eISSN: 1896-9151
ISSN: 1734-1922
Archives of Medical Science
Current issue Archive Manuscripts accepted About the journal Special issues Editorial board Abstracting and indexing Subscription Contact Instructions for authors Ethical standards and procedures
SCImago Journal & Country Rank
Share:
Share:
more
 
 
Basic research

MicroRNA-331-3p inhibits proliferation and metastasis of ovarian cancer by targeting RCC2

Gulimire Buranjiang
,
Reziya Kuerban
,
Ailikemu Abuduwanke
,
Xiaowen Li
,
Gulina Kuerban

Online publish date: 2018/08/28
Article file
- microRNA-331.pdf  [0.60 MB]
Get citation
ENW
EndNote
BIB
JabRef, Mendeley
RIS
Papers, Reference Manager, RefWorks, Zotero
AMA
APA
Chicago
Harvard
MLA
Vancouver
 
 

Introduction

Ovarian cancer is one of the most common malignancies worldwide [1, 2]. Each year approximately 140,000 people in the world die from ovarian cancer [3]. To date, staging/debulking surgery and taxane-platinum-based chemotherapy are commonly used treatment modalities for ovarian cancer [4]. Despite advances in surgical techniques and chemotherapy, the 5-year survival rate for ovarian cancer is still less than 50% [5, 6]. This poor prognosis is due to latent symptoms in the early stages, tumor resistance to chemotherapy, and a lack of effective early detection [7]. In order to improve the poor survival of ovarian cancer, a large number of studies are needed to unravel the molecular mechanism(s) responsible for the development of ovarian cancer.
MicroRNAs (miRNAs) are non-coding RNAs, composed of 20–23 nucleotides. miRNA binds to the 3-UTR of a target gene and plays a negative regulatory role by inhibiting mRNA translation or inducing mRNA degradation [8, 9].
Accumulating studies indicated that miRNAs were involved in the progression of ovarian cancer and therefore could be used for cancer detection [10]. miRNAs have been reported to inhibit tumorigenesis of ovarian cancer by suppressing cell proliferation [11], migration [12], invasion [13, 14], and epithelial-to-mesenchymal transition [15]. In addition, miRNAs were also expected to be an important diagnostic tool [16, 17] as well as therapeutic targets for ovarian cancer [18, 19]. Although a few studies have suggested that miR-331-3p can promote the growth of liver cancer [20, 21], miR-331-3p was taken as a suppressor in a variety of other human cancers. Studies showed that miR-331-3p could inhibit cervical cancer [22], gastric cancer [23], renal cell carcinoma [24], and colorectal cancer [25] by promoting apoptosis, suppressing cell proliferation, and inhibiting migration and invasion of tumor cells. However, the mechanism of miR-331-3p is still unclear in ovarian cancer.
The results of the current study showed that by inhibiting the target gene RCC2, miR-331-3p could significantly suppress tumor metastasis in an ovarian cancer cell line.

Material and methods

Cell lines

Of six human ovarian cancer cell lines, A2780 and SKOV3 were purchased from ATCC (Manassas, USA), and CAOV3, OVCAR3, ES-2, and COC1 were purchased from the Cell Center of Chinese Academy of Medical Sciences (Beijing, China). Two immortalized ovarian surface epithelial (IOSE) cells, IOSE80 and IOSE144, were purchased from BeNa Culture Collection (Suzhou, China). The 293T cell line was purchased from ATCC (Manassas, USA). Cells were cultured in appropriate growth media supplemented with 10% FBS and antibiotics at 37°C in presence of 5% CO2 following the manufacturer’s instructions.
Reverse transcription-PCR (RT-PCR)
The total RNA was extracted from cells using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The primers for miR-331-3p and U6 were provided by the miRNA Real-Time PCR Assay kit (Genepharm, China). The primers for GAPDH and RCC2 were obtained from Sangon Biotech (Shanghai, China): GAPDH, forward, 5-GCACCGT­CAAGGCTGAGAAC-3, reverse, 5-ATGGTGGTGAAGACGCCAGT-3; RCC2 forward 5-TTCCTTTGGGTGCCCTGAA-3, reverse 5-GGCAGAATCTGTCCATC­TTTCG-3. The expression of RCC2 was detected by qRT-PCR (SYBR Green PCR kit, Bio-Rad). The relative expression level was determined by the 2–Ct method. U6 and GAPDH were used as controls. Each experiment was performed 3 times.

Transfection of oligonucleotide

Negative control (NC) mimics, miR-331-3p mimics, and miR-inhibitors were designed and synthesized by Genepharm (Shanghai, China). Cells were transfected with NC mimics, miR-331-3p mimics or miR-inhibitors using transfection reagent (pufei biotech, China) following the manufacturer’s instructions.

RCC2 overexpressing stable cells

The RCC2 encoding sequence (without 3-UTR) was cloned and inserted into the pcDNA3.1(+) vector (Invitrogen). Cell transduction was performed with Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol, and RCC2 overexpressing stable cells were selected in the presence of 500 µg/ml of G418. The pcDNA3.1(+) vector was transfected into SKOV3 and A2780 cells and stable cell lines were selected as controls as described above.

Cell proliferation

Cell proliferation was measured with the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). The absorbance at 450 nm was measured at 0 h, 24 h, 48 h, and 72 h; and then the growth curves were established to evaluate proliferation rates. Each experiment was performed 3 times.

Wound-healing assay

Cells were cultured in six-well plates and grown to 90% confluency. A 10 µl pipette tip was used to make a wound on cell monolayers and then the cells were cultured in medium supplemented with 10% FBS. Wound healing was observed using a microscope (Leica, German) at 0 h, 24 h, 48 h, and 72 h. The wound healing rate was calculated using image pro plus 6.0 software.
Migration and invasion assay
Cell migration and invasion were measured by transwell assay. Briefly, cells were trypsinized, washed and seeded on standard 24-well chambers (Corning Costar) at a seeding density of 1 × 105 cells per well, and FBS was added to the bottom chamber as a chemo-attractant. After 24 h, chambers were fixed and stained with crystal violet, and the migrated cells were counted using a microscope. The invaded cells were counted after 48 h. Data are shown as the mean value ± SD from triplicate experiments.

Luciferase assays

A psiCHECK-2 vector (Promega) which contained the luciferase reporter gene and the 3-UTR of RCC2 (or its mutant sequence) was transfected into the 293T cell, and then miR-331-3p mimics or the control mimics were transfected into the same cells. After 48 h, the cells were cracked and the firefly and Renilla fluorescence was detected by the Dual-Luciferase Reporter Assay System (Promega). Renilla fluorescence reflected the expression of 3-UTR of RCC2. The experiments were performed independently in triplicate.

Western blot

The total protein was extracted from cells using NP-40 lysis buffer, supplemented with protease inhibitor cocktail (Roche Applied Science). A BCA protein quantitative kit (Beyotime) was used to detect protein concentration. The primary antibodies anti-RCC2 (Abcam) and anti-GAPDH (Abcam) were used in the western blot assays. All the experiments were performed in triplicate independently.

Statistical analysis

Data from three repeated experiments are presented as mean ± SD. Student’s t-test or ANOVA was conducted for statistical analysis. A p-value less than 0.05 was considered as statistically significant.

Results

miR-331-3p is down-regulated in ovarian cancer

To examine the expression of miR-331-3p in human ovarian cancer cells, total RNAs were isolated from six ovarian cancer cell lines (CAOV3, OVCAR3, ES-2, COC1, A2780 and SKOV3), and two immortalized ovarian surface epithelial cell lines (IOSE80 and IOSE144). Quantitative-PCR was performed to measure miR-331-3p expression. The expression levels of miR-331-3p were down-regulated in ovarian cancer cell lines, compared to that of the normal ovarian surface epithelial cells. Decreased miR-331-3p expression in ovarian cancer cell lines suggested miR-331-3p to be a tumor suppressor in ovarian cancer (Figure 1).

miR-331-3p inhibits cell proliferation, migration and invasion

In order to confirm the impact of miR-331-3p in ovarian cancer, miR-331-3p mimics were transfected into ovarian cancer cell lines A2780 and SKOV3. The expression of miR-331-3p was detected by RT-PCR. The results showed that miR-331-3p was upregulated in transfected cells (Figure 2 A). Proliferation assays showed that miR-331-3p expression significantly inhibited cell proliferation in A2780 and SKOV3 cells (Figure 2 B). Transwell assays were utilized to examine the impact of miR-331-3p on EOC cell migration and invasion. The results showed that miR-331-3p mimics dramatically inhibited the migration abilities of SKOV3 and A2780 cells (Figure 2 C). Similarly, the invasion abilities of SKOV3 and A2780 cells were suppressed significantly after transfection with miR-331-3p mimics compared to that of cells transfected with NC mimics (Figure 2 D). Cell mobility in A2780 and SKOV3 was determined by wound-healing assays (Figure 2 E). The results showed that miR-331-3p mimics suppressed the cell mobility significantly in A2780 and SKOV3 cells. Images of transwell and wound healing assays are shown in Figure 3. These data suggested that miR-331-3p suppresses cell proliferation, migration and invasion of ovarian cancer.

miR-331-3p directly targets RCC2

In order to characterize the molecular mechanism by which miR-331-3p suppresses EOC growth, migration and invasion, we searched three target scan databases – TargetScan, DIANA and miRanda – which predicted RCC2 as a direct target of miR-331-3p (Figure 4 A). To confirm that miR-331-3p can target and suppress RCC2, a luciferase reporter vector containing the binding site of RCC2 3‑UTR, wild type (WT) or the mutant type (MUT) was constructed and transfected into 293T cells. The results of luciferase assays are shown in Figure 4 B. In the WT group, miR-331-3p significantly suppressed the relative luciferase activities, whereas in the mutant type group, no significant difference in the relative luciferase activity was detected between NC and miR-331-3p treated cells. The results of the luciferase assay suggested that miR-331-3p suppresses RCC2 by identifying and binding specific sequences of RCC2 3-UTR. The mRNA level (Figure 4 C) and protein level (Figure 4 D) of RCC2 in miR-331-3p transfected A2780 and SKOV3 were detected by RT-PCR and western blot respectively. The results showed that the expression of RCC2 was significantly inhibited by
miR-331-3p at both the mRNA level and the protein level
. Restoration of RCC2 expression rescues the impact of miR-331-3p on EOC cells To explore whether RCC2 is the target of miR-331-3p in ovarian cancer; RCC2 was overexpressed in A2780 and SKOV3 cells by transfection with recombinant vectors carrying RCC2 cDNA (without 3-UTR) (Figure 5 A). Transwell and wound healing assays were conducted, and the results showed that the subsequent reintroduction of RCC2 without an intact 3-UTR partly restored the inhibitory effects of miR-331-3p mimics on the cell proliferation, migration and invasion ability of A2780 and SKOV3 cells (Figures 5 B–E, 6). Taken together, the data indicate that RCC2 acts as a functional target of miR-331-3p in EOC cells.

Discussion

Ovarian cancer is one of the world’s most common gynecological malignant tumors [5, 26]. Despite recent advances in detection and cytotoxic therapies, more than 70% of ovarian cancer cases were diagnosed at an advanced stage, and late diagnosis led to poor prognosis [27, 28]. Furthermore, patients with ovarian cancer are faced with a serious risk of recurrence, and the current standard treatment is unable to cure the disease [6, 29]. Therefore, ovarian cancer still remains the leading cause of mortality in gynecologic cancer, which accounts for nearly 140 thousand deaths per year worldwide [5, 26]. Thus, there is an urgent demand to develop new diagnostic and therapeutic targets for this devastating disease. In recent years, more and more miRNAs have been found to play an important role in the occurrence and development of ovarian cancer. miR-331-3p was reported to suppress a wide variety of human cancers by inhibiting different target genes. In human renal carcinoma cells, miR-331-3p inhibits cell proliferation by targeting HER2 and NRP2 [24]; miR-331-3p has also been reported to inhibit cell proliferation, block the cell cycle and induce apoptosis in cervical cancer by regulating NRP2 [22], while in prostate cancer, miR-331-3p suppresses cell proliferation and migration by directly targeting RALA [30].
Our study found that miR-331-3p was significantly reduced in ovarian cancer cells compared to immortalized ovarian surface epithelial cell lines (Figure 1). These results suggested that miR-331-3p might act as a suppressor in ovarian cancer. In order to verify this hypothesis, miR-331-3p mimics were transfected into EOC cells (Figure 2 A). CCK8, transwell, and wound healing assays were conducted, and the results indicated that miR-331-3p suppressed EOC cell proliferation, migration, and invasion (Figures 2 B–E). In addition, regulator of chromosome condensation 2 (RCC2) was predicted to be a novel target of miR-331-3p.
The luciferase assay verified that miR-331-3p negatively regulated RCC2 gene expression via specifically binding to the 3-UTR (Figures 4 A, B). Results of RT-PCR and western blot suggested that the expression of RCC2 was downregulated by miR-331-3p in EOC cells (Figures 4 C, D). In addition, upregulated RCC2 was able to restore the cell proliferation, migration, and invasion in miR-331-3p transfected cells (Figure 5). The results in Figure 5 suggested that RCC2 plays an important role in the miR-331-3p suppressing ovarian cancer. RCC2 was previously reported to be an important component of the chromosomal passenger complex (CPC); and CPC was regarded as an important regulator of chromosome and cytoskeleton formation as well as mitosis [31, 32]. In recent years, RCC2 has been reported as a tumor promoter in various cancers. RCC2 was shown to be upregulated in gastric carcinoma tissues, which can regulate the cell cycle process to promote the proliferation of cancer cells [33, 34]. Another study reported that RCC2 knockdown arrested the cell cycle at prometaphase in HeLa cells, suggesting RCC2 to be a negative regulator of the cell cycle which was found to be driven by regulating chromosomal separation and cell division [35–37]. The molecular mechanism of RCC2 in ovarian cancer will be further verified in our future work.
In conclusion, our study revealed that miR-331-3p could significantly inhibit the proliferation, migration and invasion of EOC cells, and that RCC2 is the direct target of miR-331-3p in ovarian cancer. The findings from our study further clarify the role of miR-331-3p in ovarian carcinoma tumorigenesis, and demonstrate that the miR-331-3p /RCC2 signaling axis could be a novel tumor detection marker as well as a potential therapeutic target for ovarian carcinoma.

Acknowledgments

Gulimire Buranjiang and Reziya Kuerban are co-first authors this article.

Conflicts of interest

The authors declare no conflict of interest.

References

1. Berek JS, Crum C, Friedlander M. Cancer of the ovary, fallopian tube, and peritoneum. Int J Gynaecol Obstet 2012; 119 Suppl 2: S118-29.
2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin 2015; 65: 5-29.
3. Dahiya N, Morin PJ. MicroRNAs in ovarian carcinomas. Endocr Relat Cancer 2010; 17: F77-89.
4. Feng Z, Wen H, Bi R, Yang W, Wu X. Prognostic impact of the time interval from primary surgery to intravenous chemotherapy in high grade serous ovarian cancer. Gynecol Oncol 2016; 141: 466-70.
5. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin 2016; 66: 7-30.
6. Vargas-Hernandez VM, Moreno-Eutimio MA, Acosta-Altamirano G, Vargas-Aguilar VM. Management of recurrent epithelial ovarian cancer. Gland Surg 2014; 3: 198-202.
7. Iorio MV, Visone R, Di Leva G, et al. MicroRNA signatures in human ovarian cancer. Cancer Res 2007; 67: 8699-707.
8. Ambros V. The functions of animal microRNAs. Nature 2004; 431: 350-5.
9. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281-97.
10. Deb B, Uddin A, Chakraborty S. miRNAs and ovarian cancer: an overview. J Cell Physiol 2018; 233: 3846-54.
11. Dong R, Liu X, Zhang Q, et al. miR-145 inhibits tumor growth and metastasis by targeting metadherin in high-grade serous ovarian carcinoma. Oncotarget 2014; 5: 10816-29.
12. Zhang H, Wang Q, Zhao Q, Di W. MiR-124 inhibits the migration and invasion of ovarian cancer cells by targeting SphK1. J Ovarian Res 2013; 6: 84.
13. Yeh YM, Chuang CM, Chao KC, Wang LH. MicroRNA-138 suppresses ovarian cancer cell invasion and metastasis by targeting SOX4 and HIF-1alpha. Int J Cancer 2013; 133: 867-78.
14. Zhang Y, Zhao FJ, Chen LL, et al. MiR-373 targeting of the Rab22a oncogene suppresses tumor invasion and metastasis in ovarian cancer. Oncotarget 2014; 5: 12291-303.
15. Guo F, Cogdell D, Hu L, et al. MiR-101 suppresses the epithelial-to-mesenchymal transition by targeting ZEB1 and ZEB2 in ovarian carcinoma. Oncol Rep 2014; 31: 2021-8.
16. Shell S, Park SM, Radjabi AR, et al. Let-7 expression defines two differentiation stages of cancer. Proc Natl Acad Sci USA 2007; 104: 11400-5.
17. Shen J, Ambrosone CB, DiCioccio RA, Odunsi K, Lele SB, Zhao H. A functional polymorphism in the miR-146a gene and age of familial breast/ovarian cancer diagnosis. Carcinogenesis 2008; 29: 1963-6.
18. Kinose Y, Sawada K, Nakamura K, Kimura T. The role of microRNAs in ovarian cancer. Biomed Res Int 2014; 2014: 249393.
19. Pecot CV, Rupaimoole R, Yang D, et al. Tumour angiogenesis regulation by the miR-200 family. Nat Commun 2013; 4: 2427.
20. Cao Y, Chen J, Wang D, et al. Upregulated in hepatitis B virus-associated hepatocellular carcinoma cells, miR-331-3p promotes proliferation of hepatocellular carcinoma cells by targeting ING5. Oncotarget 2015; 6: 38093-106.
21. Chang RM, Yang H, Fang F, Xu JF, Yang LY. MicroRNA-331-3p promotes proliferation and metastasis of hepatocellular carcinoma by targeting PH domain and leucine-rich repeat protein phosphatase. Hepatology 2014; 60: 1251-63.
22. Fujii T, Shimada K, Asano A, et al. MicroRNA-331-3p suppresses cervical cancer cell proliferation and E6/E7 expression by targeting NRP2. Int J Mol Sci 2016; 17: 1351.
23. Guo X, Guo L, Ji J, et al. miRNA-331-3p directly targets E2F1 and induces growth arrest in human gastric cancer. Biochem Biophys Res Commun 2010; 398: 1-6.
24. Qi J, Zhao D, Cui M, Liu Q, Fang Q. MiR-331-3p is down-regulated in renal cell carcinoma and inhibits cell proliferation and invasion by targeting HER2 and NRP2. Int J Clin Exp Med 2017; 10: 16224-30.
25. Zhao D, Sui Y, Zheng X. miR-331-3p inhibits proliferation and promotes apoptosis by targeting HER2 through the PI3K/Akt and ERK1/2 pathways in colorectal cancer. Oncol Rep 2016; 35: 1075-82.
26. Cannistra SA. Cancer of the ovary. N Engl J Med 2004; 351: 2519-29.
27. Bell DA. Origins and molecular pathology of ovarian cancer. Mod Pathol 2005; 18 Suppl 2: S19-32.
28. Pongsavee M. Effects of 744ins20 – ter240 BRCA1 mutation on breast/ovarian carcinogenesis and the role of curcumin in telomerase inhibition. Arch Med Sci Civil Dis 2017; 2: 125-9.
29. Vaughan S, Coward JI, Bast RC Jr, et al. Rethinking ovarian cancer: recommendations for improving outcomes. Nat Rev Cancer 2011; 11: 719-25.
30. Epis MR, Giles KM, Beveridge DJ, et al. miR-331-3p and Aurora Kinase inhibitor II co-treatment suppresses prostate cancer tumorigenesis and progression. Oncotarget 2017; 8: 55116-34.
31. Pang B, Wu N, Guan RW, et al. Overexpression of RCC2 enhances cell motility and promotes tumor metastasis in lung adenocarcinoma by inducing epithelial-mesenchymal transition. Clin Cancer Res 2017; 23: 5598-610.
32. Cao Q, Li B, Wang X, Sun K, Guo Y. Therapeutic inhibition of CXC chemokine receptor 2 by SB225002 attenuates LPS-induced acute lung injury in mice. Arch Med Sci 2018; 14: 635-44.
33. Matsuo M, Nakada C, Tsukamoto Y, et al. MiR-29c is downregulated in gastric carcinomas and regulates cell proliferation by targeting RCC2. Mol Cancer 2013; 12: 15.
34. Kang K, Huang YH, Li HP, Guo SM. Expression of UCA1 and MALAT1 long-chain non-coding RNAs in esophageal squamous cell carcinoma tissues is predictive of patient prognosis. Arch Med Sci 2018; 14: 752-9.
35. Mollinari C, Reynaud C, Martineau-Thuillier S, et al. The mammalian passenger is an RCC1 family member protein TD-60 with an essential role in prometaphase to metaphase progression. Dev Cell 2003; 5: 295-307.
36. Li JY, Liu CP, Shiao WC, et al. Inhibitory effect of PDGF-BB and serum-stimulated responses in vascular smooth muscle cell proliferation by hinokitiol via up-regulation of p21 and p53. Arch Med Sci 2018; 14: 579-87.
37. Zhang Y, Wang Z, Zhang L, et al. Impact of connexin 43 coupling on survival and migration of multiple myeloma cells. Arch Med Sci 2017; 13: 1335-46.
Copyright: © 2018 Termedia & Banach. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
FEATURED PRODUCTS
Quick links
© 2019 Termedia Sp. z o.o. All rights reserved.
Developed by Bentus.
PayU - płatności internetowe