- Email: [email protected]
miR-140-5p inhibits ovarian cancer growth partially by repression of PDGFRA
G Model BIOPHA 3722 No. of Pages 6
Biomedicine & Pharmacotherapy xxx (2015) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article
miR-140-5p inhibits ovarian cancer growth partially by repression of PDGFRA Hong Lana , Wangsheng Chenb,* , Guoli Hea , Shuying Yanga a b
Department of Gynecology, People's Hospital of Hainan Province, Haikou, China Department of Radiology, People's Hospital of Hainan Province, 19 Xinhua Road, Haikou 570311, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 April 2015 Accepted 26 July 2015
Dysregulation of miRNAs is a common feature in human cancers, and miR-140-5p has been found to be down-regulated in cancer. However its role in ovarian cancer remains unclear. miR-140-5p was underexpressed in HCC tissues and cell lines compared with their normal controls. Additionally, PDGFRA was predicted the target gene of miR-140-5p. PDGFRA was inversely correlated with the expression of miR-140-5p in ovarian cancer cells. Importantly, we demonstrate that the over expression of miR-140-5p significantly inhibits ovarian cancer cell proliferation and induces apoptosis. Our results suggest the existence of a novel miR-140-5p-PDGFRA pathway and indicate that miR-140-5p acts as a tumor suppressor during ovarian carcinogenesis. These results may provide a promising alterative strategy for the therapeutic treatment of ovarian cancer. ã 2015 Published by Elsevier Masson SAS.
Keywords: miR-140-5p PDGFRA Ovarian cancer
1. Introduction Ovarian cancer is the most lethal gynecologic malignancy and the most common cause of death in women [1,2]. Recurrence and metastasis seriously affect the prognosis of ovarian cancer. The advance stage at the time of diagnosis and relapse due to chemoresistance are the major reasons for poor prognosis [3]. Using molecular methods, a number of genes and pathways playing critical roles in the pathogenesis of ovarian cancer have been identified. Many of them may act as the targets of ovarian cancer therapy. microRNAs (miRNAs) are one of them. miRNAs are approximately 22-nucleotide noncoding RNAs with high conservation and involved in gene regulation on posttranscriptional level. miRNAs negatively regulate genes expression by binding to the 30 -untranslated region (UTR) of target mRNAs [4]. A miRNA can regulate many mRNAs which may regulate only a target gene [6]. miRNAs involve in various processes of tumor progression including development, differentiation, metabolism, proliferation, cell cycle, and metastasis [5–7]. They play oncogenes or tumor suppressor. RNAs have been definitively linked to cancer development, including ovarian cancer. There is a report suggested that miR-140-5p is involved in cancer progression [8], but its role in ovarian cancer is still unknown.
* Corresponding author. E-mail address: [email protected] (W. Chen).
In this present study, we set out to investigate mechanism and function of miR-140-5p via altering its target gene expression in human ovarian cancer. Here we report the PDGFRA expression in ovarian cancer is regulated by miR-140-5p. We show that miR-1405p is down-regulated in ovarian cancer cells, resulting in increased PDGFRA expression and enhance ovarian cancer cell proliferation, migration and reduced apoptosis. 2. Materials and methods 2.1. Ovarian cancer samples Cancer tissues and their paired normal tissues were obtained from the patients with ovarian cancer. The patients were from People’s Hospital of Hainan Province (Haikou, China), who did not receive either radiotherapy or chemotherapy. All the patients were diagnosed as ovarian cancer and the samples were histologically confirmed by pathologist. Informed consent was obtained from each patient, and the research protocols were approved by the Ethics Committee of the hospital. 2.2. Cell culture Human ovarian cancer cells SKOV3, A2780, 3AO, OVCAR, COC1, HRA and HO-8910 were obtained primarily from the American Type Culture Collection (ATCC) and were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/mL of penicillin
http://dx.doi.org/10.1016/j.biopha.2015.07.035 0753-3322/ ã 2015 Published by Elsevier Masson SAS.
Please cite this article in press as: H. Lan, et al., miR-140-5p inhibits ovarian cancer growth partially by repression of PDGFRA, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.035
G Model BIOPHA 3722 No. of Pages 6
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and 100 mg/mL of streptomycin. Cells were cultured at 37 C in a humidified atmosphere of 5% CO2.
3. Results 3.1. Loss of miR-140-5p in human ovarian cancer
2.3. MiRNA and siRNA transfection Lentiviral vectors mediated miR-14-5p were constructed and production was performed according to the protocol (Invitrogen). Transduction was performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Lentivirus-mediated silencing of miR-140-5p was examined by qRT-PCR. 2.4. Real-time RT-PCR
To determine miR-140-5p expression in ovarian cancer tissues, we examined its level in 38 samples from the patients by qRT-PCR method. miR-140-5p levels were detected in human ovarian cancer tissues and reduced in ovarian cancer tissues compared with the control (Fig. 1A and B). The result was further verified in seven human ovarian cancer cell lines (SKOV3, A2780, 3AO, OVCAR, COC1, HRA, HO-8910) compared with the normal cells (ISOE80) (Fig. 1C). The results indicated that miR-140-5p was downregulated tissues and cell lines of ovarian cancer.
Total RNA, following the manufacturer’s instructions, was isolated from the cells using Trizol reagent (Invitrogen). To measure mRNA expression, real-time RT-PCR was performed using a sequence detector (ABI-Prism, Applied Biosystems). Primers were purchased from Invitrogen. The relative expression levels were calculated by comparing Ct values of the samples with those of the reference, all data normalized to the internal control GAPDH. 2.5. MTT method Cell proliferation was assayed in ovarian cancer cells with miRNA transfection and the growth curve was delineated. Briefly, the cells were incubated at 37 C, 5% CO2 until cells cover the bottom of the well (a flat-bottom 96-well plate), and then the cells were cultured. Cells were treated with MTT solution (5 mg/ml, 0.5% MTT). The cells were continued to culture for 4 h and 150 ml DMSO was added. The ELISA reader was used to measure the absorbance at 570 nm. 2.6. Colony formation assay Ovarian cancer cells were transfected with miR-485-5p or other plasmids and were seeded to 6-well plates (200 cells/well) followed by incubation at 37 C in an environment with saturated humidity and 5% CO2 for 24 h. Non-adherent cells were removed. After culture for 10–14 days, colonies were present and counted. 2.7. Western blot analysis In brief, cells were lysed in RIPA buffer containing 1X protease inhibitor cocktail, and protein concentrations were determined using the Bradford assay (Bio-Rad, Philadelphia, PA). Proteins were separated by SDS-PAGE and then transferred to membranes (Millipore, Bedford, MA) at 90 V for 1.5 h at 4 C. After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies overnight at 4 C then incubated with secondary antibodies for 1 h at room temperature. Membranes were washed again in PBST for three times at room temperature. Protein bands were visualized on X-ray film using an enhanced chemiluminescence detection system. 2.8. Statistical analysis Each experiment was performed independently atleast three with similar results; one representative experiment was presented. All statistical analyses were performed using the SPSS 13.0 statistical software package (SPSS, Chicago, IL, USA). The significance of the data was determined using Student’s t test. Oneway ANOVA was used to compare gene expression in different groups. All the statistical tests were two-sided, and a P value <0.05 was considered significant.
Fig. 1. miR-140-5p is down-regulated in human ovarian cancer and cell lines. (A) miR-140-5p expression levels were down-regulated in ovarian cancer tissues (n = 28) and their adjacent normal tissues (n = 28). miR-140-5p expression was examined by qRT-PCR and normalized to U6 snRNA. (B) Relative miR-140-5p expression levels in ovarian cancer tissues and their corresponding adjacent normal tissues. (C) The relative expression levels of miR-140-5p were determined by qRTPCR in human ovarian cancer cell lines. *p < 0.05, **p < 0.01.
Please cite this article in press as: H. Lan, et al., miR-140-5p inhibits ovarian cancer growth partially by repression of PDGFRA, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.035
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3.2. miR-140-5p suppresses the proliferation of ovarian cancer cells The expression of miR-140-5p was deleted in ovarian cancer tissues and cell lines. To investigate the function of miR-140-5p, we restored it by induced miR-140-5p mimic in SKOV3 and A2780 cells (Fig. 2A). Proliferation of SKOV3 cells was determined by MTT assay at the indicated time (0–4 days). With time longer, difference in the proliferation rate became more significant in SKOV3 and A2780 cells with miR-140-5p restoration compared with control group (Fig. 2B and C). Cell colony formation is anothor method to examine cell proliferation. The data from colony formation assay showed the similar results to MTT assay (Fig. 2D and E). 3.3. miR-140-5p promotes ovarian cancer cell sensitive to cisplatin Chemoresistance is a characteristic of cancer including ovarian cancer. Cisplatin (DDP) resistance is a challenge in the treatment of ovarian cancer. Whether miR-140-5p is related to chemoresistance in ovarian cancer cells, SKOV3 and A2780 cells were transfected with miR-140-5p or its control and then treated with DDP to observe the cell survival rate, it was found that in the cells with miR-140-5p, cell proliferation was inhibited (Fig. 3 A). The result was verified in A2780 cells (Fig. 3B). Hoechst 33342 strainning assay showed that cell apoptosis were also increased in the cells with miR-140-5p compared with the control (Fig. 3C and D).
3
3.4. miR-140-5p down-regulates PDGFRA expression in ovarian cancer cells posttranscriptionally PDGFRA was predicted as a potential target gene of miR-140-5p (Fig. 4 A). To identify whether PDGFRA is the direct target gene of miR-140-5p, plasmids with PDGFRA 30 UTR or mutated PDGFRA 30 UTR were constructed and transfected to SKOV3 cells combined with miR-140-5p. The results showed that the luciferase activity of PDGFRA in SKOV3 cells was much lower than in control cells and luciferase activity of PDGFRA-Mut was rescued in the cells (Fig. 4B). Compared with control, endogenous PDGFRA mRNA levels (Fig. 4C) were down-regulated when SKOV3 cells were transfected with miR-140-5p, so did in A2780 cells. PDGFRA protein was downregulated in SKOV3 and A2780 cells with miR-140-5p mimics (Fig. 4D). The data suggested that PDGFRA was a target gene of miR-140-5p in ovarian cancer cells. 3.5. Relationship of miR-140-5p and PDGFRA in ovarian cancer Above data suggested that miR-140-5p inhibits ovarian cancer cell apoptosis by targeting PDGFRA. In order to investigate PDGFRA expression in ovarian cancer tissues, the data from qRT-PCR showed that PDGFRA was up-regulated in ovarian cancer tissues than their normal tissues (Fig. 5 A and B). The clinic data showed that miR-140-5p was negatively associated with PDGFRA in ovarian cancer tissues (Fig. 5C).
Fig. 2. miR-140-5p inhibits proliferation of ovarian cancer cells. (A) miR-140-5p expression was restored in SKOV3 and A2780 cells. Cells were transfected with miR-140-5p mimics, control miRNA and RNA was isolated for qRT-PCR. miR-140-5p expression was normalized to U6 snRNA. (B) Cell proliferation was examined in SKOV3 cells by MTT assay. (C) Cell proliferation was examined in A2780 cells by MTT assay. (D) Cell proliferation was examined in SKOV3 cells by colony formation assay. (E) Cell proliferation was examined in A2780 cells by colony formation assay. *p < 0.05, **p < 0.01.
Please cite this article in press as: H. Lan, et al., miR-140-5p inhibits ovarian cancer growth partially by repression of PDGFRA, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.035
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Fig. 3. miR-140-5p promotes ovarian cancer cell sensitive to DDP. (A and B) miR-140-5p promotes ovarian cancer cell sensitive to DDP in SKOV3 and A2780 cells. Cells were transfected with miR-140-5p mimics or the control and exposed to DDP for 0, 1, 3, 5 days and then cell survival rate was calculated by MTT assay. (C and D) miR-140-5p promoted apoptosis of SKOV3 and A2780 cells exposed to DDP.
Fig. 4. miR-140-5p down-regulates PDGFRA expression in ovarian cancer cells. (A) PDGFRA (NM_006206) was predicted as a target gene of miR-140-5p. (B) miR-140-5p could inhibit luciferase activity of pGL4-PDGFRA-WT in SKOV3 cells. (C) miR-140-5p inhibited PDGFRA mRNA in SKOV3 and A2780 cells. (D) miR-140-5p inhibited PDGFRA protein in SKOV3 and A2780 cells. **p < 0.01.
Please cite this article in press as: H. Lan, et al., miR-140-5p inhibits ovarian cancer growth partially by repression of PDGFRA, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.035
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Fig. 5. Relationship of miR-140-5p and PDGFRA in ovarian cancer. (A) PDGFRA expression in ovarian cancer tissues was up-regulated compared to their adjacent normal tissues by qRT-PCR assay. (B) Data analysis of (A). (C) miR-140-5p was negatively associated with PDGFRA mRNA in ovarian cancer tissues (r = 0.615, p = 0.0017). **p < 0.01.
4. Discussion Here, we investigated the mechanism and function of miR-1405p via altering its target gene expression in human ovarian cancer. It was shown that miR-140-5p is down-regulated in ovarian cancer tissues and cells and enhance ovarian cancer cell proliferation and reduced apoptosis via increasing PDGFRA expression. It has been reported that miR-140-5p plays a tumor suppressor in non-small cell lung cancer [9], tongue squamous cell carcinoma [10], hepatocellular carcinoma [11]. In lung cancer, miR-140-5p
targeting MMD inhibits cell proliferation via suppressing MAPK signal pathway [12]. In tongue squamous cell carcinoma, miR-1405p attenuates tumor progression and metastasis including migration and invasion via ADAM10 by down-regulation of LAMC1, HDAC7 and PAX6 expression [13]. In hepatocellular carcinoma, miR-140-5p suppressed HCC cell proliferation and HCC metastasis by inhibiting MAPK signaling pathway [14]. In our present study, we found that miR-140-5p expression in ovarian cancer tissues and cell lines were down-regulated compared their normal controls. Also, miR-140-5p is related to cell proliferation suppression,
Please cite this article in press as: H. Lan, et al., miR-140-5p inhibits ovarian cancer growth partially by repression of PDGFRA, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.035
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apoptosis, and promotes sensitivity to DDP. All the data indicated that miR-140-5p may play as a tumor inhibitor. Platelet-derived growth factor receptor alpha (PDGFRa), a tyrosine kinase, is involved in various developmental stages, which plays a crucial role in tumor growth and survival by inhibition of apoptosis signaling [15,16]. In ovarian carcinoma, some studies have examined the biological role of PDGFRa and its efficacy as a therapeutic target. Stimulation of PDGFRa with its ligand, such as PDGF-AA, increases cell proliferation and activation of AKT and MAPK signaling, and neutralizing PDGFRa inhibited these signaling effects [17]. PDGFRA is verified as the direct target gene of miR140-5p in ovarian cancer cells. PDGFR a and b are involved in a variety of physiological and pathological processes, such as cell growth and survival, transformation, migration, vascular permeability, stroma modulation and wound healing. The expression of PDGFR in ovarian tumors was previously reported [18–22]. PDGFRa exhibits different expression profiles in ovarian carcinoma and normal ovarian epithelium. Its expression is often linked to poor prognosis and aggressive tumor characteristics [23,24]. Here, we verified that PDGFRA is the target gene of miR-140-5p and is correlated to it in clinic. Our present study has investigated that a tumor suppressor miR-140-5p, in HCC that can negatively regulate ITGA2 and ITGA5 in vitro and in vivo. Taken together with clinical observations, our finding suggests that miR-140-5p and ITGA2 and ITGA5 are significant biomarkers for metastasis and could be targets for the development of antimetastasis strategy in the treatment of HCC. Acknowledgement The study was supported by the Hainan Key Science and Technology Project, the Hainan Natural Science Foundationfunded project (813209) and the Hainan Health Department research project approval (2012PT-09). References [1] G.M. Mantia-Smaldone, R.P. Edwards, A.M. Vlad, Targeted treatment of recurrent platinum-resistant ovarian cancer: current and emerging therapies, Cancer Manag. Res. 13 (2011) 25–38. [2] E. Lengyel, Ovarian cancer development and metastasis, Am. J. Pathol. 177 (3) (2010) 1053–1064. [3] E.J. Nam, H. Yoon, S.W. Kim, H. Kim, Y.T. Kim, J.H. Kim, et al., MicroRNA expression profiles in serous ovarian carcinoma, Clin. Cancer Res. 14 (2008) 2690–2695. [4] Y. Kinose, K. Sawada, K. Nakamura, T. Kimura, The role of microRNAs in ovarian cancer, Biomed. Res. Int. 2014 (2014) 249–393. [5] B.P. Lewis, I.-H. Shih, M.W. Jones-Rhoades, D.P. Bartel, C.B. Burge, Prediction of mammalian MicroRNA targets, Cell 115 (7) (2003) 787–798.
[6] G. Di Leva, M. Garofalo, C.M. Croce, MicroRNAs in cancer, Annu. Rev. Pathol. 9 (2014) 287–314. [7] N.S. Sokol, Small temporal RNAs in animal development, Curr. Opin. Genet. Dev. 22 (4) (2012) 368–373. [8] K. Ruan, X. Fang, G. Ouyang, MicroRNAs: novel regulators in the hallmarks of human cancer, Cancer Lett. 285 (2) (2009) 116–126. [9] W. Li, F. He, Monocyte to macrophage differentiation-associated (MMD) targeted by miR-140-5p regulates tumor growth in non-small cell lung cancer, Biochem. Biophys. Res. Commun. 450 (July (1)) (2014) 844–850. [10] Y. Kai, W. Peng, W. Ling, H. Jiebing, B. Zhuan, Reciprocal effects between microRNA-140-5p and ADAM10 suppress migration and invasion of human tongue cancer cells, Biochem. Biophys. Res. Commun. 448 (3) (2014) 308–314. [11] S. Hwang, S.K. Park, H.Y. Lee, S.W. Kim, J.S. Lee, E.K. Choi, D. You, C.S. Kim, N. Suh, miR-140-5p suppresses BMP2-mediated osteogenesis in undifferentiated human mesenchymal stem cells, FEBS Lett. 588 (17) (2014) 2957–2963. [12] H. Yang, F. Fang, R. Chang, L. Yang, MicroRNA-140-5p suppresses tumor growth and metastasis by targeting transforming growth factor b receptor 1 and fibroblast growth factor 9 in hepatocellular carcinoma, Hepatology 58 (1) (2013) 205–217. [13] Z. Shi, G. Luo, L. Fu, Z. Fang, X. Wang, X. Li, miR-9 and miR-140-5p target FoxP2 and are regulated as a function of the social context of singing behavior in zebra finches, J. Neurosci. 33 (42) (2013) 16510–16521. [14] T.A. Karlsen, R.B. Jakobsen, T.S. Mikkelsen, J.E. Brinchmann, microRNA140 targets RALA and regulates chondrogenic differentiation of human mesenchymal stem cells by translational enhancement of SOX9 and ACAN, Stem Cells Dev. 23 (3) (2014) 290–304. [15] J. Rakoczy, S.L. Fernandez-Valverde, E.A. Glazov, E.N. Wainwright, T. Sato, S. Takada, A.N. Combes, D.J. Korbie, D. Miller, S.M. Grimmond, M.H. Little, H. Asahara, J.S. Mattick, R.J. Taft, D. Wilhelm, MicroRNAs140-5p/140-3p moduate Leydig cell numbers in the developing mouse testis, Biol. Reprod. 88 (6) (2013) 143. [16] S. Yamamoto, H. Tsuda, M. Takano, T. Kita, K. Kudoh, K. Furuya, S. Tamai, O. Matsubara, Expression of platelet-derived growth factors and their receptors in ovarian clear-cell carcinoma and its putative precursors, Mod. Pathol. 21 (2) (2008) 115–124. [17] R.P. Rocconi, K.S. Matthews, K.J. Kimball, M.G. Conner, A.C. Baker, M.N. Barnes, Expression of c-kit and platelet-derived growth factor receptors in ovarian granulosa cell tumors, Reprod. Sci. 15 (7) (2008) 673–677. [18] C. Yi, L. Li, K. Chen, S. Lin, X. Liu, Expression of c-Kit and PDGFRa in epithelial ovarian tumors and tumor stroma, Oncol. Lett. 3 (2) (2012) 369–372. [19] K. Matsuo, M. Nishimura, K. Komurov, M.M. Shahzad, R. Ali-Fehmi, J.W. Roh, C. Lu, D.D. Cody, P.T. Ram, N. Loizos, R.L. Coleman, A.K. Sood, Platelet-derived growth factor receptor alpha (PDGFRa) targeting and relevant biomarkers in ovarian carcinoma, Gynecol. Oncol. 132 (1) (2014) 166–175. [20] L.S. Sleer, C.C. Taylor, Cell-type localization of platelet-derived growth factors and receptors in the postnatal rat ovary and follicle, Biol. Reprod. 76 (3) (2007) 379–390. [21] L.S. Sleer, C.C. Taylor, Platelet-derived growth factors and receptors in the rat corpus luteum: localization and identification of an effect on luteogenesis, Biol. Reprod. 76 (3) (2007) 391–400. [22] D. Matei, M. Satpathy, L. Cao, Y.C. Lai, H. Nakshatri, D.B. Donner, The plateletderived growth factor receptor alpha is destabilized by geldanamycins in cancer cells, J. Biol. Chem. 282 (1) (2007) 445–453. [23] D. Matei, R.E. Emerson, Y.C. Lai, L.A. Baldridge, J. Rao, C. Yiannoutsos, D.D. Donner, Autocrine activation of PDGFRa promotes the progression of ovarian cancer, Oncogene 25 (14) (2006) 2060–2069. [24] S.P. Wilczynski, Y.Y. Chen, W. Chen, S.B. Howell, J.E. Shively, D.S. Alberts, Expression and mutational analysis of tyrosine kinase receptors c-kit, PDGFRa, and PDGFRb in ovarian cancers, Hum. Pathol. 36 (3) (2005) 242–249.
Please cite this article in press as: H. Lan, et al., miR-140-5p inhibits ovarian cancer growth partially by repression of PDGFRA, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.035
Biomedicine & Pharmacotherapy xxx (2015) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article
miR-140-5p inhibits ovarian cancer growth partially by repression of PDGFRA Hong Lana , Wangsheng Chenb,* , Guoli Hea , Shuying Yanga a b
Department of Gynecology, People's Hospital of Hainan Province, Haikou, China Department of Radiology, People's Hospital of Hainan Province, 19 Xinhua Road, Haikou 570311, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 April 2015 Accepted 26 July 2015
Dysregulation of miRNAs is a common feature in human cancers, and miR-140-5p has been found to be down-regulated in cancer. However its role in ovarian cancer remains unclear. miR-140-5p was underexpressed in HCC tissues and cell lines compared with their normal controls. Additionally, PDGFRA was predicted the target gene of miR-140-5p. PDGFRA was inversely correlated with the expression of miR-140-5p in ovarian cancer cells. Importantly, we demonstrate that the over expression of miR-140-5p significantly inhibits ovarian cancer cell proliferation and induces apoptosis. Our results suggest the existence of a novel miR-140-5p-PDGFRA pathway and indicate that miR-140-5p acts as a tumor suppressor during ovarian carcinogenesis. These results may provide a promising alterative strategy for the therapeutic treatment of ovarian cancer. ã 2015 Published by Elsevier Masson SAS.
Keywords: miR-140-5p PDGFRA Ovarian cancer
1. Introduction Ovarian cancer is the most lethal gynecologic malignancy and the most common cause of death in women [1,2]. Recurrence and metastasis seriously affect the prognosis of ovarian cancer. The advance stage at the time of diagnosis and relapse due to chemoresistance are the major reasons for poor prognosis [3]. Using molecular methods, a number of genes and pathways playing critical roles in the pathogenesis of ovarian cancer have been identified. Many of them may act as the targets of ovarian cancer therapy. microRNAs (miRNAs) are one of them. miRNAs are approximately 22-nucleotide noncoding RNAs with high conservation and involved in gene regulation on posttranscriptional level. miRNAs negatively regulate genes expression by binding to the 30 -untranslated region (UTR) of target mRNAs [4]. A miRNA can regulate many mRNAs which may regulate only a target gene [6]. miRNAs involve in various processes of tumor progression including development, differentiation, metabolism, proliferation, cell cycle, and metastasis [5–7]. They play oncogenes or tumor suppressor. RNAs have been definitively linked to cancer development, including ovarian cancer. There is a report suggested that miR-140-5p is involved in cancer progression [8], but its role in ovarian cancer is still unknown.
* Corresponding author. E-mail address: [email protected] (W. Chen).
In this present study, we set out to investigate mechanism and function of miR-140-5p via altering its target gene expression in human ovarian cancer. Here we report the PDGFRA expression in ovarian cancer is regulated by miR-140-5p. We show that miR-1405p is down-regulated in ovarian cancer cells, resulting in increased PDGFRA expression and enhance ovarian cancer cell proliferation, migration and reduced apoptosis. 2. Materials and methods 2.1. Ovarian cancer samples Cancer tissues and their paired normal tissues were obtained from the patients with ovarian cancer. The patients were from People’s Hospital of Hainan Province (Haikou, China), who did not receive either radiotherapy or chemotherapy. All the patients were diagnosed as ovarian cancer and the samples were histologically confirmed by pathologist. Informed consent was obtained from each patient, and the research protocols were approved by the Ethics Committee of the hospital. 2.2. Cell culture Human ovarian cancer cells SKOV3, A2780, 3AO, OVCAR, COC1, HRA and HO-8910 were obtained primarily from the American Type Culture Collection (ATCC) and were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/mL of penicillin
http://dx.doi.org/10.1016/j.biopha.2015.07.035 0753-3322/ ã 2015 Published by Elsevier Masson SAS.
Please cite this article in press as: H. Lan, et al., miR-140-5p inhibits ovarian cancer growth partially by repression of PDGFRA, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.035
G Model BIOPHA 3722 No. of Pages 6
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and 100 mg/mL of streptomycin. Cells were cultured at 37 C in a humidified atmosphere of 5% CO2.
3. Results 3.1. Loss of miR-140-5p in human ovarian cancer
2.3. MiRNA and siRNA transfection Lentiviral vectors mediated miR-14-5p were constructed and production was performed according to the protocol (Invitrogen). Transduction was performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Lentivirus-mediated silencing of miR-140-5p was examined by qRT-PCR. 2.4. Real-time RT-PCR
To determine miR-140-5p expression in ovarian cancer tissues, we examined its level in 38 samples from the patients by qRT-PCR method. miR-140-5p levels were detected in human ovarian cancer tissues and reduced in ovarian cancer tissues compared with the control (Fig. 1A and B). The result was further verified in seven human ovarian cancer cell lines (SKOV3, A2780, 3AO, OVCAR, COC1, HRA, HO-8910) compared with the normal cells (ISOE80) (Fig. 1C). The results indicated that miR-140-5p was downregulated tissues and cell lines of ovarian cancer.
Total RNA, following the manufacturer’s instructions, was isolated from the cells using Trizol reagent (Invitrogen). To measure mRNA expression, real-time RT-PCR was performed using a sequence detector (ABI-Prism, Applied Biosystems). Primers were purchased from Invitrogen. The relative expression levels were calculated by comparing Ct values of the samples with those of the reference, all data normalized to the internal control GAPDH. 2.5. MTT method Cell proliferation was assayed in ovarian cancer cells with miRNA transfection and the growth curve was delineated. Briefly, the cells were incubated at 37 C, 5% CO2 until cells cover the bottom of the well (a flat-bottom 96-well plate), and then the cells were cultured. Cells were treated with MTT solution (5 mg/ml, 0.5% MTT). The cells were continued to culture for 4 h and 150 ml DMSO was added. The ELISA reader was used to measure the absorbance at 570 nm. 2.6. Colony formation assay Ovarian cancer cells were transfected with miR-485-5p or other plasmids and were seeded to 6-well plates (200 cells/well) followed by incubation at 37 C in an environment with saturated humidity and 5% CO2 for 24 h. Non-adherent cells were removed. After culture for 10–14 days, colonies were present and counted. 2.7. Western blot analysis In brief, cells were lysed in RIPA buffer containing 1X protease inhibitor cocktail, and protein concentrations were determined using the Bradford assay (Bio-Rad, Philadelphia, PA). Proteins were separated by SDS-PAGE and then transferred to membranes (Millipore, Bedford, MA) at 90 V for 1.5 h at 4 C. After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies overnight at 4 C then incubated with secondary antibodies for 1 h at room temperature. Membranes were washed again in PBST for three times at room temperature. Protein bands were visualized on X-ray film using an enhanced chemiluminescence detection system. 2.8. Statistical analysis Each experiment was performed independently atleast three with similar results; one representative experiment was presented. All statistical analyses were performed using the SPSS 13.0 statistical software package (SPSS, Chicago, IL, USA). The significance of the data was determined using Student’s t test. Oneway ANOVA was used to compare gene expression in different groups. All the statistical tests were two-sided, and a P value <0.05 was considered significant.
Fig. 1. miR-140-5p is down-regulated in human ovarian cancer and cell lines. (A) miR-140-5p expression levels were down-regulated in ovarian cancer tissues (n = 28) and their adjacent normal tissues (n = 28). miR-140-5p expression was examined by qRT-PCR and normalized to U6 snRNA. (B) Relative miR-140-5p expression levels in ovarian cancer tissues and their corresponding adjacent normal tissues. (C) The relative expression levels of miR-140-5p were determined by qRTPCR in human ovarian cancer cell lines. *p < 0.05, **p < 0.01.
Please cite this article in press as: H. Lan, et al., miR-140-5p inhibits ovarian cancer growth partially by repression of PDGFRA, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.035
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3.2. miR-140-5p suppresses the proliferation of ovarian cancer cells The expression of miR-140-5p was deleted in ovarian cancer tissues and cell lines. To investigate the function of miR-140-5p, we restored it by induced miR-140-5p mimic in SKOV3 and A2780 cells (Fig. 2A). Proliferation of SKOV3 cells was determined by MTT assay at the indicated time (0–4 days). With time longer, difference in the proliferation rate became more significant in SKOV3 and A2780 cells with miR-140-5p restoration compared with control group (Fig. 2B and C). Cell colony formation is anothor method to examine cell proliferation. The data from colony formation assay showed the similar results to MTT assay (Fig. 2D and E). 3.3. miR-140-5p promotes ovarian cancer cell sensitive to cisplatin Chemoresistance is a characteristic of cancer including ovarian cancer. Cisplatin (DDP) resistance is a challenge in the treatment of ovarian cancer. Whether miR-140-5p is related to chemoresistance in ovarian cancer cells, SKOV3 and A2780 cells were transfected with miR-140-5p or its control and then treated with DDP to observe the cell survival rate, it was found that in the cells with miR-140-5p, cell proliferation was inhibited (Fig. 3 A). The result was verified in A2780 cells (Fig. 3B). Hoechst 33342 strainning assay showed that cell apoptosis were also increased in the cells with miR-140-5p compared with the control (Fig. 3C and D).
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3.4. miR-140-5p down-regulates PDGFRA expression in ovarian cancer cells posttranscriptionally PDGFRA was predicted as a potential target gene of miR-140-5p (Fig. 4 A). To identify whether PDGFRA is the direct target gene of miR-140-5p, plasmids with PDGFRA 30 UTR or mutated PDGFRA 30 UTR were constructed and transfected to SKOV3 cells combined with miR-140-5p. The results showed that the luciferase activity of PDGFRA in SKOV3 cells was much lower than in control cells and luciferase activity of PDGFRA-Mut was rescued in the cells (Fig. 4B). Compared with control, endogenous PDGFRA mRNA levels (Fig. 4C) were down-regulated when SKOV3 cells were transfected with miR-140-5p, so did in A2780 cells. PDGFRA protein was downregulated in SKOV3 and A2780 cells with miR-140-5p mimics (Fig. 4D). The data suggested that PDGFRA was a target gene of miR-140-5p in ovarian cancer cells. 3.5. Relationship of miR-140-5p and PDGFRA in ovarian cancer Above data suggested that miR-140-5p inhibits ovarian cancer cell apoptosis by targeting PDGFRA. In order to investigate PDGFRA expression in ovarian cancer tissues, the data from qRT-PCR showed that PDGFRA was up-regulated in ovarian cancer tissues than their normal tissues (Fig. 5 A and B). The clinic data showed that miR-140-5p was negatively associated with PDGFRA in ovarian cancer tissues (Fig. 5C).
Fig. 2. miR-140-5p inhibits proliferation of ovarian cancer cells. (A) miR-140-5p expression was restored in SKOV3 and A2780 cells. Cells were transfected with miR-140-5p mimics, control miRNA and RNA was isolated for qRT-PCR. miR-140-5p expression was normalized to U6 snRNA. (B) Cell proliferation was examined in SKOV3 cells by MTT assay. (C) Cell proliferation was examined in A2780 cells by MTT assay. (D) Cell proliferation was examined in SKOV3 cells by colony formation assay. (E) Cell proliferation was examined in A2780 cells by colony formation assay. *p < 0.05, **p < 0.01.
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Fig. 3. miR-140-5p promotes ovarian cancer cell sensitive to DDP. (A and B) miR-140-5p promotes ovarian cancer cell sensitive to DDP in SKOV3 and A2780 cells. Cells were transfected with miR-140-5p mimics or the control and exposed to DDP for 0, 1, 3, 5 days and then cell survival rate was calculated by MTT assay. (C and D) miR-140-5p promoted apoptosis of SKOV3 and A2780 cells exposed to DDP.
Fig. 4. miR-140-5p down-regulates PDGFRA expression in ovarian cancer cells. (A) PDGFRA (NM_006206) was predicted as a target gene of miR-140-5p. (B) miR-140-5p could inhibit luciferase activity of pGL4-PDGFRA-WT in SKOV3 cells. (C) miR-140-5p inhibited PDGFRA mRNA in SKOV3 and A2780 cells. (D) miR-140-5p inhibited PDGFRA protein in SKOV3 and A2780 cells. **p < 0.01.
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Fig. 5. Relationship of miR-140-5p and PDGFRA in ovarian cancer. (A) PDGFRA expression in ovarian cancer tissues was up-regulated compared to their adjacent normal tissues by qRT-PCR assay. (B) Data analysis of (A). (C) miR-140-5p was negatively associated with PDGFRA mRNA in ovarian cancer tissues (r = 0.615, p = 0.0017). **p < 0.01.
4. Discussion Here, we investigated the mechanism and function of miR-1405p via altering its target gene expression in human ovarian cancer. It was shown that miR-140-5p is down-regulated in ovarian cancer tissues and cells and enhance ovarian cancer cell proliferation and reduced apoptosis via increasing PDGFRA expression. It has been reported that miR-140-5p plays a tumor suppressor in non-small cell lung cancer [9], tongue squamous cell carcinoma [10], hepatocellular carcinoma [11]. In lung cancer, miR-140-5p
targeting MMD inhibits cell proliferation via suppressing MAPK signal pathway [12]. In tongue squamous cell carcinoma, miR-1405p attenuates tumor progression and metastasis including migration and invasion via ADAM10 by down-regulation of LAMC1, HDAC7 and PAX6 expression [13]. In hepatocellular carcinoma, miR-140-5p suppressed HCC cell proliferation and HCC metastasis by inhibiting MAPK signaling pathway [14]. In our present study, we found that miR-140-5p expression in ovarian cancer tissues and cell lines were down-regulated compared their normal controls. Also, miR-140-5p is related to cell proliferation suppression,
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apoptosis, and promotes sensitivity to DDP. All the data indicated that miR-140-5p may play as a tumor inhibitor. Platelet-derived growth factor receptor alpha (PDGFRa), a tyrosine kinase, is involved in various developmental stages, which plays a crucial role in tumor growth and survival by inhibition of apoptosis signaling [15,16]. In ovarian carcinoma, some studies have examined the biological role of PDGFRa and its efficacy as a therapeutic target. Stimulation of PDGFRa with its ligand, such as PDGF-AA, increases cell proliferation and activation of AKT and MAPK signaling, and neutralizing PDGFRa inhibited these signaling effects [17]. PDGFRA is verified as the direct target gene of miR140-5p in ovarian cancer cells. PDGFR a and b are involved in a variety of physiological and pathological processes, such as cell growth and survival, transformation, migration, vascular permeability, stroma modulation and wound healing. The expression of PDGFR in ovarian tumors was previously reported [18–22]. PDGFRa exhibits different expression profiles in ovarian carcinoma and normal ovarian epithelium. Its expression is often linked to poor prognosis and aggressive tumor characteristics [23,24]. Here, we verified that PDGFRA is the target gene of miR-140-5p and is correlated to it in clinic. Our present study has investigated that a tumor suppressor miR-140-5p, in HCC that can negatively regulate ITGA2 and ITGA5 in vitro and in vivo. Taken together with clinical observations, our finding suggests that miR-140-5p and ITGA2 and ITGA5 are significant biomarkers for metastasis and could be targets for the development of antimetastasis strategy in the treatment of HCC. Acknowledgement The study was supported by the Hainan Key Science and Technology Project, the Hainan Natural Science Foundationfunded project (813209) and the Hainan Health Department research project approval (2012PT-09). References [1] G.M. Mantia-Smaldone, R.P. Edwards, A.M. Vlad, Targeted treatment of recurrent platinum-resistant ovarian cancer: current and emerging therapies, Cancer Manag. Res. 13 (2011) 25–38. [2] E. Lengyel, Ovarian cancer development and metastasis, Am. J. Pathol. 177 (3) (2010) 1053–1064. [3] E.J. Nam, H. Yoon, S.W. Kim, H. Kim, Y.T. Kim, J.H. Kim, et al., MicroRNA expression profiles in serous ovarian carcinoma, Clin. Cancer Res. 14 (2008) 2690–2695. [4] Y. Kinose, K. Sawada, K. Nakamura, T. Kimura, The role of microRNAs in ovarian cancer, Biomed. Res. Int. 2014 (2014) 249–393. [5] B.P. Lewis, I.-H. Shih, M.W. Jones-Rhoades, D.P. Bartel, C.B. Burge, Prediction of mammalian MicroRNA targets, Cell 115 (7) (2003) 787–798.
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