STAT3 regulated miR-216a promotes ovarian cancer proliferation and cisplatin resistance

Cisplatin is the first-line treatment for ovarian cancer. However, the clinical outcome of cisplatin treatment in ovarian cancer is hindered by cancer resistance. Here we aim to explore the role and mechanism of miR-216a in the cisplatin resistance of ovarian cancer. The effects of miR-216a overexpression and inhibition on ovarian cell proliferation, colony formation, and cisplatin resistance were investigated by MTT assay and soft agar colony formation assay. Bioinformatics analyses using TargetScan and rVista, qPCR, and luciferase assay were also used to explore and verify downstream effectors and regulators of miR-216a. Proliferation, colony formation, and cisplatin resistance of ovarian cancer cells are promoted by miR-216a overexpression but inhibited by miR-216a inhibition. PTEN is a direct target of miR-216a and PTEN expression antagonizes the tumor-promoting function of miR-216a. STAT3 is a regulator of miR-216a, and PTEN is also regulated by STAT3. miR-216a up-regulation is associated with cisplatin resistance in ovarian cancer and this effect is mediated by PTEN. STAT3 is a regulator of miR-216a. Strategies that inhibit miR-216a is a potential strategy for overcoming the cisplatin resistance in ovarian cancer.


Introduction
Ovarian cancer is the fifth most common cancer amongst women in the United States. According to the estimation by American Cancer Society, approximately 22240 women will be newly diagnosed of ovarian cancer and approximately 14070 women will die from ovarian cancer in 2018. Despite progresses made in ovarian cancer therapy, the survival of patients with advanced ovarian cancer still remains dismal, due to the lack of effective treatment strategies to impede ovarian cancer metastasis and recurrence. Cisplatin, the platinum-based treatment, has been used as the first-line chemotherapy drug for ovarian cancer since more than 30 years ago [1,2]. However, little improvement has been made to the survival of ovarian cancer by cisplatin treatment, and metastatic and recurrent ovarian cancer frequently develop resistance, which primarily accounts for the poor clinical outcome of this treatment. It is imperative to develop novel methods to attenuate or reverse the cisplatin resistance in ovarian cancer.
Recent studies have implicated a plethora of factors involved in the ovarian cancer cisplatin resistance [3,4], including BRCA1 and BRCA2 mutations [5]. These discoveries, whereas, have yet to be translated into clinical practices. Human genome research unveiled a surprisingly high proportion of non-coding genes that play an indispensable role in many physiological processes. miRNAs (miRs) are a class of non-coding RNAs with the length of approximately 22 nts. Emerging evidences indicate that miRNAs are important regulators of cancer. Dysregulated miRNA expression substantially contribute to tumorigenesis and are associated with the poor clinical outcome of patients. A couple of miRNAs, such as miR-214, miR-130a-3p, and miR- 25-3p have been identified as biomarkers of cancer cisplatin resistance [6][7][8][9]. These findings qualify miRNAs as a powerful toolkit for prevention, early detection, and therapy of cancers.
In ovarian cancer, the essential role of a number of miRNAs have been reported [10,11]. Altered expression of miRNA has been recognized as an important constituent of ovarian cancer [12,13]. PTEN is a putative tumor suppressor that regulates the oncogenic PI3K/Akt pathway [14]. Previous evidences have identified that miR-214 is responsible for the enhanced cell survival and cisplatin resistance of ovarian cancer by targetting PTEN [6]. miR-216a is amongst the most investigated miRNAs in cancer [15][16][17][18]. It has been found that miR-216a is also an important mediator of PTEN [19].
Herein, we strive to clarify the correlation between miR-216a expression and malignant ovarian cancer and explore the mechanism of miR-216a in regulating cisplatin resistance in ovarian cancer. The effects of miR-216a up-regulation or inhibition on the phenotypical changes in two commonly used ovarian cancer cell lines, SKOV3 and OVCA433, were investigated. We are particularly interested in the downstream effector and upstream regulator of miR-216a. The results reported in the present study could provide clear evidence on the functional role of miR-216a and potentially provide an opportunity for the diagnosis, treatment of cisplatin-resistant ovarian cancers.

Materials and methods
Cell culture SKOV3 and OVCA433 were acquired from American Type Culture Collection (ATCC, Rockville, MD, U.S.A.). Cells were cultured in RPMI-1640 medium supplemented with 10% FBS, in an incubator maintained at 37 • C and 5% CO 2 . The cisplatin resistant SKOV3 CR cells were acquired by maintaining SKOV3 cells in the presence of cisplatin over a 10-month period. The cisplatin resistance sustained when SKOV3 CR cells were grown in the absence of cisplatin for 30 passages.

Transfection of RNAs and plasmids
The miRNAs were purchased from GenePharma (Shanghai, China). MiR-216a mimics and miR-216a inhibitors (miR-216a siRNA), were transfected into ovarian cancer cells to induce miR-216a up-regulation and down-regulation, respectively. The PTEN plasmid was cloned into pcDNA3.1 plasmid and transfected into the cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, U.S.A.) according to manufacturer's protocols.

Proliferation and soft agar colony formation assay
MTT assay was used to assess proliferation of cells. In brief, 2000 cells were seeded into 96-well plates at 24 h after treatment, the proliferation was monitored for 5 days. On each day, MTT reagent was added to a well and incubated for 2 h. Then, medium of the well was removed and 100 μl of DMSO was added and the absorbance was measured at 562 nm. Soft agar colony formation assay was performed according to a previously published protocol [20].

Western blot
Western blot analysis was used to analyze PTEN expression. Proteins in SKOV3 cells were first extracted after cells' lysis. Protein lysates of 20 μg were then used for SDS/PAGE, followed by transferring on to PVDF membranes. Rabbit anti-PTEN primary antibody was purchased from Abcam. The HRP-conjugated anti-rabbit secondary antibody was purchased from Abcam. PVDF membranes were blocked with 1% BSA for 1 h at room temperature. The anti-PTEN primary antibody diluted at 1:1000 in PBS-T (Tween 20, 0.1% v/v) was then added to the membrane and incubated at RT for 1 h with gentle shaking. After extensive washing with PBS-T, HRP-conjugated anti-rabbit antibody (1:20000 dilution) was added to the membrane and incubated at RT for another hour.

Bioinformatics analysis and luciferase assay
TargetScan and rVista 2.0 were used for bioinformatics analysis. The prediction of effective miRNA target sites in mammalian mRNAs was performed using TargetScan using a previously published protocol [21]. The evolutionary analysis of transcription factor binding sites was performed using rVista according to previously published protocols [22]. Luciferase assay was used to confirm the interaction between two miRNAs and 3 -UTR of genes as previously described [16].

Statistical analysis
SPSS was used for statistical analysis. Data were represented as mean + − S.D. Student's t test was used to compare differences between two groups, and two-way ANOVA was used for comparison amongst three groups or between two groups with two factors. P-values of less than 0.05 were considered statistically significant.

MiR-216a promotes ovarian cancer cells proliferation
To clarify the role of miR-216a in ovarian cancer, miR-216a mimics or inhibitors were transfected into the SKOV3 and OVCA433 ovarian cells, followed by monitoring proliferation by MTT assay. As shown in Figure 1A-D, miR-216a mimics promoted the proliferation of SKOV3 ( Figure 1A) and OVCA433 ( Figure 1B), while miR-216a inhibitor attenuated proliferation of SKOV3 ( Figure 1C) and OVAC433 ( Figure 1D). This unveiled the tumor-promoting role of miR-216a. As another verification, colony formation assay, as shown in Figure 1E-H, demonstrated that miR-216a increased the number of colonies formed by SKOV3 ( Figure 1E,F) and OVCA433 ( Figure 1G,H) cells in soft agar. These data confirmed that miR-216a adopts a tumor-promoting role in ovarian cancer and the cancer cell proliferation is enhanced by miR-216a.

miR-216a increases cisplatin resistance in ovarian cancer cells
We next examined the correlation between miR-216a and cisplatin resistance. Here we compared the miR-216a level in SKOV3 cells and that in cisplatin-resistant SKOV3 cells (SKOV3 CR). As shown in Figure 2A, miR-216a was markedly up-regulated in SKOV3 CR. Further, SKOV3 cells were treated with cisplatin (4 μg/ml) for varied duration (0-72 h), and consequently, an increasing miR-216a levels were seen with treatment duration ( Figure 2B). Therefore, miR-216a up-regulation is also closely associated with cisplatin resistance. In-line with this, transfecting miR-216a inhibitor sensitized SKOV3 cells to cisplatin, as evidenced by lower survival with increasing cisplatin concentration ( Figure 2C). Conversely, up-regulation of miR-216a through transfecting miR-216a mimics increased the resistance of SKOV3 to cisplatin ( Figure 2D). Moreover, colony formation assay was performed for SKOV3, SKOV3 CR, and SKOV3 CR transfected with miR-216a inhibitor. As shown in Figure 2E,F, miR-216a inhibitor dramatically reduced the number of colonies. Consequently, inhibition of miR-216a was effective in attenuating cisplatin resistance.

PTEN is a direct target of miR-216a
To unravel the factors involved in the cancer-regulatory role of miR-216a, we used TargetScan to predict targets of miR-216a and acquired 372 potential targets. We then performed a qPCR analysis of a panel of cancer-related genes in SKOV3 and OVCA433 cells transfected with non-coding miRNA or miR-216a. Figure 3A is a heatmap that represents the mRNA expression profile of eleven putative oncogenes, amongst which six showed down-regulation. Further, we cloned the 3 -UTR of those six genes to psi-check2 plasmid to perform dual-luciferase reporter assay. As shown in Figure 3B, we showed that only PTEN was significantly down-regulated by miR-216a mimic transfection (P<0.01). TargetScan study further identified the binding sites of miR-216a on PTEN ( Figure 3C). To corroborate the binding site of miR-216a on PTEN, we mutated the binding site on the 3 -UTR of PTEN and consequently abrogated the down-regulation of PTEN by miR-216a ( Figure 3D). In addition, Western blot analysis was performed, showing that PTEN was down-regulated by miR-216a mimics transfection ( Figure 3E).

PTEN is the functional downstream effector of miR-216a
We further explored whether miR-216a promotes ovarian cancer proliferation and metastasis through PTEN. To this end, we induced PTEN overexpression in SKOV3 cells. qPCR analysis was used to confirm the overexpression of PTEN ( Figure 4A). After transfection with miR-216a and/or PTEN for 24 h, the cell proliferation was determined by MTT and soft agar assay. Consistent with the role of miR-216a in down-regulating PTEN, overexpression of PTEN in cells transfected with miR-216a resulted in retarded cell proliferation ( Figure 4B) and colony formation ( Figure 4C). More importantly, we showed that cisplatin-resistant cells were characterized by dramatic down-regulation of PTEN (P<0.001) ( Figure  4D), and PTEN down-regulation became increasingly prominent with the increasing duration (1-3 days) of cisplatin treatment ( Figure 4E). At day 3, the level of PTEN was less than 50% of control cells (P<0.001). These data implied that PTEN down-regulation is correlated with cisplatin-resistance. As another verification, we overexpressed PTEN in SKOV3 cells and observed an increased cell survival with increasing dose of cisplatin ( Figure 4F). Furthermore, miR-216a decreased the sensitivity to cisplatin in SKOV3 cells, while overexpressing PTEN reversed this effect ( Figure 4G). Taken together, PTEN down-regulation, induced by miR-216a up-regulation, is indispensable for cisplatin resistance of ovarian cancer cells.

MiR-216a is regulated by STAT3
We next strived to elucidate what contributes to the up-regulation of miR-216a in ovarian cancer. Using rVista 2.0 for binding region prediction, we uncovered that miR-216a promoter contains a binding region for STAT3 (Figure 5A). To validate the interaction between STAT3 and miR-216a, we overexpressed STAT3 in SKOV3 cells, which was confirmed by qPCR ( Figure 5B), followed by evaluating miR-216a levels in STAT3 overexpressing cells. As shown in Figure 5C, we also observed a marked up-regulation of miR-216a in cells with high STAT3 expression. Dual-luciferase reporter assay verified that while STAT3 overexpression induced substantial up-regulation luciferase activity ( Figure  5D), mutation of miR-216a promoter region abrogated this effect. Consistent with the aforementioned antagonizing effects between miR-216a and PTEN, STAT3 overexpression also significantly reduced PTEN levels significantly (P<0.001) ( Figure 5E). These data clearly suggested that STAT3 governs the overexpression of miR-216a, which consequently reduces PTEN expression.

Discussion
Here we demonstrate the important role of miR-216a in cisplatin resistance of ovarian cancer. Thus far, miR-216a has been reported as both a tumor inducer and a tumor suppressor [16,17,23] and the role of miR-216a in ovarian cancer still remains elusive. Additionally, the link between miR-216a and cancer resistance has rarely been established. Previously, Xia et al. [16] showed that miR-216a/217 induces epithelial-to-mesenchymal transition (EMT) through targetting PTEN, thereby promoting drug resistance and recurrence of liver cancer. The up-regulation of miR-216a, which can be detected by measuring miR-216a levels in plasma, has also demonstrated potential as a diagnostic marker in pancreatic cancer [24]. However, to our knowledge, our study represents the first evidence that miR-216a increases cisplatin resistance in ovarian cancer. What has not been demonstrated here is whether miR-216a possesses diagnostic value in ovarian cancer. In this context, the measurement of plasma miR-216a levels can also be adopted to study the correlation of miR-216a up-regulation and clinical outcomes of ovarian cancer patients. No effective screening method is available to detect early-stage ovarian cancer with high sensitivity and specificity. The close alliance between miR-216a up-regulation and cisplatin may allow to identify the patients who do not respond to cisplatin therapy, consequently facilitating timely and efficient treatment planning for this highly lethal disease.
Our study provides a new avenue for overcoming cisplatin resistance to improve the clinical outcomes of patient with ovarian cancers at advanced stages. Currently, the overall 5-year survival rates of patients diagnosed at stages III and IV of this disease are 32 and 18%, respectively [2]. A large population of ovarian cancer patients, who initially respond to standard chemotherapeutic regimens, inevitably relapse with recurrence, metastasis, and drug resistance. Because of this, conventional cytotoxic chemotherapy with cisplatin, even in combination with paclitaxel, have not had a significant impact on overall survival of ovarian cancer in last several decades. Our efforts in elucidating the mechanisms of cisplatin resistance and devising approaches to overcome the resistance are juxtaposed with numerous studies that focus on miRNAs as promising cancer targets [6,[25][26][27]. The inhibition of these miRNAs offers a precise and potent tool to throttle oncogenic pathways that drive cisplatin resistance. Here we utilized siRNA specific to miR-216a to inhibit miR-216a expression, which is a clinically applicable approach. Fueled by recent advances in moving siRNA therapeutics from bench to clinics [28], possible therapeutic strategy can also be developed by suppressing miR-216a expression. It should be noted that cisplatin resistance may also be ascribed to other mechanisms, such as reduced accumulation of the drug [29], increased levels of metallothionein [30] and gluotathione [31], as well as enhanced DNA repair [32]. Gene predisposition, such as BRCA1 and BRCA2 mutation, also contributes significantly to ovarian cancer. Successful treatment of ovarian cancer may necessitate a comprehensive strategy based on these resistance mechanisms, in combination to cisplatin therapy.
We identified PTEN as the downstream effector of miR-216a. We also analyzed a panel of putative cancer-related genes, including CDK8, MITF, SMAD7, KLF12, AG02, CDK14, and BCL-9 [33][34][35][36][37][38][39], but only PTEN expression was shown to be significantly altered by miR-216a overexpression. The loss of PTEN has been implicated as an important mediator of EMT [40], which enhances cancer cells proliferation, migration, and metastasis, thereby leading to poor survival rates of patients. Due to the regulatory role of PTEN in PIK3/Akt pathway, and their critical role in cell apoptosis, it is quite reasonable that resultant inhibition of PTEN by miR-216a led to reduced apoptosis by cisplatin [41]. This observation is consistent with the notion that the suppressed apoptosis is a major contributing factor to cisplatin resistance in ovarian cancers [42].
To elucidate the underlying mechanism of miR-216a in ovarian cancer regulation, we also demonstrated that STAT3, a putative oncogene in ovarian cancer [43], is a regulator of miR-216a. Previous studies have suggested the interaction between miR-216a and JAK2/STAT pathway [17,18]. However, in these studies STAT3 is a target of miR-216a. STAT3 induces cellular transformation and promotes tumorigenesis by regulating a wide array of oncogenes, including c-Myc, cyclinD1 etc. [44]. In the present study, we showed that STAT3 promotes miR-216a by binding to the promoter region of miR-216a. These data are in accordance with previous evidences that STAT3 regulates the expression of a number of miRNAs [45]. By regulating miR-216a, PTEN was suppressed by STAT3 overexpression. In-line with this, the interaction of PTEN and STAT3 has been demonstrated previously and their levels of expression were shown to be negatively correlated [45,46]. Given the significant roles of STAT3 and PTEN, and the close interaction of miR-216a with both of them, miR-216a is an important molecule in cancer and further investigations are warranted to further demonstrated the clinical utility of miR-216a in ovarian cancer.

Conclusion
Here we show that miR-216a is an inducer of cisplatin resistance in ovarian cancer. Inhibition of miR-216a attenuated the cisplatin resistance. Using bioinformatics approach and further validation with qPCR and Western blot, we show that PTEN is an effector and STAT3 is a regulator of miR-216a. By elucidating the role and mechanism of miR-216a in ovarian cancer, our study provides an opportunity to overcome cisplatin resistance in ovarian cancer.

Author contribution
P.J. and Y.L. performed the experiments, analyzed and interpreted the data. R.W. wrote the manuscript. All authors read and approved the final submission.