PRMT5 promotes progression of endometrioid adenocarcinoma via ERα and cell cycle signaling pathways

Abstract

Protein arginine methyltransferase 5 (PRMT5) has previously been reported to be upregulated in many malignant tumors. This study investigated the significance of PRMT5 in endometrial carcinoma (EC) and explored its function in tumorigenesis. Immunohistochemistry was performed to evaluate PRMT5 expression in 62 EC and 66 endometrial hyperplasia samples. The functions of PRMT5 were investigated by cell counting kit-8, plate colony formation, wound healing, and transwell and flow cytometry assays. Quantitative reverse transcription-polymerase chain reaction and western blotting were used to measure the expression of PRMT5, changes in estrogen receptor α (ERα), and related functional proteins. Coimmunoprecipitation was performed to examine the interaction of PRMT5 with ERα and its coactivator steroid receptor coactivator-1 (SRC1). Compared to endometrial hyperplasia tissue, PRMT5 was overexpressed in endometrioid adenocarcinoma (EAC) but not overexpressed in mucinous EC. The main expression pattern of PRMT5 in EAC was cytoplasmic. However, the positive cases of endometrial hyperplasia showed both cytoplasmic and nuclear positivity in the endometrial glands or were mainly positive in stromal cells. Knockdown of PRMT5 significantly inhibited the growth and migration ability of EAC cells and promoted their apoptosis by regulating cyclin D1, c-myc, p53, and Bcl2 proteins. Furthermore, PRMT5 could form a complex with ERα and SRC1 to promote the expression of ERα. In conclusion, PRMT5 plays a significant role in the progression of EAC by interacting with ERα and impacting the cell cycle signaling pathways.

Keywords: ERα; PRMT5; cell function; endometrial cancer; epigenetics.

Figure 1

The expression pattern of PRMT5 in EC and a series of endometrial hyperplasia. (A) PRMT5 was mainly expressed in stromal cells and in the cytoplasm and nuclei of some glandular cells in some cases of SH; (B) a PRMT5‐positive case of SH (both glandular cytoplasmic and nuclear positivity, with stromal cell positivity); (C) PRMT5 was mainly expressed in stromal cells in some cases of CH; (D) PRMT5‐positive case of CH (both glandular nuclear and cytoplasmic positivity); (E) PRMT5‐positive case of AH (moderate intensity cytoplasmic positivity, with a small number of positive nuclei); (F) PRMT5 expression showed strong cytoplasmic staining and limited nuclear localization in EAC; (G) PRMT5 was barely expressed in MEC. AH, atypical hyperplasia; CH, complex hyperplasia; EAC, endometrioid adenocarcinoma; MEC, mucinous endometrial carcinoma; SH, simple hyperplasia.

Figure 2

Knockdown of PRMT5 inhibited EAC cell proliferation, migration, and invasion in vitro. (A) CCK‐8 assay showed that the proliferation of Ishikawa cells was significantly inhibited after 48 h; (B) plate colony formation assay showed that silencing PRMT5 impaired the colony formation ability of Ishikawa cells after 18 days; (C) wound‐healing assays revealed that the width of wound gaps in PRMT5‐knockdown cells was markedly wider than that in SC cells at 48 h; (D) transwell assays indicated that the number of cells passing through the chamber (or matrix for the invasion test) was significantly reduced after transfection for 22 h; (E) the number of apoptotic cells increased significantly when PRMT5 was stably suppressed; (F) no significant difference was found for the effect of PRMT5 knockdown on the cell cycle by flow cytometry. SC, scrambled control; shPRMT5, short hairpin PRMT5; *p < 0.05 and **p < 0.01.

Figure 3

PRMT5 forms a complex with ERα and SRC1, and knockdown of PRMT5 inhibits ERα expression. (A) ERα transcription is suppressed at both the mRNA and protein levels after PRMT5 knockdown. PRMT5^a and GAPDH^b: the bands of PRMT5 and GAPDH (SC and shPRMT5) shown here are the same as those in Figure 4 but for different purposes; we aim to show the relationship between PRMT5 and ERα here. (B) Using PRMT5 and ERα as bait proteins, PRMT5, SRC1, and ERα could form a complex in EAC. (C) PRMT5 expression decreased with increasing E2 (β‐estradiol) dosage concentration (10⁻⁷–10⁻³ mm). SC, scrambled control; shPRMT5, short hairpin PRMT5.

Figure 4

The changes of related proteins after PRMT5 knockdown. Cyclin D1, Bcl‐2, and c‐myc proteins were significantly reduced, while p53 expression was increased after knockdown of PRMT5 in Ishikawa cells. PRMT5^a and GAPDH^b: the bands of PRMT5 and GAPDH (SC and shPRMT5) shown here are the same as those in Figure 3A but for different purposes; we aim to show the relationship between PRMT5 and involved signaling pathways here. SC, scrambled control; shPRMT5, short hairpin PRMT5.