RAD6 Positively Affects Tumorigenesis of Esophageal Squamous Cell Carcinoma by Regulating Histone Ubiquitination of CCNB1

Abstract

Objective Esophageal carcinoma (ESCA) is deadly cancer worldwide with unknown etiology. This study aimed to investigate the impact and mechanism of RAD6 on the development of Esophageal squamous cell carcinoma (ESCC).Expressions of RAD6A and RAD6B in ESCA were investigated from TCGA dataset and their expressions in tissue sample of ESCA patients and cells were determined. Functional experiments were conducted to explore the impact of RAD6A and RAD6B on malignant characteristics of several kinds of ESCC cells. Animal experiment was established and injected with RAD6A and RAD6B shRNA to evaluate the effect on tumor growth.RAD6A and RAD6B were up-regulated in ESCC cells and tissues. Overexpressed RAD6A and RAD6B similarly increased ESCC cell proliferation, invasion and migration and silencing of RAD6 exerted opposite effects. Knockdown of RAD6A suppressed tumor growth and decreased the level of H2B, as data demonstrated positive correlation between RAD6A and CCNB1 in ESCC tissues.Collectively, this study elucidates that RAD6 is up-regulated in ESCC and promotes the progression of ESCC through up-regulation of CCNB1 to enhance H2B ubiquitination. These evidence provide a novel insight into the pathogenesis of ESCC and might contribute to the development of targeted therapy.

Keywords: CCNB1; Esophageal squamous cell carcinoma; RAD6A; RAD6B; Ubiquitin.

Fig. 1

RAD6A and RAD6B are highly expressed in EC. A RAD6A and RAD6B expression were evaluated in TCGA database; B Expression of RAD6A and RAD6B in different tumor stages; C: Survival analysis for RAD6A and RAD6B in ESCA; D Expression of RAD6A and RAD6B in different histological types; E mRNA expression of RAD6A and RAD6B were evaluated in 50 ESCC patients; F Representative pictures of RAD6 staining in ESCC patients. *** P < 0.001, **** P < 0.0001, vs Normal

Fig. 2

Overexpression of RAD6A and RAD6B promote cell proliferation, migration and invasion of KYSE180 cells in vitro. A mRNA expression of RAD6A and RAD6B were evaluated in ESCC cells. B-D KYSE180 cells were infected with lentiviruses expressing RAD6A or RAD6B or control. B Western blot analysis of RAD6; C CCK-8 assay was used to assess the effect of cell proliferation after overexpressing RAD6A and RAD6B; D and E Transwell assays were used to evaluate the effect of cell migration and invasion after overexpressing RAD6A and RAD6B. ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs OE-NC

Fig. 3

Knockdown RAD6A and RAD6B inhibit cell proliferation, migration and invasion of TE8 cells in vitro. TE8 cells were infected with lentiviruses expressing shRNA targeting RAD6A or RAD6B or shRNA control. A Western blot analysis of RAD6; B CCK-8 assays was used to assess the effect of cell proliferation after knocking down RAD6A and RAD6B; C and D Transwell assays were used to evaluate the effect of cell migration and invasion after knocking down RAD6A and RAD6B. * P < 0.05, *** P < 0.001, **** P < 0.0001, vs sh-NC

Fig. 4

Suppression of RAD6A expression inhibits ESCC growth in vivo. TE8 cells with or without depletion of RAD6A were injected into nude mice. A Volumes of xenograft tumors were measured and plotted; B weights of xenograft tumors; C Representative HE staining in tumor; D Representative immunohistochemical detection of RAD6A in tumor. ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs sh-NC

Fig. 5

RAD6A regulates CCNB1 through pro-transcription histone modifications. A and B Western blot analysis of cell cycle-related proteins; C qRT-PCR was used to detect the correlation between RAD6A and CCNB1 expression in ESCC patients; D and E ChIP was used to determine if suppression of RAD6A altered the level of pro-transcription H2B-Ub in the promoters of CCNB1 genes. ** P < 0.01, *** P < 0.001, vs sh-NC