PRMT1 accelerates cell proliferation, migration, and tumor growth by upregulating ZEB1/H4R3me2as in thyroid carcinoma

Abstract

Thyroid carcinoma (TC) represents the most prevalent malignancy of the endocrine system. Protein arginine methyltransferase 1 (PRMT1) is a critical member of the protein arginine methyltransferase family in mammals and is involved in multiple biological processes. This study aimed to investigate the function of PRMT1 in TC. In the present study, human TC cell lines (8505C, CAL62, and BCPAP) and a normal human thyroid cell line Nthy‑ori 3‑1 were employed. Small interfering RNA targeting PRMT1 was used to knock down PRMT1 expression in 8505C cells, and PRMT1 overexpression plasmids were transfected into BCPAP cells. Cell viability was assessed using a CCK‑8 and colony formation assays. Apoptosis was measured using flow cytometry and TUNEL assays. Cell migration was assessed using wound healing and Transwell assays. Reverse transcription‑quantitative PCR was used to determine the mRNA expression levels of PRMT1. Western blotting was used to detect the protein expression levels of PRMT1, E‑cadherin, vimentin, H4R3me2as, and zinc‑finger E homeobox‑binding 1 (ZEB1). Notably, PRMT1 expression was elevated in TC (P<0.01). PRMT1 knockdown inhibited TC cell proliferation and migration and concurrently enhanced migration. Furthermore, PRMT1 knockdown suppressed tumor growth and metastasis in a mouse model of TC. PRMT1 downregulation increased E‑cadherin expression and decreased the expression of vimentin, H4R3me2as, and ZEB1 in the TC cells and the mouse model of TC. Conversely, PRMT1 overexpression had the opposite effect on TC malignant characteristics (P<0.05). These findings suggest that PRMT1 knockdown inhibited TC malignancy by downregulating H4R3me2as/ZEB1, thereby highlighting novel therapeutic targets and diagnostic markers for the management of TC.

Keywords: H4R3me2; ZEB1; malignancy; protein arginine methyltransferase 1; thyroid carcinoma.

Figure 1.

PRMT1 expression is upregulated in TC cells. Western blotting was used to detect the expression of PRMT1 in human TC cell lines 8505C and CAL62, and in the human normal thyroid cell line Nthy-ori 3-1. **P<0.01 vs. Nthy-ori 3-1. PRMT1, protein arginine methyltransferase 1; TC, thyroid carcinoma.

Figure 2.

PRMT1 downregulation inhibits cell proliferation and migration and promotes cell apoptosis in TC. (A and B) RT-qPCR and western bolting were used to determine the expression of PRMT1 in 8505C cells following various treatments. (C and D) CCK-8 and colony formation assays were used to assess cell viability and proliferation, respectively, of treated 8050C cells. (E) Flow cytometry was used to determine apoptosis of treated 8505C cells. (F and G) Wound healing (magnification, ×100) and Transwell (magnification, ×200) assays were used to assess cell migration of the treated 8505C cells. *P<0.05, **P<0.01 vs. si-NC; ^#P<0.05, ^##P<0.01 vs. si-PRMT1; ^{^^}P<0.01 vs. 10 µM AMI-1. PRMT1, protein arginine methyltransferase 1; TC, thyroid carcinoma; si-NC, small interfering RNA negative control.

Figure 3.

PRMT1 knockdown suppresses cell metastasis and downregulates expression of ZEB1 and H4R3me2as in TC. (A) IF analyzed the contents of E-cadherin and vimentin in 8505C cells, magnification=400×. (B) Western blotting was used to assess the protein expression levels of E-cadherin, vimentin, H4R3me2as, and ZEB1 in 8505C cells. The 8505C cells were transfected with si-NC or si-PRMT1. *P<0.05, **P<0.01 compared with si-NC; ^##P<0.01 compared with si-PRMT1; ^{^^}P<0.01 compared with 10 µM AMI-1. Each experiment was conducted at least thrice. PRMT1, protein arginine methyltransferase 1; ZEB1, zinc finger e-box binding homeobox1, H4R3me2as, asymmetric demethylation of H4 at the third arginine residue; TC, thyroid carcinoma; si-NC, small interfering RNA negative control.

Figure 4.

PRMT1 overexpression accelerates TC progression and upregulates the expression of ZEB1 and H4R3me2as. (A and B) RT-qPCR and western bolting were used to assess the expression of PRMT1 in BCPAP cells. (C and D) The CCK-8 and colony formation analyses were used to determine BCPAP cell viability and proliferation, respectively. (E) Flow cytometry was used to analyze BCPAP cell apoptosis. (F and G) Wound healing (magnification, ×100) and Transwell (magnification, ×200) assays were used to assess cell migration of BCPAP cells. (H) IF was used to assess E-cadherin and vimentin expression in BCPAP cells. Magnification, ×400. (I) Western blotting was used to assess the protein expression of E-cadherin, vimentin, H4R3me2as, and ZEB1 in BCPAP cells. BCPAP cells were transfected with oe-PRMT1. *P<0.05, **P<0.01 vs. with VE. PRMT1, protein arginine methyltransferase 1; ZEB1, zinc finger e-box binding homeobox1; H4R3me2as, asymmetric demethylation of H4 at the third arginine residue; TC, thyroid carcinoma; oe-PRMT1, overexpression-PRMT1; VE, vector.

Figure 5.

PRMT1 overexpression accelerates TC progression through the activation of ZEB1/H4R3me2as. (A) RT-qPCR was used to assess the expression of ZEB1 in BCPAP cells transfected with si-ZEB1. (B) mRNA expression of PRMT1 and ZEB1 in multiple group of cells. (C and D) CCK-8 and colony formation assays were used to test BCPAP cell viability and proliferation, respectively. (E and F) Wound healing and Transwell assays were used to assess cell migration of BCPAP cells. Scale bar, 50 µM. (G) Western blotting was used to measure the protein expression levels of E-cadherin, vimentin, and H4R3me2as in BCPAP cells. BCPAP cells were transfected with oe-PRMT1 and/or si-ZEB1. **P<0.01 vs. VE group, ^##P<0.01 vs. oe-PRMT1 + si-NC group. PRMT1, protein arginine methyltransferase 1; ZEB1, zinc finger e-box binding homeobox1; H4R3me2as, asymmetric demethylation of H4 at the third arginine residue; TC, thyroid carcinoma; oe-PRMT1, overexpression-PRMT1; VE, vector.

Figure 6.

PRMT1 knockdown reduces tumor growth and metastasis by inhibiting ZEB1/H4R3me2as in a xenograft model. (A) PRMT1 protein expression in TC tissues (B) Excised tumors from the mouse model of TC. (C) H&E staining of TC tissues. Magnification, ×200. (D) Image of pulmonary nodules and number of pulmonary nodules. (E) TUNEL assays were used to assess apoptosis in xenograft model mice. Magnification, ×400. (F) Western blotting was to measure the protein expression levels of E-cadherin, vimentin, H4R3me2as, and ZEB1 in TC tissues. BALB/C nude mice were subcutaneously injected with treated 8505C cells. *P<0.05, **P<0.01 vs. si-NC; ^#P<0.05, ^##P<0.01 vs. si-PRMT1; ^{^}P<0.05; ^{^^}P<0.01 vs. 10 µM AMI-1. PRMT1, protein arginine methyltransferase 1; ZEB1, zinc finger e-box binding homeobox1; H4R3me2as, asymmetric demethylation of H4 at the third arginine residue; TC, thyroid carcinoma; si-NC, small interfering RNA negative control.