POLE2 silencing inhibits the progression of colorectal carcinoma cells via wnt signaling axis

Abstract

Colorectal cancer (CRC) is one of the most common malignant carcinoma worldwide. DNA polymerase epsilon 2, accessory subunit (POLE2) participates in DNA replication, repair, and cell cycle control, but its association with CRC development remains unclear. In the present study, the differentially expressed genes (DEGs) in CRC were screened from bioinformatics analysis based on GEO database. RT-qPCR was used to assess mRNA expression. CCK-8 and colony formation assays were applied for the evaluation of cell proliferation. Wound healing and transwell assays were used to detect cell migration and invasion. Protein levels were determined by Western blotting assay. We found that POLE2 was highly expressed in CRC tissues and cell lines. Inhibition of POLE2 suppressed the proliferation, migration and invasion of CRC cells. Mechanistically, Wnt/β-catenin signaling pathway was inactivated by inhibition of POLE2. Activation of Wnt/β-catenin pathway can reverse the function of POLE2 knockdown on CRC cells. In vivo studies demonstrated that POLE2 silencing could notably inhibit the growth of tumors, which was consistent with the results in vitro. In conclusion, we found POLE2 as a novel oncogene in CRC, providing a potential therapeutic or diagnostic target in CRC.

Keywords: Colorectal cancer; POLE2; Wnt/β-catenin; invasion; proliferation.

Figure 1.

POLE2 is a highly expressed gene in colorectal cancer (CRC). (a) Venn diagram of genes identified to participate in progress of CRC. (b) IL12RB1, POLE2, MRE11 and POT1 expressions in colon adenocarcinoma (COAD) tissues from GEPIA database. (c) POT1 expressions in COAD tissues from StarBase database. The mRNA levels of POLE2 in (d) CRC tissues and (e) cell lines were evaluated by rt-qPCR. *p < .05, **p < .01, ***p < .001 vs NC or FHC group.

Figure 2.

Knockdown of POLE2 inhibits cell proliferation, migration and invasion of CRC cells. (a) mRNA and (b) protein expression of POLE2 in RKO and SW480 cells after transfection were detected by qPCR and western blot, respectively. (c) CCK-8 and (d–e) colony formation assays were combined to evaluate cell proliferation. (f-h) transwell assay was performed to determine cell invasion and migration. (i) The protein expression of vimentin, N-cadherin and E-cadherin was evaluated by western blot. *p < .05, **p < .01, ***p < .001.

Figure 3.

POLE2 knockdown inhibited the activation of Wnt/β-catenin pathway. (a) The cell lysates of RKO cells before and after transfection were analyzed using the human phospho-kinase array. (c, d) the activation of PI3K/AKT, p38 MAPK, and Wnt/β-catenin pathways was evaluated by western blot.

Figure 4.

POLE2 promotes the aggressive behaviors of CRC cells through the Wnt/β-catenin signaling pathway. (a) CCK-8 and (b-c) colony formation assays were combined to evaluate cell proliferation. (d-e) transwell assay was performed to determine cell invasion and migration. **p <.01 vs sh-control group, #p <.05 vs sh-POLE2 group, ##p<.01 vs sh-POLE2 group.

Figure 5.

Knockdown of POLE2 suppressed tumor growth of CRC in vivo. (a) The photographs, (b) tumor weight, and (c) tumor volume of xenograft tumors from control, knockdown of POLE2 groups. (d) Representative immunohistochemistry staining images of tumors indicated the levels of POLE2 and Ki67. (e) The activation of Wnt/β-catenin pathway was evaluated by western blot. **p <.01 vs sh-control group.