Background
Mesenchymal stem cells are adult, self-renewing multipotent progenitors that construct the stromal compartment [
1,
2]. Mesenchymal stromal/stem cell population (MSCs) is a population of stromal cells that demonstrate stem cell capabilities isolated from the bone marrow and from other diverse human tissues (like adipose, cartilage, muscle) [
3‐
7]. Bone marrow mesenchymal stem cells (BM-MSCs) support tumor progression through immune suppression, epithelial-to-mesenchymal transition, angiogenesis, and serving as cancer stromal cells [
8‐
12]. In contrast, BM-MSCs also suppress cancer by downregulating cancer survival signaling pathways involving WNT/β-catenin and/or AKT [
8]. Ovarian cancer is the most common cancer from women, however, the effects of BM-MSCs on ovarian cancer are still unclear. It is to necessary to investigate the mechanisms underlying the contradictory roles of BM-MSCs on ovarian cancer cell biological functions.
In this study, we hypothesized that human BM-MSCs might have important influence on the regulation of ovarian cancer cell proliferation and glycolysis. Hence, we investigated the influence of BM-MSCs from miR-1180 on ovarian cancer cell glycolysis and cell proliferation. Our results showed that BM-MSCs treatment promoted cell glycolysis and cell proliferation of ovarian cancer cells. We also found that up-regulation of miR-1180 decreased SFRP1 expression, which activated Wnt signaling in ovarian cancer cells. Our results suggest that miR-1180 may be a therapeutic target in ovarian cancer.
Methods
Cell culture
All the ovarian cancer cell lines used in the study were primarily obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured according to the standard protocols. IOSE80 (normal ovarian epithelial cell line) cells were cultured in DMEM-F12 with 10% fetal bovine serum with penicillin (100 U/ml), streptomycin sulfate (100 µg/ml), EGF and insulin. The cells were incubated in a humidified incubator at 37 °C with 5% CO2.
BM-MSCs isolation
BM was harvested from the sternum or iliac crest of seven healthy volunteers. Bone marrow was flushed out with 1 ml DMEM/F12 medium. The bone marrow was repeatedly washed to generate a single-cell suspension that was centrifuged at 1000 rpm for 5 min. The supernatant was removed, and cells were washed with DMEM/F12 and centrifuged for an additional 5 min. Finally, the supernatant was removed, and cells were resuspended in DMEM/F12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Cells isolated from one hind limb were plated in a 25-cm2 dish and incubated at 37 °C with 5% CO2, which was defined as passage 0 (P0). After 24 h, cells were washed with PBS twice to remove non-adherent cells. When cell confluency was greater than 90%, the cells were secondarily cultured, and the passage number was increased by one.
Conditioned medium preparation
Normal BM-MSCs (control) or the BM-MSCs co-cultured with ovarian cancer (BM-MSCs) were cultured in DMEM/F12 media with 10% FBS for 24 h, and then washed for three times with PBS and finally cultured in 3 ml serum free DMEM/F12 media for 2 h. Conditioned medium was collected and filtered through a 0.22-μm filter (Merck Millipore, Massachusetts, USA) to remove cellular debris for treating ovarian cancer cells.
RNA isolation and miRNA array
The conditioned medium from Normal BM-MSCs (control) or the BM-MSCs co-cultured with ovarian cancer (BM-MSCs) was collected for total RNA extraction using TRIzol (Roche Applied Science). A three-step procedure was performed to profile the miRNAs. First, for cDNA synthesis from the miRNAs, 30 ng of total RNA was subjected to RT (reverse transcription) using a TaqMan® microRNA Reverse Transcription Kit (Applied Biosystems) and Megaplex RT primers (Applied Biosystems) following the manufacturer’s protocol. RT was performed on a Mastercycler Epgradient thermocycler (Eppendorf) with the following cycling conditions: 40 cycles at 16 °C for 2 min, 42 °C for 1 min and 50 °C for 1 s followed by a final step of 80 °C for 5 min to inactivate reverse transcriptase. The expression profile of miRNAs was determined using the TaqMan® Universal Master Mix II (Life Technologies, Applied Biosystems) in an Applied Biosystems 7900HT thermal cycler using the manufacturer’s recommended program. Finally, all the raw data from each array were retrieved from the 7900HT and run on Data Assist Software ver.3.1 (Applied Biosystems).
Cell proliferation
Ovarian cancer cells were seeded in 6-well plates and transfected with miRNAs or treated with BM-MSCs conditioned medium and cultured in the normal condition. Cell survival ability was tested by the method of MTT assay.
Ovarian cancer cells were seeded in 6-well plates. Cells were transfected miR-1180 in the present of BM-MSCs-CM and cultured in the normal condition. The cells were cultured for 10 days, washed with 1 × PBS, fixed with 70% ethanol for 5 min and stained with 0.5% crystal violet for 3 min at room temperature. The colonies (> 50 cells) were counted. All experiments were performed at least three times.
ECAR
ECAR was measured in purified ovarian cancer cells following a 6-day culture in the presence or absence of stromal contact under basal conditions, in response to glucose, and upon blocking the mitochondrial ATP generation by oligomycin. The resulting (compensatory) effects on ECAR following the interference with the mitochondrial energy metabolism represent the maximal glycolytic capacity and are shown as a percentage of the baseline measurement (set as 100%).
ATP levels
Adenosine triphosphate (ATP) concentration was assessed using a colorimetric ATP Assay Kit (Abcam, Cambridge, UK).
RNA extraction and real-time PCR analysis
Ovarian cancer cells were transfected with miR-1180, anti-miR-1180 or the controls for 48 h and then total RNA was isolated for Real time RT-PCR analysis. The expression level of miRNAs was defined based on the threshold cycle (Ct), and relative expression levels were calculated using the 2−ΔΔCt method, using the expression level of the U6 snRNA as a reference gene.
Western blotting
Cultured cells were harvested and lysed with RIPA buffer containing the protease inhibitors on ice for 30 min. Equal protein was separated by SDS-PAGE. The protein was transferred onto nitrocellulose membrane using and probed with primary antibodies and then horseradish peroxidase-labeled secondary antibodies. The protein band signals were visualized using an ECL.
Statistical analysis
The data were analyzed using the SPSS 18.0 (SPSS, Chicago, IL, USA) or Excel. Every experiment was completed independently at least three times. A p value < 0.05 was considered significant.
Discussion
MSCs in tumor microenvironment are from bone marrow or other tissues. MSCs integrate into the tumor stroma and function in a paracrine manner to promote ovarian cancer progression. The secreting paracrine molecule is a main functional way in tumor stroma [
5‐
8]. Although there are studies indicating the important roles of BM-MSCs in ovarian cancer, the roles of MSCs in cell proliferation and glycolysis is still unclear. The molecular mechanisms mediating ovarian cancer cell glycolysis need to be investigated. In this study, our purpose is to investigate the effects of BM-MSCs on ovarian cancer cell glycolysis. We found that MSCs could promote ovarian cell survival ability and glycolysis via the up-regulation of miR-1180/Wnt signaling pathway.
The reported studies show that MSCs exert a positive effect on cancer cell growth [
1,
2,
13,
14]. Unlimited cell proliferation is a most characteristics of malignant tumor. So, BM-MSCs were isolated and co-cultured with ovarian cancer cell to observe cell proliferation. It was found that BM-MSCs promoted ovarian cancer cell proliferation. The Warburg effect is a characteristic of cancer. We measured ECAR and ATP production of ovarian cancer cells with BM-MSCs conditioned medium. The data clearly demonstrated that MSCs increased ovarian cancer cell glycolysis.
MiRNAs play great important roles in ovarian cancer glycolysis. MiR-1180 is reported as a tumor suppressive miRNA in bladder cancer cells and inhibits cell proliferation and tumorigenicity by inhibiting cell cycle related proteins including CDK4, CDK6, cyclinD1, cyclinA2 expression and up-regulating p21 expression [
15]. However, another report showed that miR-1180 is up-regulated in hepatocellular carcinoma [
16,
17]. MiR-1180 promotes hepatocellular carcinoma cell proliferation by down-regulating TNIP2 expression [
16] and induces apoptosis-resistance by activating NF-κB signaling pathway [
17]. So, miR-1180 is a tumor promoter or a tumor suppressor depending on the cancer phenotype and genetics background. Our study showed that miR-1180 expression in BM-MSCs conditioned medium was up-regulated. Further study identified that SFRP1 was a direct target gene of miR-1180 in MSCs. We found that miR-1180 promoted glycolysis via targeting SFRP1. SFRP1 is reported as a tumor suppressor in breast cancer [
18,
19]. Wnt signal pathway is activated in cancers by loss of SFRP1 expression [
20,
21]. In addition, Wnt signal pathway is related to cancer cell drug resistance [
22‐
26] and glycolysis [
27‐
30]. Our findings indicated that BM-MSCs promoted ovarian cancer cell proliferation and glycolysis by miR-1180 activating Wnt pathway.
In the present study, our findings indicated that BM-MSCs promoted ovarian cancer cell proliferation and glycolysis by miR-1180. MiR-1180 functioned as onco-miRNA by activating Wnt pathway. There needs further research on the mechanisms of MSCs derived miR-1180 in ovarian cancer progression. Our study for the first time verified that miR-1180 functioned as an onco-miRNA in ovarian cancer cell by targeting SFRP1 expression in ovarian cancer cells.
Conclusion
The data from our study showed that BM-MSCs treatment promoted cell glycolysis and cell proliferation of ovarian cancer cells. We also found that up-regulation of miR-1180 decreased SFRP1 expression, which activated Wnt signaling in ovarian cancer cells. Our results suggest that miR-1180 may be a therapeutic target in ovarian cancer.
Authors’ contributions
JH and WZ conceived and designed experiments. JH, WZ and YH interpreted data. WZ, YH, ZW, TJ and LW performed and experiments. JH and LW wrote the manuscript. All authors read and approved the final manuscript.
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