IGF2BP3 is upregulated in endometrial cancer and tightly regulates the growth of drug-resistant endometrial cancer cells via HMGA1

Abstract

Objective: Endometrial cancer (EC) is a malignant tumor with various histological subtypes and molecular phenotypes. The evaluation of drug resistance is important for cancer treatment. Progesterone resistance is the major challenge in EC. Knowledge of drug resistance in EC is important in the development of novel therapies.

Methods: In this study, ten paracancerous and ten tumor tissues were collected to measure the expression of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) and high-mobility group protein 1 (HMGA1). AN3CA and Ishikawa cells were used to explore the effects of IGF2BP3 on EC.

Results: The expression levels of IGF2BP3 and HMGA1 were higher in EC tumor tissues than in paracancerous tissues. IGF2BP3 and HMGA1 are highly expressed in cisplatin-resistant EC cells. IGF2BP3 knockdown decreased the growth of cisplatin-resistant EC cells. Knockdown of IGF2BP3 decreased the level of HMGA1 protein. HMGA1 knockdown decreased the growth of cisplatin-resistant EC cells.

Discuss and conclusions: The findings demonstrate that IGF2BP3 is upregulated in EC and closely regulates the growth of drug-resistant EC cells via HMGA1. The findings will inform the development of novel therapies for EC.

Keywords: HMGA1; IGF2BP3; drug resistance; endometrial cancer.

Figure 1.

mRNA expression level of IGF2BP3 and HMGA1 is higher in EC tumor tissue compared to paracancerous tissue detected by qRT-PCR. (A) mRNA expression level of HMGA1 is significantly higher in EC tumor tissue compared to paracancerous tissue detected by qRT-PCR (n = 10, ****p < 0.0001). (B) mRNA expression level of IGF2BP3 is significantly higher in EC tumor tissue compared to paracancerous tissue detected by qRT-PCR (n = 10, ****p < 0.0001). NOTE: fold of change was normalized to paracancerous tissue.

Figure 2.

mRNA expression level of IGF2BP3 and HMGA1 is higher in cisplatin-resistant EC cell lines detected by qRT-PCR. (A) cisplatin-resistance was confirmed in AN3CA cell line; (B) cisplatin-resistance was confirmed in Inshikawa cell line; (C) mRNA expression level of IGF2BP3 is higher in cisplatin-resistant AN3CA cell line detected by qRT-PCR. (D) mRNA expression expression level of IGF2BP3 is higher in cisplatin-resistant Inshikawa cell line detected by qRT-PCR. (E) mRNA expression expression level of HMGA1 is higher in cisplatin-resistant AN3CA cell line detected by qRT-PCR. (F) mRNA expression expression level of HMGA1 is higher in cisplatin-resistant Ishikawa cell line detected by qRT-PCR.

Figure 3.

Knockdown of IGF2BP3 by siRNA potently inhibited growth of cisplatin-resistant AN3CA cells. (A) IGF2BP3 was successfully knock down on AN3CA cells using siRNA against IGF2BP3 detected by western blot. (B) IGF2BP3 knockdown significantly decreased colony formation on AN3CA cell line. (C) quantification of colony formation from (B). (D) IGF2BP3 knockdown significantly decreased migration of cisplatin-resistant AN3CA cells detected by wound healing assay. (E) Quantification of distance of cellular migration in isplatin-resistant AN3CA cells.

Figure 4.

Knockdown of IGF2BP3 by siRNA potently inhibited growth of cisplatin-resistant Ishikawa cells. (A) IGF2BP3 was successfully knockdown by siRNA against the gene on Ishikawa cells detected by western blot. (B) IGF2BP3 knockdown significantly decreased colony formation on Ishikawa cell line detected by western blot. (C) Quantification of colony formation of IGF2BP3 knockdown affecting Ishikawa cell line. (D) IGF2BP3 knockdown by siRNA against the gene significantly decreased migration of Ishikawa cells measured by wound healing assay. (E) Quantification of migration of Ishikawa cells affected by IGF2BP3 knockdown (n = 4, *p < 0.05).

Figure 5.

Knockdown of HMGA1 by siRNA potently inhibited growth of cisplatin-resistant AN3CA cells. (A) knockdown of IGF2BP3 inhibited protein level of HMGA1 detected by western blot assay. (B) quantification of protein level of HMGA1 detected by western blot assay. (C) siRNA against HMGA1 knockdown HMGA1 on AN3CA cells detected by western blot assay. (D) quantification of HMGA1 protein on HMGA1 knockdown AN3CA cells. (E) Co-IP study indicated that IGF2BP had interaction with HMGA1 on AN3CA cells with IGF2BP3 antibody. (F) Co-IP study indicated that IGF2BP had interaction with HMGA1 on AN3CA cells with HMGA1 antibody. (G) HMGA1 knockdown by siRNA significantly decreased colony formation on AN3CA cell line detected by colony formation assay. (H) Quantification of colony formation of HMGA1 knockdown affecting AN3CA cell line. (I) HMGA1 knockdown significantly decreased migration of AN3CA cells measured by wound healing assay. (J) Quantification of migration of AN3CA cells affected by HMGA1 knockdown (n = 4, **p < 0.01).

Figure 6.

Knockdown of HMGA1 by siRNA potently inhibited growth of cisplatin-resistant Ishikawa cells. (A) Knockdown of IGF2BP3 inhibited protein level of HMGA1 detected by western blot. (B) quantification of protein level of HMGA1 detected by western blot assay. (C) HMGA1 was successfully knock down on Ishikawa cells detected by western blot. (D) quantification of HMGA1 protein on HMGA1 knockdown AN3CA cells. (E) Co-IP study indicated that IGF2BP had interaction with HMGA1 on Ishikawa cells with IGF2BP antibody. (F) Co-IP study indicated that IGF2BP had interaction with HMGA1 on Ishikawa cells with IGF2BP antibody. (G) HMGA1 knockdown by siRNA significantly decreased colony formation on Ishikawa cell line detected by colony formation assay. (H) Quantification of colony formation of HMGA1 knockdown affecting Ishikawa cell line (**p < 0.01). (I) HMGA1 knockdown significantly decreased migration of Ishikawa cells measured by wound healing assay. (J) Quantification of migration of Ishikawa cells affected by HMGA1 knockdown (n = 4, *p < 0.05).

Figure 7.

HMGA1 over-expression compromised effects of IGF2BP3 on growth of EC cells. (A) Knockout of AN3CA cell line was confirmed using western blot assay. (B) HMGA1 over-expression on top of IGF3BP3 knockout in AN3CA cell line was confirmed. (C) HMGA1 over-expression compromised inhibitory effects of IGF2BP3 knockdown on AN3CA cell line. (D) Knockout of Ishikawa cell line was confirmed using western blot assay. (E) HMGA1 over-expression on top of IGF3BP3 knockout in Ishikawa cell line was confirmed. (F) HMGA1 over-expression compromised inhibitory effects of IGF2BP3 knockdown on Ishikawa cell line.