FOXC1 Regulation of miR-31-5p Confers Oxaliplatin Resistance by Targeting LATS2 in Colorectal Cancer

Abstract

Colorectal cancer (CRC) is the second leading cause of cancer-related illness worldwide and one of the most common malignancies. Therefore, colorectal cancer research and cases have gained increasing attention. Oxaliplatin (OXA) is currently used in first-line chemotherapy to treat stage III and stage IV metastatic CRC. However, patients undergoing chemotherapy often develop resistance to chemo drugs being used. Evidence has confirmed that microRNAs regulate downstream genes in cancer biology and thereby have roles related to tumor growth, proliferation, invasion, angiogenesis, and multi-drug resistance. The aim of our study is to establish whether miR-31-5p is an oncogene in human colorectal cancers that are resistant to OXA and further confirm its malignant phenotype-associated target molecule. From the results of miRNA microarray assay, we establish that miR-31-5p expression was upregulated in oxaliplatin-resistant (OR)-LoVo cells compared with parental LoVo cells. Moreover, through in vitro and in vivo experiments, we demonstrate that miR-31-5p and large tumor suppressor kinase 2 (LATS2) were inversely related and that miR-31-5p and Forkhead box C1 (FOXC1) were positively correlated in the same LoVo or OR-LoVo cells. Importantly, we reveal a novel drug-resistance mechanism in which the transcription factor FOXC1 binds to the miR-31 promoter to increase the expression of miR31-5p and regulate LATS2 expression, resulting in cancer cell resistance to OXA. These results suggest that miR-31-5p may be a novel biomarker involved in drug resistance progression in CRC patients. Moreover, the FOXC1/miR31-5p/LATS2 drug-resistance mechanism provides new treatment strategies for CRC in clinical trials.

Keywords: Forkhead box C1 (FOXC1); colorectal cancer (CRC); drug-resistance mechanism; large tumor suppressor kinase 2 (LATS2); microRNAs; oxaliplatin.

Figure 1

Cell properties differ between LoVo cells and oxaliplatin resistance (OR)-LoVo cells. (A) The MTT assay result indicates the survival rate of OR-LoVo cells after treatment with or without oxaliplatin (OXA) (45 μM at 24 h) compared with the LoVo cell control group. ** p < 0.01 vs. the LoVo cell control group; *** p < 0.001 vs. the LoVo cell control group. (B) Expression of cell proliferation- and cell cycle checkpoint proteins in LoVo cells and OR-LoVo cells by Western blotting. (C) Quantification of the protein expression of Ki-67, α-SMA, p-Akt, p-ERK, p21, and p27 (n = 3). ** p < 0.01 vs. LoVo cells; *** p < 0.001 vs. LoVo cells.

Figure 1

Cell properties differ between LoVo cells and oxaliplatin resistance (OR)-LoVo cells. (A) The MTT assay result indicates the survival rate of OR-LoVo cells after treatment with or without oxaliplatin (OXA) (45 μM at 24 h) compared with the LoVo cell control group. ** p < 0.01 vs. the LoVo cell control group; *** p < 0.001 vs. the LoVo cell control group. (B) Expression of cell proliferation- and cell cycle checkpoint proteins in LoVo cells and OR-LoVo cells by Western blotting. (C) Quantification of the protein expression of Ki-67, α-SMA, p-Akt, p-ERK, p21, and p27 (n = 3). ** p < 0.01 vs. LoVo cells; *** p < 0.001 vs. LoVo cells.

Figure 2

MicroRNA expression in LoVo and OR-LoVo cells. (A) MiRNA microarray data analysis, with the red bar indicating upregulated expression and the green bar indicating downregulated expression. (B) Detailed miRNA microarray data analysis lists the hsa-miR-31-5p C, RL, or RL/C (C is LoVo cells; RL is OR-LoVo cells) value. C is LoVo cells; RL is OR-LoVo cells. (C) Results of the qRT-PCR analysis of the expression levels of miR-31-5p are shown by the bar. *** p < 0.001 vs. LoVo cells.

Figure 3

The effect of the overexpression or knockdown of miR-31-5p and treatment with or without OXA on cell survival and cell death of LoVo or OR-LoVo cells in vitro. (A) MiR-31-5p expression in LoVo or OR-LoVo cells transfected with a miR-31-5p mimic (40 nM), inhibitor (40 nM), or scrambled miRNA (40 nM) (mimic negative control (NC) or inhibitor NC) or treatment with OXA (45 μM). MiR-31-5p fold increase which was calculated by the ratio miR-31-5p/U6, being U6 snRNA constitutively expressed. (B) Cell survival fold changed of LoVo cells or OR-LoVo cells determined by the MTT assay. The TUNEL assay was used to detect cell death in LoVo cells (C) and OR-LoVo cells (D). Fluorescein staining was used to indicate apoptotic cells, and DAPI staining was used to determine the number of nuclei and assess the gross cellular morphology. LoVo_scrambled represents the miRNA mimic NC, LoVo_mimic represents the miR-31-5p mimic (overexpression of miR-31-5p), OR_scrambled represents the miRNA inhibitor NC, OR_inhibitor represents the miR-31-5p inhibitor (knockdown of miR-31-5p), and OXA represents oxaliplatin. * p < 0.05 vs. LoVo cells control group; ** p < 0.01 vs. LoVo cells control group; *** p < 0.001 vs. LoVo cells control group. # p < 0.05 vs. OR-LoVo cells control group; ## p < 0.01 vs. OR-LoVo cells control group; ### p < 0.001 vs. OR-LoVo cells control group.

Figure 3

The effect of the overexpression or knockdown of miR-31-5p and treatment with or without OXA on cell survival and cell death of LoVo or OR-LoVo cells in vitro. (A) MiR-31-5p expression in LoVo or OR-LoVo cells transfected with a miR-31-5p mimic (40 nM), inhibitor (40 nM), or scrambled miRNA (40 nM) (mimic negative control (NC) or inhibitor NC) or treatment with OXA (45 μM). MiR-31-5p fold increase which was calculated by the ratio miR-31-5p/U6, being U6 snRNA constitutively expressed. (B) Cell survival fold changed of LoVo cells or OR-LoVo cells determined by the MTT assay. The TUNEL assay was used to detect cell death in LoVo cells (C) and OR-LoVo cells (D). Fluorescein staining was used to indicate apoptotic cells, and DAPI staining was used to determine the number of nuclei and assess the gross cellular morphology. LoVo_scrambled represents the miRNA mimic NC, LoVo_mimic represents the miR-31-5p mimic (overexpression of miR-31-5p), OR_scrambled represents the miRNA inhibitor NC, OR_inhibitor represents the miR-31-5p inhibitor (knockdown of miR-31-5p), and OXA represents oxaliplatin. * p < 0.05 vs. LoVo cells control group; ** p < 0.01 vs. LoVo cells control group; *** p < 0.001 vs. LoVo cells control group. # p < 0.05 vs. OR-LoVo cells control group; ## p < 0.01 vs. OR-LoVo cells control group; ### p < 0.001 vs. OR-LoVo cells control group.

Figure 4

MiR-31-5p regulates LATS2 mRNA and protein expression by targeting the 3’UTR in LoVo cells and OR-LoVo cells. (A) We combined the miRTarBase, miRDB, and TargetScanHuman databases to predict the putative targets of miR-31-5p, and there were 18 predicted targets that overlapped among the three online databases. (B) Real-time RT-PCR analysis was used to confirm the mRNA expression levels of LATS2 in the two cell lines. *** p < 0.001 vs. OR-LoVo cells. (C) Protein expression of LATS2 in LoVo cells and OR-LoVo cells by Western blotting. Quantification of LATS2 protein expression (n = 3, the 3 lanes for each cell line loaded in the Western blot which were harvested from the total protein of the three different passages). *** p < 0.001 vs. OR-LoVo cells. (D) Protein expression of LATS2, p21, and p27 in LoVo or OR-LoVo cells transfected with the miR-31-5p mimic (40 nM), inhibitor (40 nM), or scrambled miRNA (40 nM) (mimic NC or inhibitor NC), or treatment with or without OXA (45 μM) by Western blotting. The experiments were performed in triplicate. (E) Luciferase activity assays of the activity of luciferase vectors containing the LATS2 3′-UTR were performed following transfection with miR-31-5p or negative control (NC) for 24 h using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). ** p < 0.01 vs. LoVo cells LATS2 3’UTR group.

Figure 4

MiR-31-5p regulates LATS2 mRNA and protein expression by targeting the 3’UTR in LoVo cells and OR-LoVo cells. (A) We combined the miRTarBase, miRDB, and TargetScanHuman databases to predict the putative targets of miR-31-5p, and there were 18 predicted targets that overlapped among the three online databases. (B) Real-time RT-PCR analysis was used to confirm the mRNA expression levels of LATS2 in the two cell lines. *** p < 0.001 vs. OR-LoVo cells. (C) Protein expression of LATS2 in LoVo cells and OR-LoVo cells by Western blotting. Quantification of LATS2 protein expression (n = 3, the 3 lanes for each cell line loaded in the Western blot which were harvested from the total protein of the three different passages). *** p < 0.001 vs. OR-LoVo cells. (D) Protein expression of LATS2, p21, and p27 in LoVo or OR-LoVo cells transfected with the miR-31-5p mimic (40 nM), inhibitor (40 nM), or scrambled miRNA (40 nM) (mimic NC or inhibitor NC), or treatment with or without OXA (45 μM) by Western blotting. The experiments were performed in triplicate. (E) Luciferase activity assays of the activity of luciferase vectors containing the LATS2 3′-UTR were performed following transfection with miR-31-5p or negative control (NC) for 24 h using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). ** p < 0.01 vs. LoVo cells LATS2 3’UTR group.

Figure 5

MiR-31-5p or OXA regulates tumor growth and tumor death in LoVo or OR-LoVo tumors in vivo. (A) The tumoral growth of LoVo cell xenografted on nude mice in five different groups. Treatment was administered every 3 days from day 0 to 15. (B) The tumoral growth of LoVo cell xenografted on nude mice in five different groups. Treatment was administered every 3 days from day 0 to 15. Tumors of LoVo or OR-LoVo cell were subcutaneously implanted. (C) Representative pictures of tumoral masses isolated at the moment of sacrifice of mice. The nude mice were sacrificed at day 15. (D) Expression of LATS2, p21, and p27 in LoVo and OR-LoVo tumor tissue by Western blotting. (E) MiR-31-5p expression in LoVo and OR-LoVo tumor tissue was measured by qRT-PCR. U6 was used as a loading control. Scale bar: 100 μm (F) IHC staining of LATS2 protein in LoVo or OR-LoVo tumor tissue samples. Scale bar: 100 μm. (G) IHC staining of Ki-67 protein in LoVo or OR-LoVo tumor tissue samples. Scale bar: 100 μm. (H) Lung metastasis in mice bearing LoVo and OR-LoVo cells. Upper panel: representative HE-stained sections of lungs from mice with colorectal cancer cell (CRC) metastasis. Scale bar: 100 μm. * p < 0.05 vs. vs. LoVo cells control group; ** p < 0.01 vs. LoVo cells control group; *** p < 0.001 vs. LoVo cells control group. ## p < 0.01 vs. OR-LoVo cells control group; ### p < 0.001 vs. OR-LoVo cells control group.

Figure 5

MiR-31-5p or OXA regulates tumor growth and tumor death in LoVo or OR-LoVo tumors in vivo. (A) The tumoral growth of LoVo cell xenografted on nude mice in five different groups. Treatment was administered every 3 days from day 0 to 15. (B) The tumoral growth of LoVo cell xenografted on nude mice in five different groups. Treatment was administered every 3 days from day 0 to 15. Tumors of LoVo or OR-LoVo cell were subcutaneously implanted. (C) Representative pictures of tumoral masses isolated at the moment of sacrifice of mice. The nude mice were sacrificed at day 15. (D) Expression of LATS2, p21, and p27 in LoVo and OR-LoVo tumor tissue by Western blotting. (E) MiR-31-5p expression in LoVo and OR-LoVo tumor tissue was measured by qRT-PCR. U6 was used as a loading control. Scale bar: 100 μm (F) IHC staining of LATS2 protein in LoVo or OR-LoVo tumor tissue samples. Scale bar: 100 μm. (G) IHC staining of Ki-67 protein in LoVo or OR-LoVo tumor tissue samples. Scale bar: 100 μm. (H) Lung metastasis in mice bearing LoVo and OR-LoVo cells. Upper panel: representative HE-stained sections of lungs from mice with colorectal cancer cell (CRC) metastasis. Scale bar: 100 μm. * p < 0.05 vs. vs. LoVo cells control group; ** p < 0.01 vs. LoVo cells control group; *** p < 0.001 vs. LoVo cells control group. ## p < 0.01 vs. OR-LoVo cells control group; ### p < 0.001 vs. OR-LoVo cells control group.

Figure 5

MiR-31-5p or OXA regulates tumor growth and tumor death in LoVo or OR-LoVo tumors in vivo. (A) The tumoral growth of LoVo cell xenografted on nude mice in five different groups. Treatment was administered every 3 days from day 0 to 15. (B) The tumoral growth of LoVo cell xenografted on nude mice in five different groups. Treatment was administered every 3 days from day 0 to 15. Tumors of LoVo or OR-LoVo cell were subcutaneously implanted. (C) Representative pictures of tumoral masses isolated at the moment of sacrifice of mice. The nude mice were sacrificed at day 15. (D) Expression of LATS2, p21, and p27 in LoVo and OR-LoVo tumor tissue by Western blotting. (E) MiR-31-5p expression in LoVo and OR-LoVo tumor tissue was measured by qRT-PCR. U6 was used as a loading control. Scale bar: 100 μm (F) IHC staining of LATS2 protein in LoVo or OR-LoVo tumor tissue samples. Scale bar: 100 μm. (G) IHC staining of Ki-67 protein in LoVo or OR-LoVo tumor tissue samples. Scale bar: 100 μm. (H) Lung metastasis in mice bearing LoVo and OR-LoVo cells. Upper panel: representative HE-stained sections of lungs from mice with colorectal cancer cell (CRC) metastasis. Scale bar: 100 μm. * p < 0.05 vs. vs. LoVo cells control group; ** p < 0.01 vs. LoVo cells control group; *** p < 0.001 vs. LoVo cells control group. ## p < 0.01 vs. OR-LoVo cells control group; ### p < 0.001 vs. OR-LoVo cells control group.

Figure 6

Experiments to identify the role of FOXC1 in colorectal cancer cells. (A) The FOXC1 mRNA level after treatment with or without OXA (45 μM) in LoVo and OR-LoVo cells in vitro. (B) Protein expression of FOXC1 following treatment with or without OXA (45 μM) in LoVo and OR-LoVo cells in vitro. (C) FOXC1 mRNA expression in the tumor formation assay in vivo. (D) Protein expression of FOXC1 following treatment with or without OXA in LoVo and OR-LoVo tumors in vivo. (E) FOXC1 nuclear translocation in LoVo cells and OR-LoVo cells in vitro. * p < 0.05 vs. LoVo cells control group; ** p < 0.01 vs. LoVo cells control group; *** p < 0.01 vs. LoVo cells control group.

Figure 7

FOXC1 transcription factor binds to the miR-31 promoter and induces high miR-31-5p expression. (A) FOXC1 protein levels after the transfection of two different FOXC1 siRNAs in OR-LoVo cells. (B) MiR-31-5p levels in FOXC1-siRNA-transfected OR-LoVo cells. (C) FOXC1 mRNA expression following transfection of two doses of the FOXC1 overexpression construct (1 and 3 μg) in LoVo cells. The fold increase is calculated by the ratio FOXC1/GAPDH, being GAPDH snRNA constitutively expressed (D) FOXC1 protein levels resulting from two doses of the FOXC1 overexpression construct (1 and 3 μg) in LoVo cells. (E) MiRNA-31-5p expression resulting from two doses of the FOXC1 overexpression construct (1 and 3 μg) in LoVo cells. The fold increase is calculated by the ratio miR-31-5p/U6, being U6 snRNA constitutively expressed. (F) The luciferase reporter assay confirmed that LoVo cells were co-transfected with the miR-31 promoter (1 μg) and FOXC1 overexpression construct (1 μg). (G) The luciferase reporter assay confirmed that OR-LoVo cells were co-transfected with the miR-31 promoter (2 μg) and various concentrations of FOXC1-siRNA 1409 (0, 10, 20, and 40 nM). * p < 0.05 vs. control group; ** p < 0.01 vs. control group; *** p <0.001 vs. control group.

Figure 7

FOXC1 transcription factor binds to the miR-31 promoter and induces high miR-31-5p expression. (A) FOXC1 protein levels after the transfection of two different FOXC1 siRNAs in OR-LoVo cells. (B) MiR-31-5p levels in FOXC1-siRNA-transfected OR-LoVo cells. (C) FOXC1 mRNA expression following transfection of two doses of the FOXC1 overexpression construct (1 and 3 μg) in LoVo cells. The fold increase is calculated by the ratio FOXC1/GAPDH, being GAPDH snRNA constitutively expressed (D) FOXC1 protein levels resulting from two doses of the FOXC1 overexpression construct (1 and 3 μg) in LoVo cells. (E) MiRNA-31-5p expression resulting from two doses of the FOXC1 overexpression construct (1 and 3 μg) in LoVo cells. The fold increase is calculated by the ratio miR-31-5p/U6, being U6 snRNA constitutively expressed. (F) The luciferase reporter assay confirmed that LoVo cells were co-transfected with the miR-31 promoter (1 μg) and FOXC1 overexpression construct (1 μg). (G) The luciferase reporter assay confirmed that OR-LoVo cells were co-transfected with the miR-31 promoter (2 μg) and various concentrations of FOXC1-siRNA 1409 (0, 10, 20, and 40 nM). * p < 0.05 vs. control group; ** p < 0.01 vs. control group; *** p <0.001 vs. control group.

Figure 8

Schematic representation of the entire signaling pathway. The FOXC1 transcription factor regulates miR-31-5p expression in LoVo cells or OR-LoVo cells. The low nuclear localization of FOXC1 causes low miR-31-5p expression and high LATS2 expression, leading to apoptosis and increased OXA-based chemosensitivity in LoVo cells. By contrast, high miR-31-5p expression regulates the chemoresistance to OXA after FOXC1 binds to the miR-31 promoter of miR-31-5p, which targets LATS2, leading to cancer growth and suppression of apoptosis in OR-LoVo cells.