POLE2 knockdown reduce tumorigenesis in esophageal squamous cells

Abstract

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most frequent malignant tumors originated from digestive system around the world and the treatment was limited by the unclear mechanism. DNA polymerase epsilon 2, accessory subunit (POLE2) is involved in DNA replication, repair, and cell cycle control, whose association with ESCC is still not clear.

Methods: In this study, the expression level of POLE2 in ESCC tissues was detected by IHC. The POLE2 knockdown cell line was constructed, identified by qPCR and western blot and used for detecting cellular functions and constructing xenotransplantation mice model. MTT Assay, colony formation assay, flow cytometry, wound-healing assay and Transwell assay were used to detected cell proliferation, apoptosis and migration.

Results: We firstly identified that the expression of POLE2 was overexpressed in ESCC. Moreover, the high expression of POLE2 can predict the tumor deterioration and poor prognosis of ESCC patients. Additionally, downregulation of POLE2 was involved in ESCC progression by promoting proliferation, migration, and inhibiting apoptosis in vitro. In vivo studies proved that POLE2 was positively correlated with ESCC tumor formation, which was consistent with the results in vitro. We also illuminated that POLE2 knockdown upregulated pro-apoptotic proteins (Bax, Caspase3, CD40L, FasL, IGFBP-5 and P21) and downregulated anti-apoptotic proteins (CLAP-2, IGF-I and sTNF-R2). In addition, POLE2 was involved in ESCC via targeting PI3K/Akt, Cyclin D1 signaling pathway.

Conclusions: Therefore, POLE2 was proved to be involved in the development of ESCC, which may be a potential therapeutic target and bring new breakthroughs in the treatment of ESCC.

Keywords: Cell apoptosis; Cell migration; Cell proliferation; ESCC; POLE2.

Fig. 1

POLE2 is highly expressed in ESCC tissues and the construction of POLE2 knockdown cell model. a Expression levels of POLE2 in ESCC tumor tissues and normal skin tissues were detected by IHC staining. b POLE2 expression and overall survival of ESCC by Kaplan–Meier survival analysis. c Transfection efficiency for Eca-109 and TE-1 cells was evaluated by expression of green fluorescent protein 72 h post-infection. d, e The specificity and validity of the lentivirus-mediated shRNA knockdown of POLE2 expression was verified by qPCR (d) and western blot analysis (e). The data were presented as the mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Knockdown of POLE2 inhibits cell proliferation, promotes apoptosis in ESCC cells. a Cell proliferation of Eca-109 and TE-1 cells with or without knockdown of POLE2 was evaluated by MTT assay. b Colony formation was evaluated for Eca-109 and TE-1 cells with or without POLE2 knockdown. c Flow cytometry analysis based on Annexin V-APC staining was utilized to detect the percentage of early apoptotic cell for Eca-109 and TE-1 cells. The X axis indicated the cell apoptosis while the Y axis indicated the green fluorescence detected from the GFP tagged on lentivirus (shPOLE2 or shCtrl). The data were expressed as mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Knockdown of POLE2 inhibits cell migration in ESCC cells. a Cell migration of Eca-109 and TE-1 cells with or without knockdown of POLE2 was evaluated by wound-healing assay. b Cell migration of Eca-109 and TE-1 cells with or without knockdown of POLE2 was evaluated by Transwell assay. The data were expressed as mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Exploration of downstream molecular mechanism of POLE2 in ESCC cells. a Human apoptosis antibody array analysis was performed in Eca-109 cells with or without POLE2 knockdown. b Differences in human apoptotic antibody array were analyzed in Eca-109 cells regardless of POLE2 knockdown. c Densitometric analysis was performed and the gray values of differentially expressed proteins were shown. d The expression of pathway was observed by western blot in Eca-109 cells. The data were expressed as mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Knockdown of MEX3A inhibits tumor growth in mice xenograft models. a The volume of tumors in shCtrl group and shPOLE2 group was measured post-injection. b The average weight of tumors in shCtrl group and shPOLE2 group. c The picture of tumors taken from mice in shCtrl group and shPOLE2 group. d The total bioluminescent intensity of tumors in shCtrl group and shPOLE2 group. e The bioluminescence imaging of tumors in shCtrl group and shPOLE2 group. f The Ki67 staining of tumor tissues in shPOLE2 group and shPOLE2 group. The data were expressed as mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001