HOXD3 was negatively regulated by YY1 recruiting HDAC1 to suppress progression of hepatocellular carcinoma cells via ITGA2 pathway

Abstract

Objectives: HOXD3 is associated with progression of multiple types of cancer. This study aimed to identify the association of YY1 with HOXD3-ITGA2 axis in the progression of hepatocellular carcinoma.

Materials and methods: Bioinformatics assay was used to identify the effect of YY1, HOXD3 and ITGA2 expression in HCC tissues. The function of YY1 and HOXD3 in HCCs was determined by qRT-PCR, MTT, apoptosis, Western blotting, colony formation, immunohistochemistry, and wound-healing and transwell invasion assays. The relationship between YY1 and HOXD3 or HOXD3 and ITGA2 was explored by RNA-Seq, ChIP-PCR, dual luciferase reports and Pearson's assays. The interactions between YY1 and HDAC1 were determined by immunofluorescence microscopy and Co-IP.

Results: Herein, we showed that the expression of YY1, HOXD3 and ITGA2 associated with the histologic and pathologic stages of HCC. Moreover, YY1, recruiting HDAC1, can directly target HOXD3 to regulate progression of HCCs. The relationship between YY1 and HOXD3 was unknown until uncovered by our present investigation. Furthermore, HOXD3 bound to promoter region of ITGA2 and up-regulated the expression, thus activating the ERK1/2 signalling and inducing HCCs proliferation, metastasis and migration in the vitro and vivo.

Conclusions: Therefore, HOXD3, a target of YY1, facilitates HCC progression via activation of the ERK1/2 signalling by promoting ITGA2. This finding provides a new whole way to HCC therapy by serving YY1-HOXD3-ITGA2 regulatory axis as a potential therapeutic target for HCC therapy.

Keywords: HOXD3; ITGA2; YY1; cell progression; hepatocellular carcinoma.

FIGURE 1

YY1 inhibited the progression and induced apoptosis of HCCs. A, TCGA database showed YY1 expression in HCC tissues and their normal tissues. B, The expression of YY1 connected with clinicopathologic characteristics of HCC patients. C, YY1 mRNA expression in HCC tissues vs counterparts' tissues. D, E, YY1 expression was identified in HCC tissues and their counterparts' tissues at the protein level by using Western blotting and immunohistochemistry assays. F, MTT assay was performed to determine the growth of HCCs treated with YY1 overexpression construct or negative control. G, The colony formation assay was performed several days after the transfection of HCCs with YY1 or negative control. H, Apoptosis was determined in HCCs at 48 h after transfection with YY1. I, Transwell analysis of HCCs after transfected with YY1‐Ctrl and YY1. Quantitative analysis of the invasion rates by solubilization of crystal violet and spectrophotometric reading at OD 570 nm. J, Wound‐healing assays with HCCs treated with YY1‐Ctrl and YY1. The relative wound closure (100%) represents the metastasis capacity of HCCs (the results are expressed as mean ± SEM; *P < .05, **P < .01)

FIGURE 2

Inhibitor of YY1 increased progression and reduced apoptosis of HCCs. A, MTT assay of Huh7/MHCC‐97H cells after inhibiting the expression of YY1. B, The growth of Huh7/MHCC‐97H cells was detected by colony formation. The colony formation assay was determined in HCCs transfected with siYY‐1, siYY1‐2 or negative control (siYY1‐Ctrl). C, Apoptosis was determined in HCCs transfected with siYY‐1, siYY1‐2 or a negative control. D, E, Transwell and wound‐healing analysis represented the migration and metastasis capacity of HCCs (the results are expressed as mean ± SEM; *P < .05, **P < .01)

FIGURE 3

HOXD3 induced the progression and induced apoptosis of HCCs A, The expression of HOXD3 in HCC tissues and their counterparts at RNA level. B, C, HOXD3 protein expression in HCC tissues vs counterparts' tissues was confirmed by using Western blotting and immunohistochemistry assays. D, The effects of HOXD3 on HCC proliferation were determined by MTT assay at 24, 48 and 72 h. E, Representative results of colony formation of HCCs after HOXD3 overexpression. F, Cell apoptosis determined in HCCs 48 h after transfection. G, Transwell analysis of HCCs after transfected with HOXD3‐Ctrl and HOXD3. Quantitative analysis of the invasion rates by solubilization of crystal violet and spectrophotometric reading at OD 570 nm. H, Wound‐healing assays with HCCs treated with HOXD3‐Ctrl and HOXD3. The relative wound closure (100%) represents the metastasis capacity of HCCs (the results are expressed as mean ± SEM; *P < .05, **P < .01)

FIGURE 4

Inhibition of HOXD3 decreased the HCC proliferation, migration and increased apoptosis. A‐C, MTT, colony formation and cell apoptosis assays were performed to determine the impact of HCCs treated with siHOXD3‐1, siHOXD3‐2 or negative control. D, E, Transwell and wound‐healing analysis represented the migration and metastasis capacity of HCCs transfected with siHOXD3‐Ctrl, siHOXD3‐1 and siHOXD3‐2 (the results are expressed as mean ± SEM; *P < .05, **P < .01)

FIGURE 5

YY1 activates HOXD3 expression directly. A, The relationship between YY1 and HOXD3 was assayed by Pearson's r. B, The expression levels of HOXD3 were determined by qRT‐PCR in HCCs transfected with YY1 or siYY1. C, Schematic diagram of the putative HOXD3 promoter with one potential YY1 response element. D, The interaction of YY1 with HOXD3 was shown using ChIP assays with control (rat IgG) or anti‐YY1 antibody. E, qRT‐PCR analysis was performed with primers spanning predicted YY1 of HOXD3. F, Luciferase assays were performed in Huh7 cells transfected with wt or mut promoter. G, The expression of HOXD3, ITGA2, p‐MEK, p‐ERK, Bcl‐xL, Bad, E‐cadherin and N‐cadherin was detected by Western blotting. H, The expression of YY1 and HDAC1 was detected by immunofluorescence staining. I, The expression of YY1 and HDAC1 was detected using Co‐IP (the results are expressed as mean ± SEM; *P < .05, **P < .01)

FIGURE 6

YY1 rescues HOXD3 induced cellular invasion in HCCs. A‐C, MTT, colony formation and cell apoptosis assays were performed to determine the impact of HCCs treated with HOXD3 and YY1 or negative control. D, E, Transwell and wound‐healing analysis represented the migration and metastasis capacity of HCCs co‐transfected with HOXD3 and YY1‐Ctrl or YY1 (the results are expressed as mean ± SEM; *P < .05, **P < .01)

FIGURE 7

ITGA2 higher expressed in HCC tissues and connected with tumour migration. A, Heat map diagram of differential mRNA expression profiles between HOXD3‐ctrl and HOXD3. B, Differential mRNA expression profiles between HOXD3‐ctrl and HOXD3. Red = genes with higher expression, green = genes with lower expression and grey = gene with not different expression. C, The expression of ITGA2 in HCC tissues and adjacent noncancerous liver tissues at RNA in TCGA database. D‐F, The expression of ITGA2 connected with pathologic and histologic grade of HCC patients. G, The expression of ITGA2 connected with survival rate of HCC patients

FIGURE 8

HOXD3 targets the promoter region of ITGA2 directly and regulates the expression of ITGA2. A, The expression of ITGA2 in 80 pairs HCC tissues and adjacent noncancerous liver tissues at RNA level. B, C, ITGA2 protein expression in HCC tissues vs counterparts was identified by using immunohistochemistry and Western blotting assays. D, E, Pearson's r was used to test the relationship between HOXD3 and ITGA2, YY1 and ITGA2. F, G, The expression of ITGA2 in HCCs transfected with HOXD3/YY1 or siHOXD3/si‐YY1. H, Schematic diagram of the putative ITGA2 promoter with one potential HOXD3 response element. I, J, The interaction of HOXD3 with ITGA2 was shown using ChIP‐qRT‐PCR assays with control (IgG) or anti‐HOXD3 antibody. K, Luciferase activity relative to Renilla control was measured in Huh7 cells. L, The expression of ITGA2, p‐MEK, p‐ERK, Bcl‐xL, Bad, N‐cadherin and E‐cadherin was detected by Western blotting (the results are expressed as mean ± SEM; *P < .05, **P < .01)

FIGURE 9

Silencing HOXD3 expression inhibits hepatocellular carcinoma progression in vivo. A, Gross morphology of tumours injected with either LV‐ctrl or LV‐shHOXD3 cells after 28 d. B, Small animal imaging analysis was used to assess tumour volume in situ at day 28 during tumour development. C, Morphology of excised tumours from nude mice. D, Growth curves of tumour volume were generated every 3 d for 28 d. E, Tumour weight. F, G, The expression levels of HOXD3 and ITGA2 were analysed by RT‐PCR and Western blotting in tissues from the animal. H, The metastasis of huh7 cells suppressing HOXD3 was shown. Down‐expression of HOXD3 significantly inhibited the metastasis of huh7 cells to the lung. I, Proposed model for the effects of YY1‐mediated HOXD3 on HCC progression via regulation of the ITGA2–ERK1/2 signalling pathway (the results are expressed as mean ± SEM; *P < .05, **P < .01)