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MicroRNA-27a regulates the proliferation, chemosensitivity and invasion of human ovarian cancer cell lines by targeting Cullin 5
Archives of Biochemistry and Biophysics 668 (2019) 9–15
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
MicroRNA-27a regulates the proliferation, chemosensitivity and invasion of human ovarian cancer cell lines by targeting Cullin 5
T
Lihui Sia, Yan Jiaa, Ruixin Linb, Wenwen Jiana, Qing Yua, Shuli Yanga,∗ a b
Department of Gynecology and Obstetrics, The Second Hospital of Jilin University, Changchun, Jilin, 130041, China Department of Hepatopancreatobiliary Surgery, The Second Hospital of Jilin University, Changchun, Jilin, 130041, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Ovarian cancer MicroRNA Cell cycle arrest Invasion Proliferation
Reports suggest that microRNAs have implications in the development of several diseases including cancer. It is therefore believed that miRs may act as therapeutic targets for cancer treatment. The treatment of ovarian cancer is mainly obstructed by lack of biomarkers and efficient drug targets. Against this backdrop, this study was undertaken to unveil the therapeutic implications of miR-27a in ovarian cancer. The results showed that the expression of miR-27a was significantly elevated in ovarian cancer tissues and cell lines. Inhibition of miR-27a expression resulted in the decrease of proliferation rate and colony formation potential of the SK-OV-3 and OVACAR-3 cells via G2/M arrest. The miR-27a inhibition triggered G2/M arrest of SK-OV-3 and OVACAR-3 cells was accompanied with depletion of cyclin A and B1 expression levels. TargetScan analysis together with dual reporter assay revealed that miR-27a exerts its effects via modulation of CUL5 expression. The CUL5 was shown to be suppressed in the ovarian cancer tissues and cell lines and suppression of miR-27a expression caused upregulation of CUL5 expression. Overexpression of CUL5 caused inhibition of SK-OV-3 and OVACAR-3 cell proliferation via induction of G2/M arrest, similar to that of miR-27a inhibition. Interestingly, CUL5 overexpression reversed the effects of miR-27a inhibition on the viability of SK-OV-3 cells. Finally, the suppression of miR-27a could enhance the chemosensitivity of the SK-OV-3 cells to cisplatin and docetaxel anticancer drugs and also decreased their migration and invasion. The findings of this study revealed that miR-27a suppression inhibits the growth, chemosensitivity and invasion of ovarian cancer and may prove beneficial in the ovarian cancer management.
1. Introduction Accumulating evidences have indicated the involvement of microRNAs (miRs) in almost all types of cancers and malignancies [1]. The advent of microarray and next generation sequencing has greatly allowed to understand the relation between miRs and cancer [2]. The miRs are generally 22 nucleotides in length non-coding RNAs molecules that regulate the expression of genes post-transcriptionally [3]. They are evolutionarily conserved and perform their functions in almost all biological processes [4]. It has been reported that more than 2500 miRs have been identified so far in human and the number is still growing [5]. The expression of miRs has been shown to be dysregulated in cancers. Generally the expression of miRs is repressed in cancerous tissues, however, may miRs have also been reported to be overexpressed in cancer [6]. It has been reported that therapeutic application of miRs may prove an amazing and essential strategy to interfere with molecular mechanisms underlying cancer [7]. Therefore, several
∗
miRs have been studied therapeutic potential and miR-27a is one such candidate. The miR-27a has been shown to promote the growth and development of gastric adenocarcinoma, colon and pancreatic cancer [8–10]. It has been shown to target MAP2K4 to suppress the proliferation and metastasis of osteosarcoma [11]. Moreover, several anticancerous molecules have been shown to decrease the survival of cancer cells by targeting miR-27a expression. For example genistein and Betulinic acid inhibit the proliferation of human uveal melanoma and ovarian cancer cells respectively via suppression of miR-27a expression [12,13]. Nonetheless, therapeutic implications of miR-27a have not been thoroughly investigated in ovarian cancer. Ovarian cancer is one of the common gynaecological malignancies in women across the globe. Accounting for 2.5% of all malignancies in women, ovarian cancer is responsible for 5% of all cancer related deaths in women [14]. Although there has been improvement in ovarian cancer treatment but the clinical outcome is still far from decent [15]. It has been reported that in United states. Approximately more than twenty
Corresponding author. E-mail address: [email protected] (S. Yang).
https://doi.org/10.1016/j.abb.2019.04.009 Received 1 February 2019; Received in revised form 13 April 2019; Accepted 27 April 2019 Available online 29 April 2019 0003-9861/ © 2019 Elsevier Inc. All rights reserved.
Archives of Biochemistry and Biophysics 668 (2019) 9–15
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2.5. Flow cytometric cell cycle analysis
two thousand new ovarian cancer cases and fourteen thousand ovarian cancer deaths are recorded annually [16]. The late diagnosis and dearth of therapeutic targets impose hurdles in the treatment of ovarian cancer. Improvement of prevention through early detection and identification of the therapeutic targets may prove beneficial to curb ovarian cancer related mortalities [17]. The present study explored the therapeutic potential of miR-27a in ovarian cancer and herein we report that miR-27a regulates the growth, chemosensitivity and invasion of the human ovarian cancer by targeting Cullin 5 (CUL5). To sum up, the findings of the present study reveal that miR-27a regulated the growth, chemosensitivity and invasion of ovarian cancer and may prove essential in its treatment.
After transfection the SK-OV-3 cells were subjected to culturing for 24 h at 37 ○C for. The SK-OV-3 cells were harvested and the PBS washed. Afterwards, the SK-OV-3 cells were stained with propidium Iodide (PI) and the distributed of the cells in cell cycle phases was assessed by FACS flow cytometer. 2.6. Dual-luciferase reporter assay The miR-27a target was identified by TargetScan online software (http://www.targetscan.org). The miR-27a inhibitor or miR-NC were co-transfected with Plasmid pGL3-CUL5-3′-UTR-WT or pGL3-CUL5-3′UTR-MUT into SK-OV-3 cells. Dual-luciferase reporter assay (Promega) was carried out at 48 h after transfection. Renilla luciferase used for normalization.
2. Materials and methods 2.1. Tissue samples and cell lines
2.7. Chemosensitivity assay
The snap-frozen malignant ovarian tissues (C1 to C8) and adjacent normal tissues (N1 to N8) were collected from Department of Gynecology and Obstetrics, The Second Hospital of Jilin University, Changchun, Jilin, China, after obtaining informed consent from the patients. The Histological classification of all the tissues is presented in Supplementary Table S1. The study was approved by the Research ethics committee of the institute under approval number HJU675/2A/ 2018. The human ovarian cell lines (Caov-4, SK-OV-3, OVACAR-3 and UWB1.289) and non-cancerous ovarian surface epithelial cell line (SV40, DoTc24510, GH329 and UACC-3247) were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) containing penicillin (100 U/mL), streptomycin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal bovine serum (FBS; Gibco) at 37 °C in 5% CO2.
To determine the effects of miR-27a inhibition on the sensitivity of the SK-OV-3 cells to docetaxel and cisplatin, the SK-OV-3 cells were transfected miR-NC, or miR-27a inhibitor, or treated with docetaxel (0.25 μM) or cisplatin (1 μM) or transfected with miR-27a inhibitor plus treated with docetaxel (0.25 μM) or transfected with miR-27a inhibitor plus treated with cisplatin (1 μM). All these groups of SK-OV-3 cells were then subjected to CCK-8 assay as mentioned above. 2.8. Transwell assay The effects of miR-27a suppression on the invasion ability of SK-OV3 cells was determined by transwell chambers (8 mm pore size, Corning, NY, USA) with Matrigel (Millipore, Billerica, USA) The SK-OV-3 cells were transfected with miR-27a inhibitor and miR-NC and around 200 ml cell cultures were placed onto the upper chambers and only medium was placed in the bottom wells. After 24 h of incubation, the cells were removed from the upper chamber and the cells that invaded via the chambers were subjected to fixation with methyl alcohol and subsequently stained with crystal violet. Inverted microscope was used to count the number of invaded cells at 200× magnification.
2.2. Expression analysis The TRIzol reagent (Invitrogen) was used for the extract of RNA from the tissues and cell lines. This was followed by purification of the RNA by RNeasy Mini Kit (Qiagen). The complementary DNA was then synthesized with the help of miScript Reverse Transcription Kit (Qiagen). Afterwards the cDNA was amplified by using SYBR Premix Ex Taq™ (TaKaRa, Otsu, Shiga, Japan). The expression was estimated by 2−ΔΔCt method and actin was used as an internal control. Heat map was generated by the online heatmaper software (http://www2. heatmapper.ca/).
2.9. Wound-healing assay After 24 h of miR-27a inhibitor and miR-NC of transfection into SKOV-3 cells, the medium was removed and cells were subjected to PBS washing. A sterile pipette tip was employed to scratch a wound in each well and cells were subjected washing again and a picture was taken. The plates were subjected to culturing at 24 h and a picture was taken again under an inverted microscope (Leica, Germany).
2.3. Cell transfection The miR-27a Inhibitor and miR-NC were synthesized by RiboBio (Guangzhou, China). The transfection was then carried out by the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) as per the manufacturer's instructions. As the SK-OV-3 cells reached 80%, the appropriate concentrations of miR-27a inhibitor or miR-NC was transfected into SK-OV-3 cells.
2.10. Western blotting After culturing the SK-OV-3 cells for 24 cells, the cells were harvested by centrifugation and subjected to washing with ice-cold PBS. The cell pellet was then suspended in RIPA lysis buffer. The protein content of cell lysates was determined by Bradford assay. From each sample 30 μg of protein was on SDS-PAGE before being shifted to polyvinylidene fluoride membrane. The membranes were then subjected to treatment with TBS and then exposed to primary antibodies at 4 ○C. Thereafter, the cells were treated with appropriate secondary antibodies and the proteins of interest were visualised by enhanced chemiluminescence reagent.
2.4. Cell viability assay The CCK-8 assay was used for the determination of the cell viability. In brief, the transfected SK-OV-3 cells were cultured in 96-well plates and incubated at 37○C for 24 h and subjected to treatment 10 microtiters of CCK-8 solution. The cells were then again subjected to incubation for 2 h at 37○C in a humidifier (5% CO2/95% O2). OD450 was taken at different time intervals (0, 12, 24, 48 and 96 h) with the help of a microplate reader. The effect of miR-27a suppression on the colony formation of the SK-OV-3 cells was determined by colony formation assay [18].
2.11. Statistical analysis Data are shown as mean ± SD. Statistical analysis was done using Students t-test with GraphPad prism 7 software. Values of p < 0.05 10
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Fig. 1. Expression of miR-27a is upregulated in ovarian cancer tissues and cell lines (A) Expression of different miRs in normal (N1 to N8) and ovarian cancer (C1 to C8) tissues (B) Expression of miR-27a in normal and ovarian cancer tissues (C) Expression of miR-27a in normal and ovarian cancer cell lines. The experiments represent mean of three biological replicates ± SD (*P < 0.01).
Fig. 2. Suppression of miR-27a triggers G2/M arrest of SK-OV-3 and OVACAR-3 cells (A) Expression of miR-27a in miR-NC and miR-27a inhibitor transfected SK-OV3 and OVACAR-3 cells (B) Cell viability of miR-NC and miR-27a inhibitor transfected SK-OV-3 and OVACAR-3 cells (C) Colony formation of miR-NC and miR-27a inhibitor transfected SK-OV-3 and OVACAR-3 cells (D) Cell cycle analysis of miR-NC and miR-27a inhibitor transfected SK-OV-3 and OVACAR-3 cells (E) Protein expression of cell cycle related proteins in miR-NC and miR-27a inhibitor transfected SK-OV-3 and OVACAR-3 cells. The experiments represent mean of three biological replicates ± SD (*P < 0.01).
tissues and eight normal adjacent tissues by qRT-PCR and heat map was generated. The results showed differential expression of these miRs (Fig. 1A). However miR-27a was found to be significantly overexpressed in the ovarian cancer tissues by upto 5 fold relative to the normal adjacent tissues (Fig. 1A and B). The miR-27a expression was also determined in four normal and four ovarian cancer cells lines and it was found that expression of miR-27a was significantly upregulated in
were taken as significant difference. 3. Results 3.1. miR-27a is upregulated in ovarian cancer tissues and cell lines The expression of 11 miRs was examined in eight ovarian cancer 11
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Fig. 3. miR-27a targets CUL-5 in ovarian cancer (A) TargetScan analysis showing CUL5 as the target of miR-27a (B) Dual luciferase assay (C) Expression of CUL5 in ovarian cancer and adjacent normal ovarian tissues (D) qRT-PCR expression of CUL5 in normal and ovarian cancer tissues (E) Western blot analysis showing expression of CUL5 in normal and ovarian cancer cell lines (E) Western blot analysis showing expression of CUL5 in miR-NC or miR-27a transfected SK-OV-3 cells. The experiments represent mean of three biological replicates ± SD (*P < 0.01).
all the ovarian cancer cell lines with fold upregulation ranging between 3.2 and 4.5 relative to normal cell lines (Fig. 1C). It was further found that the SK-OV-3 cell line showed the highest expression of miR-27a and was therefore selected for further investigation.
expression levels of cyclin A and B1 and upregulation of p21 and p27 in SK-OV-3 and OVACAR-3 cells (Fig. 2E).
3.2. Suppression of miR-27a inhibits the proliferation of ovarian cancer cells via G2/M arrest
The bioinformatic analysis of miR-27a with TargetScan online software showed that CUL5 could be the potential target of miR-27a F (Fig. 3A). The dual reporter assay further confirmed that miR-27a targets CUL5 (Fig. 3B). Next, CUL5 expression profile was determined in eight normal and eight ovarian cancer tissues and it was found that CUL5 is suppressed in the ovarian cancer cells (Fig. 3C). The qRT-PCR analysis revealed very low expression CUL5 (upto 8 fold downregulation) in ovarian cancer cells (Fig. 3D). The Western blot analysis also showed revealed similar results (Fig. 3E). Nonetheless, the suppression of miR-27a expression resulted in considerable depletion in the expression of CUL5 (Fig. 3F). To ascertain the effects of CUL5 in SK-OV3 cells, the expression of CUL5 was upregulated in the SK-OV-3 and OVACAR-3 cells and the results showed that ectopic expression of CUL5 resulted in the decline of proliferation and colony formation of the SK-
3.3. miR-27a targets CUL5 in ovarian cancer cells
Next to ascertain the miR-27a function in ovarian cancer, its expression was suppressed in the SK-OV-3 and OVACAR-3 cells (Fig. 2A). The results revealed that inhibition of miR-27a expression resulted in the reduction in the proliferation rate and colony formation of the SKOV-3 and OVACAR-3 cells (Fig. 2B and C). Cell cycle profile analysis revealed that suppression of the miR-27a expression caused the arrest of SK-OV-3 and OVACAR-3 cells at G2/M check point. The G2/M cells increased from 18.4% in miR-NC to around 36% in miR-27a inhibitor transfected SK-OV-3 cells. While as in case of OVACAR-3 cells the G2/M phase cells increased from 22.2 to 52.7% upon miR-27a inhibition (Fig. 2D). The inhibition of miR-27a also caused depletion of the 12
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Fig. 4. Overexpression of CUL5 triggers G2/M arrest of SK-OV-3 and OVACAR-3 cells (A) Expression of CUL5 in NC and pcDNA-CUL5 transfected SK-OV-3and OVACAR-3 cells (B) Cell viability of NC and pcDNA-CUL5 transfected SK-OV-3 and OVACAR-3 cells (C) Colony formation of NC and pcDNA-CUL5 transfected SK-OV3 and OVACAR-3 cells (D) Cell cycle analysis of NC and pcDNA-CUL5 transfected SK-OV-3 and OVACAR-3 cells (E) Protein expression of cell cycle related proteins in NC and pcDNA-CUL5 transfected SK-OV-3 and OVACAR-3 cells (E) Effect of CUL5 silencing on the viability of miR-27a transfected SK-OV3 cells. The experiments represent mean of three biological replicates ± SD (*P < 0.01).
invasion of the SK-OV-3 cells by wound heal and transwell assays respectively. The results showed that miR-27a inhibition caused significant decline in the migration and invasion of the ovarian cancer cells (Fig. 5C and D).
OV-3 and OVACAR-3 cells via induction of G2/M cell cycle arrest (Fig. 4A–D). This was also accompanied by the downregulation of cyclin A and B1 and upregulation of p21 and p27 in SK-OV-3 and OVACAR-3 cells (Fig. 4E). These results were similar to that of miR-27a inhibition. Interestingly, the silencing of CUL5 in the miR-27a inhibitor transfected SK-OV3- cells could almost completely abolish the growth inhibitory effects of miR-27a suppression (Fig. 4F).
4. Discussion The treatment of ovarian cancer is mainly obstructed by dearth of therapeutic targets and biomarkers for early detection [14]. It has been reported that miRNAs modulate the expression of majority of the human genes that are involved in a wide array of biological processes [6]. Because of the implications of miRNAs in cellular and physiological processes, previous studies have revealed the potential of miRNAs as therapeutic targets [5]. This study explored the therapeutic implications of miR-27a in ovarian cancer. In this study, the expression of eleven different miRs was evaluated in normal and ovarian cancer tissues. Although, many of these miRs exhibited differential expression but the expression of miR-27a was upregulated in all the ovarian cancer tissues and therefore miR-27a was selected as the candidate miR for
3.4. Suppression of miR-27a enhances chemosensitivity and inhibits migration and invasion of SK-OV-3 cells The effects of miR-27a inhibition on the chemosensitivity of the SKOV-3 ovarian cancer cells. Henceforth, the miR-27a inhibitor transfected SK-OV-3 cells were treated with 0.25 μM concentration of docetaxel or 1 μM cisplatin and the cell viability was monitored at different time intervals by CCK-8 assay. The results showed that suppression of miR-27a caused enhancement in the chemosensitivity of SK-OV-3 cells to both docetaxel and cisplatin (Fig. 5A and B). Next the effects of miR-27a were examined on the migration and 13
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Fig. 5. Effect of miR-27a inhibition on chemosensitivity of SKOV-3 cells to (A) docetaxel and (B) cisplatin. Effect of miR-27a inhibition on (C) Cell migration and (D) cell invasion of the SK-OV3 cells. The experiments represent mean of three biological replicates ± SD (*P < 0.01).
invasion of osteosarcoma cells and chemosensitivity of bladder cancer cells [11, 26]. To sum up, miR-27a acts as an oncogene in ovarian cancer and regulates their proliferation, invasion and chemosensitivity by targeting CUL5. Therefore, miR-27a may prove beneficial in ovarian cancer treatment and drugs can be developed to target its expression.
further investigation. The qRT-PCR analysis showed miR-27a to be upregulated in both ovarian cancer tissues and cell lines. The results are also in agreement with studies carried out earlier wherein miR-27 has been reported to be overexpressed in cancer cells. For example, miR27a has been shown to be overexpressed in prostate cancer, pancreatic cancer and gastric cancer [10,19]. Next, miR-27a expression was inhibited in the SK-OV-3 and OVACAR-3 cells to explore its role in ovarian cancer. Interestingly it was revealed that miR-27a inhibition in the ovarian cancer cells results in the decline of their cell viability and colony formation via induction of G2/M cell cycle arrest. These findings are also supported by a study wherein miR-27a has been shown to prompt the arrest of the breast cancer cells at the G2/M check point [20]. The G2/M arrest triggered by the miR-27a inhibition caused depletion of cyclin A and B1 and enhancement in the expression p27 and p21. Previous studies have shown that depletion of cyclin A and B1and upregulation of p27 and p21 results in the G2/M arrest of the cells during cell division [21]. Bioinformatic analysis together with dual luciferase assay showed that miR-27a exerts its effects by modulating CUL5 expression. CUL5 has been showed to be involved in the regulation of migration of neurons as well as the angiogenesis of granulocytes [22]. Additionally CUL5 plays part in the metastasis of cervical cancer [23]. Herein, it was found that overexpression of CUL5 caused suppression of SK-OV-3 cell growth via induction of G2/M arrest, similar to that of miR-27a inhibition. This is also supported by a study wherein CUL5 has been shown to play a role in cell cycle [24]. Interestingly, the silencing of CUL5 could almost abolish the growth inhibitory effects of miR-27a knockdown on the proliferation of SK-OV3 cells. The inhibition of miR-27a also enhanced the chemosensitivity of SK-OV-3 to docetaxel and cisplatin and suppressed their migration and invasion. These observations are also supported by previous studies wherein miR-27a has been reported to regulate the migration and
Conflicts of interest The authors declare that there are no conflicts of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.abb.2019.04.009. References [1] R.W. Carthew, E.J. Sontheimer, Origins and mechanisms of miRNAs and siRNAs, Cell 136 (4) (2009) 642–655. [2] O. Slaby, M. Svoboda, P. Fabian, T. Smerdova, D. Knoflickova, M. Bednarikova, R. Nenutil, R. Vyzula, Altered expression of miR-21, miR-31, miR-24 and miR-145 is related to clinicopathologic features of colorectal cancer, Oncology 72 (5–6) (2007) 397–402. [3] J. Tao, D. Wu, B. Xu, W. Qian, P. Li, Q. Lu, C. Yin, W. Zhang, microRNA-133 inhibits cell proliferation, migration and invasion in prostate cancer cells by targeting the epidermal growth factor receptor, Oncol. Rep. 27 (6) (2012 Jun 1) 1967–1975. [4] S. Wang, Q. Li, K. Wang, Y. Dai, J. Yang, S. Xue, F. Han, Q. Zhang, J. Liu, W. Wu, Decreased expression of microRNA-31 associates with aggressive tumor progression and poor prognosis in patients with bladder cancer, Clin. Transl. Oncol. 15 (10) (2013 Oct 1) 849–854. [5] A. Ramassone, S. Pagotto, A. Veronese, R. Visone, Epigenetics and microRNAs in cancer, Int. J. Mol. Sci. 19 (2) (2018 Feb 3) 459. [6] I. Vannini, F. Fanini, M. Fabbri, Emerging roles of microRNAs in cancer, Curr. Opin. Genet. Dev. 48 (2018 Feb) 128–133. [7] N. Bastos, S.A. Melo, Quantitative analysis of precursors MicroRNAs and their respective mature MicroRNAs in cancer exosomes overtime, InMicroRNA Protocols,
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[16] K.B. Mathieu, D.G. Bedi, S.L. Thrower, A. Qayyum, R.C. Bast Jr., Screening for ovarian cancer: imaging challenges and opportunities for improvement, Ultrasound Obstet. Gynecol. 51 (3) (2018 Mar) 293–303. [17] C. La Vecchia, Ovarian cancer: epidemiology and risk factors, Eur. J. Cancer Prev. 26 (1) (2017 Jan 1) 55–62. [18] Z.F. Gu, Z.T. Zhang, J.Y. Wang, B.B. Xu, Icariin exerts inhibitory effects on the growth and metastasis of KYSE70 human esophageal carcinoma cells via PI3K/AKT and STAT3 pathways, Environ. Toxicol. Pharmacol. 54 (2017 Sep 1) 7–13. [19] X. Zhao, L. Yang, J. Hu, Down-regulation of miR-27a might inhibit proliferation and drug resistance of gastric cancer cells, J. Exp. Clin. Cancer Res. 30 (1) (2011 Dec) 55. [20] S.U. Mertens-Talcott, S. Chintharlapalli, X. Li, S. Safe, The oncogenic microRNA-27a targets genes that regulate specificity protein transcription factors and the G2-M checkpoint in MDA-MB-231 breast cancer cells, Cancer Res. 67 (22) (2007 Nov 15) 11001–11011. [21] F. Hua, C.H. Li, X.G. Chen, X.P. Liu, Daidzein exerts anticancer activity towards SKOV3 human ovarian cancer cells by inducing apoptosis and cell cycle arrest, and inhibiting the Raf/MEK/ERK cascade, Int. J. Mol. Med. 41 (6) (2018 Jun 1) 3485–3492. [22] N.A. Forrester, G.G. Sedgwick, A. Thomas, A.N. Blackford, T. Speiseder, T. Dobner, P.J. Byrd, G.S. Stewart, A.S. Turnell, R.J. Grand, Serotype-specific inactivation of the cellular DNA damage response during adenovirus infection, J. Virol. 85 (5) (2011 Mar 1) 2201–2211. [23] X. Dong, B. Lv, Y. Li, Q. Cheng, C. Su, G. Yin, MiR-143 regulates the proliferation and migration of osteosarcoma cells through targeting MAPK7, Arch. Biochem. Biophys. 630 (2017 Sep 15) 47–53. [24] F. Okumura, A. Joo-Okumura, K. Nakatsukasa, T. Kamura, The role of cullin 5containing ubiquitin ligases, Cell Div. 11 (1) (2016 Mar) 1.
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Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
MicroRNA-27a regulates the proliferation, chemosensitivity and invasion of human ovarian cancer cell lines by targeting Cullin 5
T
Lihui Sia, Yan Jiaa, Ruixin Linb, Wenwen Jiana, Qing Yua, Shuli Yanga,∗ a b
Department of Gynecology and Obstetrics, The Second Hospital of Jilin University, Changchun, Jilin, 130041, China Department of Hepatopancreatobiliary Surgery, The Second Hospital of Jilin University, Changchun, Jilin, 130041, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Ovarian cancer MicroRNA Cell cycle arrest Invasion Proliferation
Reports suggest that microRNAs have implications in the development of several diseases including cancer. It is therefore believed that miRs may act as therapeutic targets for cancer treatment. The treatment of ovarian cancer is mainly obstructed by lack of biomarkers and efficient drug targets. Against this backdrop, this study was undertaken to unveil the therapeutic implications of miR-27a in ovarian cancer. The results showed that the expression of miR-27a was significantly elevated in ovarian cancer tissues and cell lines. Inhibition of miR-27a expression resulted in the decrease of proliferation rate and colony formation potential of the SK-OV-3 and OVACAR-3 cells via G2/M arrest. The miR-27a inhibition triggered G2/M arrest of SK-OV-3 and OVACAR-3 cells was accompanied with depletion of cyclin A and B1 expression levels. TargetScan analysis together with dual reporter assay revealed that miR-27a exerts its effects via modulation of CUL5 expression. The CUL5 was shown to be suppressed in the ovarian cancer tissues and cell lines and suppression of miR-27a expression caused upregulation of CUL5 expression. Overexpression of CUL5 caused inhibition of SK-OV-3 and OVACAR-3 cell proliferation via induction of G2/M arrest, similar to that of miR-27a inhibition. Interestingly, CUL5 overexpression reversed the effects of miR-27a inhibition on the viability of SK-OV-3 cells. Finally, the suppression of miR-27a could enhance the chemosensitivity of the SK-OV-3 cells to cisplatin and docetaxel anticancer drugs and also decreased their migration and invasion. The findings of this study revealed that miR-27a suppression inhibits the growth, chemosensitivity and invasion of ovarian cancer and may prove beneficial in the ovarian cancer management.
1. Introduction Accumulating evidences have indicated the involvement of microRNAs (miRs) in almost all types of cancers and malignancies [1]. The advent of microarray and next generation sequencing has greatly allowed to understand the relation between miRs and cancer [2]. The miRs are generally 22 nucleotides in length non-coding RNAs molecules that regulate the expression of genes post-transcriptionally [3]. They are evolutionarily conserved and perform their functions in almost all biological processes [4]. It has been reported that more than 2500 miRs have been identified so far in human and the number is still growing [5]. The expression of miRs has been shown to be dysregulated in cancers. Generally the expression of miRs is repressed in cancerous tissues, however, may miRs have also been reported to be overexpressed in cancer [6]. It has been reported that therapeutic application of miRs may prove an amazing and essential strategy to interfere with molecular mechanisms underlying cancer [7]. Therefore, several
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miRs have been studied therapeutic potential and miR-27a is one such candidate. The miR-27a has been shown to promote the growth and development of gastric adenocarcinoma, colon and pancreatic cancer [8–10]. It has been shown to target MAP2K4 to suppress the proliferation and metastasis of osteosarcoma [11]. Moreover, several anticancerous molecules have been shown to decrease the survival of cancer cells by targeting miR-27a expression. For example genistein and Betulinic acid inhibit the proliferation of human uveal melanoma and ovarian cancer cells respectively via suppression of miR-27a expression [12,13]. Nonetheless, therapeutic implications of miR-27a have not been thoroughly investigated in ovarian cancer. Ovarian cancer is one of the common gynaecological malignancies in women across the globe. Accounting for 2.5% of all malignancies in women, ovarian cancer is responsible for 5% of all cancer related deaths in women [14]. Although there has been improvement in ovarian cancer treatment but the clinical outcome is still far from decent [15]. It has been reported that in United states. Approximately more than twenty
Corresponding author. E-mail address: [email protected] (S. Yang).
https://doi.org/10.1016/j.abb.2019.04.009 Received 1 February 2019; Received in revised form 13 April 2019; Accepted 27 April 2019 Available online 29 April 2019 0003-9861/ © 2019 Elsevier Inc. All rights reserved.
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2.5. Flow cytometric cell cycle analysis
two thousand new ovarian cancer cases and fourteen thousand ovarian cancer deaths are recorded annually [16]. The late diagnosis and dearth of therapeutic targets impose hurdles in the treatment of ovarian cancer. Improvement of prevention through early detection and identification of the therapeutic targets may prove beneficial to curb ovarian cancer related mortalities [17]. The present study explored the therapeutic potential of miR-27a in ovarian cancer and herein we report that miR-27a regulates the growth, chemosensitivity and invasion of the human ovarian cancer by targeting Cullin 5 (CUL5). To sum up, the findings of the present study reveal that miR-27a regulated the growth, chemosensitivity and invasion of ovarian cancer and may prove essential in its treatment.
After transfection the SK-OV-3 cells were subjected to culturing for 24 h at 37 ○C for. The SK-OV-3 cells were harvested and the PBS washed. Afterwards, the SK-OV-3 cells were stained with propidium Iodide (PI) and the distributed of the cells in cell cycle phases was assessed by FACS flow cytometer. 2.6. Dual-luciferase reporter assay The miR-27a target was identified by TargetScan online software (http://www.targetscan.org). The miR-27a inhibitor or miR-NC were co-transfected with Plasmid pGL3-CUL5-3′-UTR-WT or pGL3-CUL5-3′UTR-MUT into SK-OV-3 cells. Dual-luciferase reporter assay (Promega) was carried out at 48 h after transfection. Renilla luciferase used for normalization.
2. Materials and methods 2.1. Tissue samples and cell lines
2.7. Chemosensitivity assay
The snap-frozen malignant ovarian tissues (C1 to C8) and adjacent normal tissues (N1 to N8) were collected from Department of Gynecology and Obstetrics, The Second Hospital of Jilin University, Changchun, Jilin, China, after obtaining informed consent from the patients. The Histological classification of all the tissues is presented in Supplementary Table S1. The study was approved by the Research ethics committee of the institute under approval number HJU675/2A/ 2018. The human ovarian cell lines (Caov-4, SK-OV-3, OVACAR-3 and UWB1.289) and non-cancerous ovarian surface epithelial cell line (SV40, DoTc24510, GH329 and UACC-3247) were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) containing penicillin (100 U/mL), streptomycin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal bovine serum (FBS; Gibco) at 37 °C in 5% CO2.
To determine the effects of miR-27a inhibition on the sensitivity of the SK-OV-3 cells to docetaxel and cisplatin, the SK-OV-3 cells were transfected miR-NC, or miR-27a inhibitor, or treated with docetaxel (0.25 μM) or cisplatin (1 μM) or transfected with miR-27a inhibitor plus treated with docetaxel (0.25 μM) or transfected with miR-27a inhibitor plus treated with cisplatin (1 μM). All these groups of SK-OV-3 cells were then subjected to CCK-8 assay as mentioned above. 2.8. Transwell assay The effects of miR-27a suppression on the invasion ability of SK-OV3 cells was determined by transwell chambers (8 mm pore size, Corning, NY, USA) with Matrigel (Millipore, Billerica, USA) The SK-OV-3 cells were transfected with miR-27a inhibitor and miR-NC and around 200 ml cell cultures were placed onto the upper chambers and only medium was placed in the bottom wells. After 24 h of incubation, the cells were removed from the upper chamber and the cells that invaded via the chambers were subjected to fixation with methyl alcohol and subsequently stained with crystal violet. Inverted microscope was used to count the number of invaded cells at 200× magnification.
2.2. Expression analysis The TRIzol reagent (Invitrogen) was used for the extract of RNA from the tissues and cell lines. This was followed by purification of the RNA by RNeasy Mini Kit (Qiagen). The complementary DNA was then synthesized with the help of miScript Reverse Transcription Kit (Qiagen). Afterwards the cDNA was amplified by using SYBR Premix Ex Taq™ (TaKaRa, Otsu, Shiga, Japan). The expression was estimated by 2−ΔΔCt method and actin was used as an internal control. Heat map was generated by the online heatmaper software (http://www2. heatmapper.ca/).
2.9. Wound-healing assay After 24 h of miR-27a inhibitor and miR-NC of transfection into SKOV-3 cells, the medium was removed and cells were subjected to PBS washing. A sterile pipette tip was employed to scratch a wound in each well and cells were subjected washing again and a picture was taken. The plates were subjected to culturing at 24 h and a picture was taken again under an inverted microscope (Leica, Germany).
2.3. Cell transfection The miR-27a Inhibitor and miR-NC were synthesized by RiboBio (Guangzhou, China). The transfection was then carried out by the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) as per the manufacturer's instructions. As the SK-OV-3 cells reached 80%, the appropriate concentrations of miR-27a inhibitor or miR-NC was transfected into SK-OV-3 cells.
2.10. Western blotting After culturing the SK-OV-3 cells for 24 cells, the cells were harvested by centrifugation and subjected to washing with ice-cold PBS. The cell pellet was then suspended in RIPA lysis buffer. The protein content of cell lysates was determined by Bradford assay. From each sample 30 μg of protein was on SDS-PAGE before being shifted to polyvinylidene fluoride membrane. The membranes were then subjected to treatment with TBS and then exposed to primary antibodies at 4 ○C. Thereafter, the cells were treated with appropriate secondary antibodies and the proteins of interest were visualised by enhanced chemiluminescence reagent.
2.4. Cell viability assay The CCK-8 assay was used for the determination of the cell viability. In brief, the transfected SK-OV-3 cells were cultured in 96-well plates and incubated at 37○C for 24 h and subjected to treatment 10 microtiters of CCK-8 solution. The cells were then again subjected to incubation for 2 h at 37○C in a humidifier (5% CO2/95% O2). OD450 was taken at different time intervals (0, 12, 24, 48 and 96 h) with the help of a microplate reader. The effect of miR-27a suppression on the colony formation of the SK-OV-3 cells was determined by colony formation assay [18].
2.11. Statistical analysis Data are shown as mean ± SD. Statistical analysis was done using Students t-test with GraphPad prism 7 software. Values of p < 0.05 10
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Fig. 1. Expression of miR-27a is upregulated in ovarian cancer tissues and cell lines (A) Expression of different miRs in normal (N1 to N8) and ovarian cancer (C1 to C8) tissues (B) Expression of miR-27a in normal and ovarian cancer tissues (C) Expression of miR-27a in normal and ovarian cancer cell lines. The experiments represent mean of three biological replicates ± SD (*P < 0.01).
Fig. 2. Suppression of miR-27a triggers G2/M arrest of SK-OV-3 and OVACAR-3 cells (A) Expression of miR-27a in miR-NC and miR-27a inhibitor transfected SK-OV3 and OVACAR-3 cells (B) Cell viability of miR-NC and miR-27a inhibitor transfected SK-OV-3 and OVACAR-3 cells (C) Colony formation of miR-NC and miR-27a inhibitor transfected SK-OV-3 and OVACAR-3 cells (D) Cell cycle analysis of miR-NC and miR-27a inhibitor transfected SK-OV-3 and OVACAR-3 cells (E) Protein expression of cell cycle related proteins in miR-NC and miR-27a inhibitor transfected SK-OV-3 and OVACAR-3 cells. The experiments represent mean of three biological replicates ± SD (*P < 0.01).
tissues and eight normal adjacent tissues by qRT-PCR and heat map was generated. The results showed differential expression of these miRs (Fig. 1A). However miR-27a was found to be significantly overexpressed in the ovarian cancer tissues by upto 5 fold relative to the normal adjacent tissues (Fig. 1A and B). The miR-27a expression was also determined in four normal and four ovarian cancer cells lines and it was found that expression of miR-27a was significantly upregulated in
were taken as significant difference. 3. Results 3.1. miR-27a is upregulated in ovarian cancer tissues and cell lines The expression of 11 miRs was examined in eight ovarian cancer 11
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Fig. 3. miR-27a targets CUL-5 in ovarian cancer (A) TargetScan analysis showing CUL5 as the target of miR-27a (B) Dual luciferase assay (C) Expression of CUL5 in ovarian cancer and adjacent normal ovarian tissues (D) qRT-PCR expression of CUL5 in normal and ovarian cancer tissues (E) Western blot analysis showing expression of CUL5 in normal and ovarian cancer cell lines (E) Western blot analysis showing expression of CUL5 in miR-NC or miR-27a transfected SK-OV-3 cells. The experiments represent mean of three biological replicates ± SD (*P < 0.01).
all the ovarian cancer cell lines with fold upregulation ranging between 3.2 and 4.5 relative to normal cell lines (Fig. 1C). It was further found that the SK-OV-3 cell line showed the highest expression of miR-27a and was therefore selected for further investigation.
expression levels of cyclin A and B1 and upregulation of p21 and p27 in SK-OV-3 and OVACAR-3 cells (Fig. 2E).
3.2. Suppression of miR-27a inhibits the proliferation of ovarian cancer cells via G2/M arrest
The bioinformatic analysis of miR-27a with TargetScan online software showed that CUL5 could be the potential target of miR-27a F (Fig. 3A). The dual reporter assay further confirmed that miR-27a targets CUL5 (Fig. 3B). Next, CUL5 expression profile was determined in eight normal and eight ovarian cancer tissues and it was found that CUL5 is suppressed in the ovarian cancer cells (Fig. 3C). The qRT-PCR analysis revealed very low expression CUL5 (upto 8 fold downregulation) in ovarian cancer cells (Fig. 3D). The Western blot analysis also showed revealed similar results (Fig. 3E). Nonetheless, the suppression of miR-27a expression resulted in considerable depletion in the expression of CUL5 (Fig. 3F). To ascertain the effects of CUL5 in SK-OV3 cells, the expression of CUL5 was upregulated in the SK-OV-3 and OVACAR-3 cells and the results showed that ectopic expression of CUL5 resulted in the decline of proliferation and colony formation of the SK-
3.3. miR-27a targets CUL5 in ovarian cancer cells
Next to ascertain the miR-27a function in ovarian cancer, its expression was suppressed in the SK-OV-3 and OVACAR-3 cells (Fig. 2A). The results revealed that inhibition of miR-27a expression resulted in the reduction in the proliferation rate and colony formation of the SKOV-3 and OVACAR-3 cells (Fig. 2B and C). Cell cycle profile analysis revealed that suppression of the miR-27a expression caused the arrest of SK-OV-3 and OVACAR-3 cells at G2/M check point. The G2/M cells increased from 18.4% in miR-NC to around 36% in miR-27a inhibitor transfected SK-OV-3 cells. While as in case of OVACAR-3 cells the G2/M phase cells increased from 22.2 to 52.7% upon miR-27a inhibition (Fig. 2D). The inhibition of miR-27a also caused depletion of the 12
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Fig. 4. Overexpression of CUL5 triggers G2/M arrest of SK-OV-3 and OVACAR-3 cells (A) Expression of CUL5 in NC and pcDNA-CUL5 transfected SK-OV-3and OVACAR-3 cells (B) Cell viability of NC and pcDNA-CUL5 transfected SK-OV-3 and OVACAR-3 cells (C) Colony formation of NC and pcDNA-CUL5 transfected SK-OV3 and OVACAR-3 cells (D) Cell cycle analysis of NC and pcDNA-CUL5 transfected SK-OV-3 and OVACAR-3 cells (E) Protein expression of cell cycle related proteins in NC and pcDNA-CUL5 transfected SK-OV-3 and OVACAR-3 cells (E) Effect of CUL5 silencing on the viability of miR-27a transfected SK-OV3 cells. The experiments represent mean of three biological replicates ± SD (*P < 0.01).
invasion of the SK-OV-3 cells by wound heal and transwell assays respectively. The results showed that miR-27a inhibition caused significant decline in the migration and invasion of the ovarian cancer cells (Fig. 5C and D).
OV-3 and OVACAR-3 cells via induction of G2/M cell cycle arrest (Fig. 4A–D). This was also accompanied by the downregulation of cyclin A and B1 and upregulation of p21 and p27 in SK-OV-3 and OVACAR-3 cells (Fig. 4E). These results were similar to that of miR-27a inhibition. Interestingly, the silencing of CUL5 in the miR-27a inhibitor transfected SK-OV3- cells could almost completely abolish the growth inhibitory effects of miR-27a suppression (Fig. 4F).
4. Discussion The treatment of ovarian cancer is mainly obstructed by dearth of therapeutic targets and biomarkers for early detection [14]. It has been reported that miRNAs modulate the expression of majority of the human genes that are involved in a wide array of biological processes [6]. Because of the implications of miRNAs in cellular and physiological processes, previous studies have revealed the potential of miRNAs as therapeutic targets [5]. This study explored the therapeutic implications of miR-27a in ovarian cancer. In this study, the expression of eleven different miRs was evaluated in normal and ovarian cancer tissues. Although, many of these miRs exhibited differential expression but the expression of miR-27a was upregulated in all the ovarian cancer tissues and therefore miR-27a was selected as the candidate miR for
3.4. Suppression of miR-27a enhances chemosensitivity and inhibits migration and invasion of SK-OV-3 cells The effects of miR-27a inhibition on the chemosensitivity of the SKOV-3 ovarian cancer cells. Henceforth, the miR-27a inhibitor transfected SK-OV-3 cells were treated with 0.25 μM concentration of docetaxel or 1 μM cisplatin and the cell viability was monitored at different time intervals by CCK-8 assay. The results showed that suppression of miR-27a caused enhancement in the chemosensitivity of SK-OV-3 cells to both docetaxel and cisplatin (Fig. 5A and B). Next the effects of miR-27a were examined on the migration and 13
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Fig. 5. Effect of miR-27a inhibition on chemosensitivity of SKOV-3 cells to (A) docetaxel and (B) cisplatin. Effect of miR-27a inhibition on (C) Cell migration and (D) cell invasion of the SK-OV3 cells. The experiments represent mean of three biological replicates ± SD (*P < 0.01).
invasion of osteosarcoma cells and chemosensitivity of bladder cancer cells [11, 26]. To sum up, miR-27a acts as an oncogene in ovarian cancer and regulates their proliferation, invasion and chemosensitivity by targeting CUL5. Therefore, miR-27a may prove beneficial in ovarian cancer treatment and drugs can be developed to target its expression.
further investigation. The qRT-PCR analysis showed miR-27a to be upregulated in both ovarian cancer tissues and cell lines. The results are also in agreement with studies carried out earlier wherein miR-27 has been reported to be overexpressed in cancer cells. For example, miR27a has been shown to be overexpressed in prostate cancer, pancreatic cancer and gastric cancer [10,19]. Next, miR-27a expression was inhibited in the SK-OV-3 and OVACAR-3 cells to explore its role in ovarian cancer. Interestingly it was revealed that miR-27a inhibition in the ovarian cancer cells results in the decline of their cell viability and colony formation via induction of G2/M cell cycle arrest. These findings are also supported by a study wherein miR-27a has been shown to prompt the arrest of the breast cancer cells at the G2/M check point [20]. The G2/M arrest triggered by the miR-27a inhibition caused depletion of cyclin A and B1 and enhancement in the expression p27 and p21. Previous studies have shown that depletion of cyclin A and B1and upregulation of p27 and p21 results in the G2/M arrest of the cells during cell division [21]. Bioinformatic analysis together with dual luciferase assay showed that miR-27a exerts its effects by modulating CUL5 expression. CUL5 has been showed to be involved in the regulation of migration of neurons as well as the angiogenesis of granulocytes [22]. Additionally CUL5 plays part in the metastasis of cervical cancer [23]. Herein, it was found that overexpression of CUL5 caused suppression of SK-OV-3 cell growth via induction of G2/M arrest, similar to that of miR-27a inhibition. This is also supported by a study wherein CUL5 has been shown to play a role in cell cycle [24]. Interestingly, the silencing of CUL5 could almost abolish the growth inhibitory effects of miR-27a knockdown on the proliferation of SK-OV3 cells. The inhibition of miR-27a also enhanced the chemosensitivity of SK-OV-3 to docetaxel and cisplatin and suppressed their migration and invasion. These observations are also supported by previous studies wherein miR-27a has been reported to regulate the migration and
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