfected cells. #p < 0.05 vs. cells co-transfected with oe-LSD1 + sh-NC. Measurement data were expressed as mean ± standard deviation derived from at least 3 independent experiments. Paired t-test was used for the data of OS tissues and paracancerous tissues, and unpaired t-test was used for the other two groups. The data comparison between multiple groups was performed using one-way ANOVA

Fig. 5

SUV39H2 facilitates the progression of OS through suppression of CDH1 expression in vivo. The BALB/c nude mice were injected with cells transduced with lentiviral plasmids containing oe-NC, oe-SUV39H2, oe-SUV39H2 + oe-NC or oe-SUV39H2 + oe-CDH1 (n = 8). a The representative images of transplanted tumors in nude mice. b Tumor volume of nude mice. c Tumor weight of nude mice. d SUV39H2, LSD1 and CDH1 expression normalized to GAPDH in tumor tissues detected using Western blot analysis. e The expression of VEGF in each group measured using immunohistochemistry. *p < 0.05 vs. mice injected with oe-NC-infected cells. #p < 0.05 vs. mice inoculated by cells transduced with oe-SUV39H2 + oe-NC. Measurement data are expressed as mean ± standard deviation derived from at least 3 independent experiments. The data comparison between multiple groups was performed using one-way ANOVA, followed with Tukey’s post hoc test. For data comparison among groups at different time points, repeated measures ANOVA was used