Preclinical report 799
MiR-489 modulates cisplatin resistance in human ovarian cancer cells by targeting Akt3 Huijuan Wua,*, Zhenghua Xiaoc,*, Hua Zhangb, Ke Wanga, Wenxin Liua and Quan Haoa MicroRNAs are a conserved class of small noncoding RNA molecules that harbour the capacity to regulate proteincoding gene expression at the post-transcriptional level. In the current study, we show that miR-489 is downregulated in cisplatin (CDDP)-resistant ovarian cancer cells, SKOV3/ CDDP and OVCAR3/CDDP cells. MiR-489 overexpression results in an inhibition of SKOV3 and OVCAR3 cell survival and cell growth after CDDP treatment and an induction of cell apoptosis. Inhibition of miR-489 yields the opposite results. In addition, miR-489 overexpression increases the sensitivity of SKOV3/CDDP and OVCAR3/CDDP cells to CDDP and inhibits their colony number. Akt3 is validated as a direct target of miR-489 in SKOV3, OVCAR3, SKOV3/ CDDP and OVCAR3/CDDP cells. In addition, miR-489 suppresses Akt3 protein expression by binding sites on its 30 UTR. Knockdown of Akt3 results in a similar effect as that because of miR-489 overexpression; importantly, Akt3 silencing rescues the functions induced by miR-489. Furthermore, we also use the Akt3 inhibitor, MK-2206 2HCl, to determine the role of Akt3 in CDDP resistance. Our study showed that MK-2206 2HCl increased the sensitivity of SKOV3/CDDP and OVCAR3/CDDP cells to CDDP. Taken together, our results indicate that miR-489 inhibited
CDDP resistance and cell growth, and promotes apoptosis by suppressing Akt3 expression. Furthermore, the identification of a novel miR-489-based pathway in CDDP-resistant ovarian cancer will facilitate the development of therapeutic strategies. Anti-Cancer Drugs c 2014 Wolters Kluwer Health | Lippincott 25:799–809 Williams & Wilkins.
Introduction
resistance [8–10]. Depending on the role of their targets in cancer, miRNA functions as either an oncogene or as a tumour suppressor [11]. For example, miR-200b is downregulated in lung cancer and plays an important role in reducing the chemoresistance to docetaxel by targeting E2F3, functioning as a tumour suppressor [12]. MiR-30b/d promotes melanoma cell invasion by directly targeting GalNAc transferase GALNT7, acting as an oncogene [13]. Emerging evidence indicates that miRNAs are dysregulated in CDDP and paclitaxel-resistant ovarian cancer, including let-7e, miR-30c, miR-125b, miR-130a, miR-335 [14] and miR-214 [15]. In addition, our previous study found that miR-489 is downregulated in CDDP-resistant SKOV3 and OVCAR3 cells using miRNA microarrays [16]; however, the potential role of miR-489 in CDDP response has not been well investigated.
One of the most common causes for cancer treatment failure is anticancer drug resistance, including cisplatin (CDDP) resistance. Human ovarian cancer is one of the most malignant gynaecological cancers and has a high mortality. Often, patients are diagnosed at later periods [1]. CDDP is an important therapeutic drug for ovarian cancer in addition to surgery. However, only a few patients respond weakly to CDDP treatment, resulting in a low 5-year survival rate and high mortality [2]. In addition, the molecular mechanism underlying the development of CDDP resistance remains unclear. Thus, identification of novel molecules in the development of CDDP resistance is important for CDDP treatment in cancer. MicroRNAs (miRNAs) are a conserved group of endogenous, small noncoding RNA molecules that have the capacity to regulate gene expression at the post-transcriptional level, resulting in mRNA cleavage or translational repression [3,4]. Thus far, miRNAs regulate the expression of B60% [5] of protein-coding genes, which are involved in diverse biological processes, such as cell growth, differentiation, development and apoptosis [6,7], as well as processes during drug c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4973
Anti-Cancer Drugs 2014, 25:799–809 Keywords: Akt3, cisplatin resistance, microRNA, miR-489, ovarian cancer a Key Laboratory of Cancer Prevention and Therapy, Department of Gynecological Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, bDepartment of Clinical Laboratory, The Affiliated Hospital of the Logistics University of Chinese People’s Armed Police Forces, Tianjin and cDepartment of Gynecology, Yongchuan Hospital, Chongqing Medical University, Chongqing, China
Correspondence to Quan Hao, PhD, Key Laboratory of Cancer Prevention and Therapy, Department of Gynecological Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Huan-Hu-Xi Road, Ti-Yuan-Bei, He Xi District, Tianjin 300060, China Tel/fax: + 86 22 23340123; e-mail: [email protected] *Huijuan Wu and Zhenghua Xiao are the co-first authors. Received 17 October 2013 Revised form accepted 17 February 2014
In the current study, we confirmed that miR-489 was downregulated in CDDP-resistant SKOV3 and OVCAR3 cells. MiR-489 overexpression reduced the SKOV3 and OVCAR3 cell resistance to CDDP and resulted in an inhibition of cell colony formation and an induction of cell apoptosis. In addition, miR-489 increased the sensitivity DOI: 10.1097/CAD.0000000000000107
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
800
Anti-Cancer Drugs 2014, Vol 25 No 7
of SKOV3/CDDP and OVCAR3/CDDP cells to CDDP and suppressed their colony number. Then, we identified a direct target of miR-489. Our data showed a novel regulatory mechanism of miR-489 in ovarian cancer cells in response to CDDP resistance, which may facilitate the development of miR-489-based molecular therapeutic strategies in CDDP-resistant cancers.
Materials and methods Cell culture and transfection
Human ovarian cancer cells, SKOV3 and OVCAR3, were cultured in RPMI1640 (Invitrogen, Carlsbad, California, USA) supplemented with 10% foetal bovine serum and 1% PS (100 IU/ml of penicillin and 100 mg/ml of streptomycin). CDDP-resistant cell lines, SKOV3/CDDP and OVCAR3/CDDP, were obtained as described previously, which was confirmed to have a higher IC50 for CDDP and higher Akt3 levels [16]. The cells were maintained in a humidified incubator with 5% CO2 at 371C. All of the transfections were performed using lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocols. RNA isolation and quantitative real-time RT-PCR (qRT-PCR)
MiRNAs were isolated using trizol reagent (Invitrogen) from transfected cells according to the manufacturer’s instructions. The RNA concentration was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, Delaware, USA). Next, 500 ng of total RNA was used for reverse transcription (RT) using Reverse Transcriptase M-MLV (Takara, Dalian, China). Finally, the SYBR Premix Ex Taq Kit (Takara) was used for qPCR under the following conditions: 951C for 3 min, followed by 40 cycles of 951C for 30 s, 581C for 30 s and 721C for 30 s. U6 was used to normalize miR-489 expression. All primers for RT and PCR are shown as follows: miR-489 RT primer: 50 -CTCAACTGGTGTCGTGGAG TCGGCAATTCAGTTGAGAGCTGCCGT-30 ; U6 RT primer: 50 -GTCGTATCCAGTGCAGGGTCCGAGGTATT CGCACTGGATACGACAAAATATGGAAC-30 ; miR-489 forward primer: 50 -ACACTCCAGCTGGGGTGACATCACA TA-30 ; U6 forward primer: 50 -ACACTCCAGCTGGGG TGCTCGCTTCGGCAGCACA-30 ; miR-489 reverse primer: 50 -TGGTGTCGTGGAGTCG-30 ; U6 reverse primer: 50 -AGGGTCCGAGGTATTC-30 . Plasmid construct and oligonucleotides synthesis
MiR-489 mimics, antisense oligonucleotides (ASO) of miR489 and siRNA of Akt3 were synthesized. The Akt3 30 UTR was amplified and inserted downstream of the psiCHECK-2/luciferase vector. Mutant Akt3 30 UTR (a point mutation within the binding sites was generated as follows: AUGUCA to UUCUGA) was amplified using wild-type 30 UTR as a template.
Chemosensitivity assay
The transfected cells were seeded into 96-well plates (5000 cells/well) and exposed to cisplatin treatment with increasing concentrations (1.25, 2.5, 5, 10, 20 and 40 mmol/l) when the cells were adhesive. For the detection of the Akt3 inhibitor, MK-2206 2HCl, on the cell response to CDDP, the cells treated with MK-2206 2HCl or not were seeded into 96-well plates (5000 cells/well) and exposed to cisplatin treatment with increasing concentrations (12.5, 25, 50, 100, 200 and 400 mmol/l) when the cells were adhesive. After incubation for B24 h, cell viability was analysed using the MTT assay as described previously [16]. The concentration at which cisplatin produced 50% of survival inhibition was considered as IC50. Colony formation assay
The transfected cells were seeded into 12-well plates (200 cells/well). The media were refreshed every 3 days until most of the colonies contained more than 50 cells, which took B10 days. The colony was stained with 5% crystal violet and then quantified. FITC Annexin V apoptosis detection with 7-aminoactinomycin D
Cell apoptosis was measured using the Annexin V-FITC and 7-aminoactinomycin D staining kit. The cells were washed with ice-cold PBS, and then 10 ml of Annexin V-FITC solution and 20 ml of 7-aminoactinomycin D viability dye were added, which was performed in the absence of light. Finally, 400 ml of ice-cold Annexin V binding buffer was added to the above mixture. The cell preparations were then analysed within 30 min using flow cytometry. Western blotting assay
The transfected cells were collected for protein analysis 48 h after transfection. Briefly, the cells were washed with 1 PBS and lysed with RIPA buffer (50 mmol/l Tris-HCl pH 7.2, 150 mmol/l NaCl, 1% Triton X-100 and 0.1% SDS). The protein concentration was measured using the BCA method. Next, 50 mg of protein was used for the detection of Akt3 and GAPDH was used as the loading control. Rabbit monoclonal anti-Akt3 (1 : 1000 dilutions; Abcam, Cambridge, UK) and anti-GAPDH were used as primary antibodies. The secondary antibody was conjugated with horseradish peroxidase. The bound antibodies were detected using the ECL Plus Western Blotting Detection system (GE Healthcare, Pittsburgh, Pennsylvania, USA) and chemiluminescent signals were detected using a high-performance chemiluminescence film (GE Healthcare). Luciferase reporter assay
The cells were cotransfected with miR-489 mimics or ASO and wild-type or mutant Akt3 30 UTR. After transfection for B48 h, the cell lysates were measured for luciferase intensity analysis using the Dual-Luciferase
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Regulation of Akt3 by miR-489 in CDDP resistance Wu et al.
801
Fig. 1
(a) Relative miR-489 level
1.5 1.0 ∗
0.5
∗∗
0.0 OVCAR3
150
SKOV3
150 Relative cell viability
100
50
100
50
0
ol /l μm
/l ol
25 1.
2. 5
μm 5
10
μm
ol
ol
/l
/l
/l
μm
μm
ol μm 40
1.
ol
/l
/l ol
/l
μm
25
μm
ol 5
2. 5
μm
ol
/l
/l ol μm
ol
10
μm 20
40
μm
ol
/l
/l
0
20
(b)
Inhibition rate (%)
OVCAR3 OVCAR3/CDDP SKOV3 SKOV3/CDDP
CDDP MiR-489 ASO
IC50 (μmol/l)
ASO control
MiR-489 ASO
Fold change
P value
OVCAR3
9.89±1.76
1.59
MiR-489 modulates cisplatin resistance in human ovarian cancer cells by targeting Akt3 Huijuan Wua,*, Zhenghua Xiaoc,*, Hua Zhangb, Ke Wanga, Wenxin Liua and Quan Haoa MicroRNAs are a conserved class of small noncoding RNA molecules that harbour the capacity to regulate proteincoding gene expression at the post-transcriptional level. In the current study, we show that miR-489 is downregulated in cisplatin (CDDP)-resistant ovarian cancer cells, SKOV3/ CDDP and OVCAR3/CDDP cells. MiR-489 overexpression results in an inhibition of SKOV3 and OVCAR3 cell survival and cell growth after CDDP treatment and an induction of cell apoptosis. Inhibition of miR-489 yields the opposite results. In addition, miR-489 overexpression increases the sensitivity of SKOV3/CDDP and OVCAR3/CDDP cells to CDDP and inhibits their colony number. Akt3 is validated as a direct target of miR-489 in SKOV3, OVCAR3, SKOV3/ CDDP and OVCAR3/CDDP cells. In addition, miR-489 suppresses Akt3 protein expression by binding sites on its 30 UTR. Knockdown of Akt3 results in a similar effect as that because of miR-489 overexpression; importantly, Akt3 silencing rescues the functions induced by miR-489. Furthermore, we also use the Akt3 inhibitor, MK-2206 2HCl, to determine the role of Akt3 in CDDP resistance. Our study showed that MK-2206 2HCl increased the sensitivity of SKOV3/CDDP and OVCAR3/CDDP cells to CDDP. Taken together, our results indicate that miR-489 inhibited
CDDP resistance and cell growth, and promotes apoptosis by suppressing Akt3 expression. Furthermore, the identification of a novel miR-489-based pathway in CDDP-resistant ovarian cancer will facilitate the development of therapeutic strategies. Anti-Cancer Drugs c 2014 Wolters Kluwer Health | Lippincott 25:799–809 Williams & Wilkins.
Introduction
resistance [8–10]. Depending on the role of their targets in cancer, miRNA functions as either an oncogene or as a tumour suppressor [11]. For example, miR-200b is downregulated in lung cancer and plays an important role in reducing the chemoresistance to docetaxel by targeting E2F3, functioning as a tumour suppressor [12]. MiR-30b/d promotes melanoma cell invasion by directly targeting GalNAc transferase GALNT7, acting as an oncogene [13]. Emerging evidence indicates that miRNAs are dysregulated in CDDP and paclitaxel-resistant ovarian cancer, including let-7e, miR-30c, miR-125b, miR-130a, miR-335 [14] and miR-214 [15]. In addition, our previous study found that miR-489 is downregulated in CDDP-resistant SKOV3 and OVCAR3 cells using miRNA microarrays [16]; however, the potential role of miR-489 in CDDP response has not been well investigated.
One of the most common causes for cancer treatment failure is anticancer drug resistance, including cisplatin (CDDP) resistance. Human ovarian cancer is one of the most malignant gynaecological cancers and has a high mortality. Often, patients are diagnosed at later periods [1]. CDDP is an important therapeutic drug for ovarian cancer in addition to surgery. However, only a few patients respond weakly to CDDP treatment, resulting in a low 5-year survival rate and high mortality [2]. In addition, the molecular mechanism underlying the development of CDDP resistance remains unclear. Thus, identification of novel molecules in the development of CDDP resistance is important for CDDP treatment in cancer. MicroRNAs (miRNAs) are a conserved group of endogenous, small noncoding RNA molecules that have the capacity to regulate gene expression at the post-transcriptional level, resulting in mRNA cleavage or translational repression [3,4]. Thus far, miRNAs regulate the expression of B60% [5] of protein-coding genes, which are involved in diverse biological processes, such as cell growth, differentiation, development and apoptosis [6,7], as well as processes during drug c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4973
Anti-Cancer Drugs 2014, 25:799–809 Keywords: Akt3, cisplatin resistance, microRNA, miR-489, ovarian cancer a Key Laboratory of Cancer Prevention and Therapy, Department of Gynecological Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, bDepartment of Clinical Laboratory, The Affiliated Hospital of the Logistics University of Chinese People’s Armed Police Forces, Tianjin and cDepartment of Gynecology, Yongchuan Hospital, Chongqing Medical University, Chongqing, China
Correspondence to Quan Hao, PhD, Key Laboratory of Cancer Prevention and Therapy, Department of Gynecological Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Huan-Hu-Xi Road, Ti-Yuan-Bei, He Xi District, Tianjin 300060, China Tel/fax: + 86 22 23340123; e-mail: [email protected] *Huijuan Wu and Zhenghua Xiao are the co-first authors. Received 17 October 2013 Revised form accepted 17 February 2014
In the current study, we confirmed that miR-489 was downregulated in CDDP-resistant SKOV3 and OVCAR3 cells. MiR-489 overexpression reduced the SKOV3 and OVCAR3 cell resistance to CDDP and resulted in an inhibition of cell colony formation and an induction of cell apoptosis. In addition, miR-489 increased the sensitivity DOI: 10.1097/CAD.0000000000000107
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
800
Anti-Cancer Drugs 2014, Vol 25 No 7
of SKOV3/CDDP and OVCAR3/CDDP cells to CDDP and suppressed their colony number. Then, we identified a direct target of miR-489. Our data showed a novel regulatory mechanism of miR-489 in ovarian cancer cells in response to CDDP resistance, which may facilitate the development of miR-489-based molecular therapeutic strategies in CDDP-resistant cancers.
Materials and methods Cell culture and transfection
Human ovarian cancer cells, SKOV3 and OVCAR3, were cultured in RPMI1640 (Invitrogen, Carlsbad, California, USA) supplemented with 10% foetal bovine serum and 1% PS (100 IU/ml of penicillin and 100 mg/ml of streptomycin). CDDP-resistant cell lines, SKOV3/CDDP and OVCAR3/CDDP, were obtained as described previously, which was confirmed to have a higher IC50 for CDDP and higher Akt3 levels [16]. The cells were maintained in a humidified incubator with 5% CO2 at 371C. All of the transfections were performed using lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocols. RNA isolation and quantitative real-time RT-PCR (qRT-PCR)
MiRNAs were isolated using trizol reagent (Invitrogen) from transfected cells according to the manufacturer’s instructions. The RNA concentration was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, Delaware, USA). Next, 500 ng of total RNA was used for reverse transcription (RT) using Reverse Transcriptase M-MLV (Takara, Dalian, China). Finally, the SYBR Premix Ex Taq Kit (Takara) was used for qPCR under the following conditions: 951C for 3 min, followed by 40 cycles of 951C for 30 s, 581C for 30 s and 721C for 30 s. U6 was used to normalize miR-489 expression. All primers for RT and PCR are shown as follows: miR-489 RT primer: 50 -CTCAACTGGTGTCGTGGAG TCGGCAATTCAGTTGAGAGCTGCCGT-30 ; U6 RT primer: 50 -GTCGTATCCAGTGCAGGGTCCGAGGTATT CGCACTGGATACGACAAAATATGGAAC-30 ; miR-489 forward primer: 50 -ACACTCCAGCTGGGGTGACATCACA TA-30 ; U6 forward primer: 50 -ACACTCCAGCTGGGG TGCTCGCTTCGGCAGCACA-30 ; miR-489 reverse primer: 50 -TGGTGTCGTGGAGTCG-30 ; U6 reverse primer: 50 -AGGGTCCGAGGTATTC-30 . Plasmid construct and oligonucleotides synthesis
MiR-489 mimics, antisense oligonucleotides (ASO) of miR489 and siRNA of Akt3 were synthesized. The Akt3 30 UTR was amplified and inserted downstream of the psiCHECK-2/luciferase vector. Mutant Akt3 30 UTR (a point mutation within the binding sites was generated as follows: AUGUCA to UUCUGA) was amplified using wild-type 30 UTR as a template.
Chemosensitivity assay
The transfected cells were seeded into 96-well plates (5000 cells/well) and exposed to cisplatin treatment with increasing concentrations (1.25, 2.5, 5, 10, 20 and 40 mmol/l) when the cells were adhesive. For the detection of the Akt3 inhibitor, MK-2206 2HCl, on the cell response to CDDP, the cells treated with MK-2206 2HCl or not were seeded into 96-well plates (5000 cells/well) and exposed to cisplatin treatment with increasing concentrations (12.5, 25, 50, 100, 200 and 400 mmol/l) when the cells were adhesive. After incubation for B24 h, cell viability was analysed using the MTT assay as described previously [16]. The concentration at which cisplatin produced 50% of survival inhibition was considered as IC50. Colony formation assay
The transfected cells were seeded into 12-well plates (200 cells/well). The media were refreshed every 3 days until most of the colonies contained more than 50 cells, which took B10 days. The colony was stained with 5% crystal violet and then quantified. FITC Annexin V apoptosis detection with 7-aminoactinomycin D
Cell apoptosis was measured using the Annexin V-FITC and 7-aminoactinomycin D staining kit. The cells were washed with ice-cold PBS, and then 10 ml of Annexin V-FITC solution and 20 ml of 7-aminoactinomycin D viability dye were added, which was performed in the absence of light. Finally, 400 ml of ice-cold Annexin V binding buffer was added to the above mixture. The cell preparations were then analysed within 30 min using flow cytometry. Western blotting assay
The transfected cells were collected for protein analysis 48 h after transfection. Briefly, the cells were washed with 1 PBS and lysed with RIPA buffer (50 mmol/l Tris-HCl pH 7.2, 150 mmol/l NaCl, 1% Triton X-100 and 0.1% SDS). The protein concentration was measured using the BCA method. Next, 50 mg of protein was used for the detection of Akt3 and GAPDH was used as the loading control. Rabbit monoclonal anti-Akt3 (1 : 1000 dilutions; Abcam, Cambridge, UK) and anti-GAPDH were used as primary antibodies. The secondary antibody was conjugated with horseradish peroxidase. The bound antibodies were detected using the ECL Plus Western Blotting Detection system (GE Healthcare, Pittsburgh, Pennsylvania, USA) and chemiluminescent signals were detected using a high-performance chemiluminescence film (GE Healthcare). Luciferase reporter assay
The cells were cotransfected with miR-489 mimics or ASO and wild-type or mutant Akt3 30 UTR. After transfection for B48 h, the cell lysates were measured for luciferase intensity analysis using the Dual-Luciferase
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Regulation of Akt3 by miR-489 in CDDP resistance Wu et al.
801
Fig. 1
(a) Relative miR-489 level
1.5 1.0 ∗
0.5
∗∗
0.0 OVCAR3
150
SKOV3
150 Relative cell viability
100
50
100
50
0
ol /l μm
/l ol
25 1.
2. 5
μm 5
10
μm
ol
ol
/l
/l
/l
μm
μm
ol μm 40
1.
ol
/l
/l ol
/l
μm
25
μm
ol 5
2. 5
μm
ol
/l
/l ol μm
ol
10
μm 20
40
μm
ol
/l
/l
0
20
(b)
Inhibition rate (%)
OVCAR3 OVCAR3/CDDP SKOV3 SKOV3/CDDP
CDDP MiR-489 ASO
IC50 (μmol/l)
ASO control
MiR-489 ASO
Fold change
P value
OVCAR3
9.89±1.76
1.59
