miR-200b/200a/429 Cluster Stimulates Ovarian Cancer Development by Targeting ING5

Ovarian cancer is the second most common gynaecological malignancy, and microRNAs (miRNAs) play important role in the cancer development. Here, we found that the level of miR-200b/200a/429 was significantly increased in serum and tumor tissues of patients with stage-I ovarian cancer. Consistent with these results, we detected increased expression levels of miR-200b/200a/429 in ovarian cancer cell lines compared with the human nontumorigenic ovarian epithelial cell line T80. The overexpression of miR-200b/200a/429 in T80 cells stimulated proliferation and caused their growth in soft agar and tumor formation in nude mice. Furthermore, we determined that miR-200b/200a/429 targets inhibitor of growth family 5 (ING5) and that the overexpression of ING5 can block miR-200b/200a/429-induced T80 cell transformation and tumorigenesis. Our findings suggest that miR-200b/200a/429 may be a useful biomarker for the early detection of ovarian cancer and that miR-200b/200a/429 significantly contributes to ovarian cancer development through ING5.


Introduction
Ovarian cancer is one of the most common gynecologic tumors in the world, with estimated 238,700 new cases and 151,900 deaths in 2012 [1]. e 5-year survival rate of earlystage ovarian cancer patients is more than 92%; however, the 5-year survival rate of late-stage ovarian cancer patients is only 29%, suggesting that early diagnosis is crucial for patient survival [2]. Unfortunately, only approximately 20-25% of patients with ovarian cancer are diagnosed at an early stage [3] due to a lack of early diagnostic markers [2]. Additionally, the molecular mechanism of ovarian cancer development is not fully understood.
MicroRNAs (miRNAs) are short, single-stranded noncoding RNAs that inhibit gene expression at the posttranscriptional level by binding to the 3′-untranslated regions (UTRs) of target gene messenger RNAs. Accumulating evidence shows that miRNAs are aberrantly expressed in tumors and closely correlated with tumor initiation and progression. Notably, the dysregulation of even a single miRNA is sufficient to cause tumor development [4] because one miRNA can target many genes, thereby affecting a large cellular signaling network [5]. Dysregulated expression of the miR-200s family (cluster 1: miR-200b/200a/429 and cluster 2: miR-200c/141) has been indicated in several tumors [6][7][8]. Interestingly, studies have shown that miRNAs of the miR-200s family play different roles in different progression stages by targeting different genes, even in the same cancer [7]. According to Korpal et al., inhibit local invasion by targeting zinc finger E-box-binding homeobox 1/2 (ZEB1/2) but promote lung metastatic colonization by targeting Sec23a in breast cancer [7]. According to Brozovic et al., differentially regulate sensitivity to paclitaxel and carboplatin in ovarian cancer [9]. ese findings suggest that the role of the miR-200s family at different tumor progression stages is necessary to study. e dysregulated expression of miR-200s was detected previously, and its role in different stages of ovarian cancer progression has been studied, including chemoresistance and metastasis [10,11]. However, its role in ovarian cancer development is still unclear. Here

Cell Lines and Clinical Samples.
e human ovarian cancer cell lines OVCAR3 and A2780 and the human ovarian epithelial cell line T80 were maintained in DMEM supplemented with 10% fetal bovine serum. All cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO 2 .
Ovarian cancer tissues, matched adjacent normal tissues, and sera were collected from 10 patients with newly diagnosed early-stage ovarian cancer at Daping Hospital (Table 1). is research was approved by the Research Ethics Board of Daping Hospital.

RNA Analysis.
Total RNA was isolated from cells and tissues using the TRIzol reagent according to the manufacturer's protocol. Mature miR-200a, miR-200b, miR-429, and RNU6 (endogenous control) expression levels were measured by qRT-PCR. e relative expression of miRNAs was normalized to RNU6 expression using the 2 −∆Ct method. GeneChip Human Transcriptome Array 2 is used to detect genes whose expression levels are affected by miR-200s in ovarian cancer cells.

Luciferase Reporter Assay.
e 3′-UTR segments of ING5 that are predicted to interact with miR-200a/200b/429 were amplified by PCR from human genomic DNA and inserted into the Mlu I and Hind III sites of the miRNA Expression Reporter Vector. For the luciferase reporter assay, cells were seeded into 24-well cell culture plates at a concentration of 1 × 10 4 per well. e next day, the cells were transfected with the indicated reporter plasmids containing firefly luciferase and either the indicated miRNA mimics or control nucleotides. After 48 hours of transfection, luciferase activity was measured using the Dual-Luciferase Assay System according to the manufacturer's protocol. Firefly luciferase activity was normalized to that of Renilla luciferase.

Soft Agar and Proliferation Assays.
Soft agar and proliferation assays were performed as described previously [12]. e experiments were conducted for 6 months.

Statistical Analysis.
All data are presented as the mean ± standard deviation, and significant differences between treatment groups were analyzed by Student's t-test. Differences were considered statistically significant at a p value less than 0.05.

miR-200b/200a/429 Expression Was Significantly Increased in Early-Stage Ovarian Cancer.
To investigate the correlation between miR-200b/200a/429 expression and ovarian cancer development, we compared the expression level of miR-200b/200a/429 between early-stage ovarian cancer tissues and normal ovarian tissues. As shown in Figure 1(a),   (Figure 1(b)). Finally, we determined that the expression of miR-200b/200a/429 was higher in ovarian cancer cell lines than in T80 nontumorigenic ovarian epithelial cells (Figure 1(c)). ese findings suggest that the increased expression of miR-200b/200a/429 may be associated with ovarian cancer development. tumorigenesis, we performed a gene array using miR-200b/ 200a/429-overexpressing T80 cells and their control cells. As shown in Figure 3(a), we observed many genes that were downregulated by miR-200b/200a/429 in T80 cells (Figure 3(a)). en, we used miRNA target prediction algorithms (starbase.sysu.edu.cn) to determine whether the 3′-UTR of the top 5 downregulated genes (Figure 3(b)) has a sequence that can bind to miR-200b/200a/429. As shown in Figure 3(c), we determined that the 3′-UTR of ING5 contains sites that can bind to miR-200a, miR-200b, and miR-429. Next, we investigated whether miR-200b/200a/429 is directly involved in the inhibition of ING5 protein

Discussion
In reported that the overexpression of miR-200b/200a/429 can markedly enhance transformation and tumor formation in K-Ras-transformed fibroblasts [8]. ese findings clearly indicate that miR-200b/200a/429 plays role in ovarian cancer development as an oncogenic miRNA and may have potential as a biomarker for the diagnosis of early-stage ovarian cancer. However, further verification is needed in a large sample size of early-stage ovarian cancer patients.
We also clarified the oncogenic mechanism of miR-200b/200a/429 in ovarian cancer development. ING5 is a member of the ING family and is a tumor suppressor gene [13]. According to Zheng et al., the downregulation of ING5 is closely correlated with ovarian carcinogenesis, ovarian cancer metastasis, and angiogenesis [14].

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no competing interests.