lncRNA GAS5 Reverses EMT and Tumor Stem Cell-Mediated Gemcitabine Resistance and Metastasis by Targeting miR-221/SOCS3 in Pancreatic Cancer

Abstract

Dysregulated long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) mediating chemotherapeutic drug effects and metastasis in pancreatic cancer (PC) are key reasons for the poor prognosis of this disease. lncRNA growth arrest-specific 5 (GAS5) is reported to be a tumor suppressor in multiple cancers. However, the functions of GAS5 and its related miRNAs in PC are poorly understood. This study explored the potential functions and mechanisms of GAS5 in PC gemcitabine resistance and metastasis. The results show that overexpression of GAS5 suppressed the proliferation, migration, gemcitabine resistance, stem cell-like properties, and epithelial-mesenchymal transition (EMT) of PC cells by directly binding to and suppressing miR-221 expression and enhancing suppressor of cytokine signaling 3 (SOCS3) expression. The effects of miR-221 overexpression on proliferation, migration, gemcitabine resistance, stem cell-like properties, and EMT inhibition were reversed by SOCS3 overexpression in PC cells. Additionally, GAS5 promoted gemcitabine-induced tumor growth and metastasis inhibition, as determined by Ki-67 staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), bioluminescence imaging, and the detection of cell-like properties and EMT in vivo. Thus, lncRNA GAS5 functioned as a competing endogenous RNA for miR-221, and it suppressed cell growth, metastasis, and gemcitabine resistance in PC by regulating the miR-221/SOCS3 pathway mediating EMT and tumor stem cell self-renewal.

Keywords: SOCS3; gemcitabine resistance; lncRNA GAS5; miR-221; pancreatic cancer.

Figure 1

Expression of GAS5, miR-221, and SOCS3 in Human PC (A) RT-PCR of GAS5 expression in pancreatic tissues. Data are means ± SD (n = 3). ***p < 0.001 versus normal group. (B) RT-PCR of GAS5 in human normal pancreatic epithelial cells (HPDE6-C7) and five pancreatic cancer cell lines (PANC-1, AsPC-1, Capan-2, SW19990, and BxPC3). Data are means ± SD (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 versus HPDE6-C7 group. (C) RT-PCR of miR-221 in pancreatic tissues. Data are means ± SD (n = 3). ***p < 0.001 versus normal group. (D) RT-PCR of miR-221 in human normal pancreatic epithelial cells (HPDE6-C7) and PC cell lines (PANC-1, AsPC-1, Capan-2, SW19990, and BxPC3). Data are means ± SD (n = 3). ***p < 0.001 versus HPDE6-C7 group. (E) IHC staining for SOCS3 in human PC (right) and paired adjacent noncancerous tissues (left). Original magnification, 400×. (F) Western blots of SOCS3 in pancreatic tissues. Data are means ± SD (n = 3). ***p < 0.001 versus normal group. T, tumor; N, normal.

Figure 2

Overexpression of GAS5 Inhibits Xenograft Tumor Growth, Metastasis, and Gemcitabine Resistance (A) Tumor growth curves of PANC-1 cells transfected with or without a GAS5 overexpression vector, treated with gemcitabine (125 mg/kg) or saline. Data are means ± SD (n = 5). ***p < 0.001. (B) Representative images of tumors from four groups at 5 weeks after subcutaneous transplantation or when mice were euthanatized. (C) Ki67 staining shows tumor growth. (D) The relative Ki67 expression was analyzed. Data are means ± SD (n = 5). ***p < 0.001 versus control; ^###p < 0.001 versus gemcitabine treatment group. (E) The relative apoptosis rate was analyzed. (F) TUNEL analysis of four tumor xenografts groups. Data are means ± SD (n = 5). ***p < 0.001 versus control; ^###p < 0.001 versus gemcitabine treatment group. (G and H) RT-PCR for miR-221 (G) and SOCS3 (H) in tumor tissue. Data are means ± SD (n = 5). ***p < 0.001 versus control group. (I) Live imaging of the effects of GAS5 overexpression on metastasis of PANC-1 cells 5 weeks after intravenous tail injection. (J) RT-PCR detection shows the GAS5 expression in tumor tissue. Data are means ± SD (n = 5). ***p < 0.001 versus control group.

Figure 3

Overexpression of GAS5 Inhibits Cell Growth, Migration, and Chemotherapy Resistance In Vitro (A) RT-PCR of GAS5 expression after transfection with a GAS5 overexpression vector. Data are means ± SD (n = 3). ***p < 0.001 versus control group. (B) Cell proliferation was examined by CCK8 assays. Data are means ± SD. *p < 0.05 and ***p < 0.001 versus control. (C) The relative migration cells were analyzed. (D) Transwell analyses showed the effects of GAS5 on PANC-1 cell migration (magnification, 200×). Data are means ± SD. ***p < 0.001 versus control. (E) Comparison of cell viability and IC₅₀ of gemcitabine in PANC-1 cells with or without GAS5 overexpression. (F) Effect of GAS5 on gemcitabine-induced apoptosis detected using annexin V-PI double staining after exposure to 10 nM gemcitabine for 72 hr. (G) The relative apoptosis cells were calculated. Data are means ± SD. ***p < 0.001 versus control.

Figure 4

Overexpression of GAS5 Inhibits Cell EMT and Stem Cell-like Properties (A) Western blot detection shows the relative protein expression. The relative E-cadherin (B), N-cadherin (C), vimentin (D), and Snail (E) were analyzed. Data are means ± SD. ***p < 0.001 versus control. (F) Western blot detection shows the stemness marker protein. The relative protein expression of OCT4 (G), CD133 (H), Nanog (I), and SOX2 (J) were calculated. Data are means ± SD. ***p < 0.001 versus control.

Figure 5

The Relationships among GAS5, miR-221, and SOCS3 in Pancreatic PANC-1 Cells (A) Complementary sequences between miR-221 and GAS5 mRNA were obtained using publicly available algorithms. The GAS5 mutant (MUT) is also shown. (B) Relative luciferase activity by luciferase reporter assays in PANC-1 cells co-transfected with wild-type GAS5 (GAS5-WT) or GAS5-MUT and miR-221 or miR negative control (NC). Data are means ± SD. ***p < 0.001 versus other groups. (C) Expression of miR-221 in PANC-1 cells transfected with GAS5 or NC by RT-PCR. Data are means ± SD. ***p < 0.001 versus control. (D) Complementary sequences between miR-221 and the 3′ UTR of SOCS3 mRNA were obtained using publicly available algorithms. The SOCS3 mutant is also shown. (E) Relative luciferase activity by luciferase reporter assays of PANC-1 cells co-transfected with SOCS3-WT or SOCS3-MUT and miR-221 or miR-NC. Data are means ± SD. ***p < 0.001 versus other groups. (F) SOCS3 expression in PANC-1 cells transfected with miR-221 mimics or NC by RT-PCR. Data are means ± SD. ***p < 0.001 versus control. (G) GAS5 expression in PANC-1 cells transfected with miR-221 mimics or NC by RT-PCR.

Figure 6

SOCS3 Overexpression Reverses miR-221 Overexpression-Induced Cell Proliferation, Migration, EMT, Chemotherapy Resistance, and Stem Cell-like Properties in Pancreatic PANC-1 Cells (A and B) RT-PCR (A) and western blot (B) of SOCS3 after transfection with a SOCS3 overexpression vector or miR-221 mimic alone or combined. Data are means ± SD. ***p < 0.001 versus control. (C) Cell proliferation was examined by CCK8 assays. Data are means ± SD. *p < 0.05 and ***p < 0.001 versus control. (D) Transwell analyses of PANC-1 cell migration (magnification, 200×). (E) The relative migration cells were calculated. Data are means ± SD. ***p < 0.001 versus control. (F) Comparison of cell viability and IC₅₀ of gemcitabine in PANC-1 cells. (G) The relative apoptosis was analyzed. (H) Effect of miR-221 and SOCS3 on gemcitabine-induced apoptosis detected using annexin V-PI double staining after treatment with 10 nM gemcitabine for 72 hr. Data are means ± SD. ***p < 0.001 versus control. (I) E-cadherin, N-cadherin, vimentin, and Snail1 proteins by western blots. (J) Stemness markers OCT4, CD133, Nanog, and SOX2 by western blots.