The A2780 human ovarian cancer cell line was purchased from the European Collection of Cell Culture (Salisbury, UK). HeyA8, SKOV3, paclitaxel-resistant SKOV3-TR and immortalized ovarian surface epithelial (IOSE) cells were kindly provided by Dr Anil K. Sood (The University of Texas MD Anderson Cancer Center, Houston, TX, USA) and maintained in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. Paclitaxel was purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich). The cells were labeled for quantitative analysis using 0.4% trypan blue (Gibco-Thermo Fisher Scientific).

Paclitaxel-induced cytotoxicity was assessed using a WST-1 assay (EZ-Cytox cell viability assay kit; ITSBio, Seoul, Korea). The cells were seeded in a 96-well culture plate and incubated overnight. Then, the cells were treated with the indicated doses of paclitaxel for 48 h; WST-1 solution was subsequently added to each well and the cells were incubated for 0.5 h. Cell viability was determined by measuring absorbance at 450 nm using an iMark microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

The rate of cell proliferation was evaluated using the BrdU Cell Proliferation Assay kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer's instructions. Briefly, the cells were transfected with control or MSI2-expressing plasmids and incubated overnight. The transfected cells were subsequently treated with 0.5% trypsin-EDTA (Gibco-Thermo Fisher Scientific). Then, 0.5×10⁴ cells were added to each well of the 96-well plate; the cells were then incubated for 24 h and subsequently incubated in BrdU solution (1:10; Cell Signaling Technology) for 2 h. Following the incubation, the cells were washed, treated with a fixing/denaturing solution (Cell Signaling Technology), and incubated with an antibody detection solution (Cell Signaling Technology) at room temperature for 1 h. Then, the cells were washed, incubated in horseradish peroxidase (HRP)-conjugated second antibody solution (Cell Signaling Technology) for 30 min, and incubated in TMB substrate solution (Cell Signaling Technology) for 30 min. At the end of the incubation, cell proliferation was determined by measuring absorbance at 450 nm using an iMark microplate reader (Bio-Rad Laboratories).

Changes in protein levels were measured using immunoblotting assays. The cells were lysed in a radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). The proteins were separated using SDS-PAGE and subsequently transferred to a nitrocellulose membrane (Bio-Rad Laboratories). The membrane was blocked with 5% skim milk for 1 h at room temperature and incubated overnight with the indicated antibody at 4°C. HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Cell Signaling Technology) were used, followed by enhanced chemiluminescence (ECL; Pierce, Thermo Fisher Scientific) and autoradiography, to detect the proteins. The primary antibody against MSI2 was purchased from Abcam (Cambridge, UK). Anti-MDR1 and anti-Myc tag antibodies were purchased from Cell Signaling Technology. β-actin was used as a loading control and was detected using an antibody against it (Sigma-Aldrich).

Changes in mRNA levels were measured using qRT-PCR. The cells were lysed using TRIzol reagent (Invitrogen-Thermo Fisher Scientific), and total RNA was purified according to the manufacturer's instructions. cDNA was synthesized using M-MLV reverse transcriptase (Invitrogen-Thermo Fisher Scientific). PCR was performed using HOT FIREPol EvaGreen qPCR Mix Plus (Solis BioDyne, Tartu, Estonia) with a StepOnePlus Real-Time PCR System (Applied Biosystems-Thermo Fisher Scientific). The primers used to amplify human MSI2 were 5′-GTTAT CTGCGAACACAGTAGT-3′ (forward) and 5′-CTCTGTGCC TGTTGGTAG-3′ (reverse). The primers used to amplify human MDR1 were 5′-TGACATTTATTCAAAGTTAAA GCA-3′ (forward) and 5′-TAGACACTTTATGCAAACATT TCA-3′ (reverse). The primers used to amplify human β-actin were 5′-GGATTCCTATGTGGGCGACGA-3′ (forward) and 5′-CGCTCGGTGAGGATCTTCATG-3′ (reverse). The C_T-values for MSI2 and MDR1 were normalized to β-actin. The 2^−ΔΔCt method was used to calculate the relative expression levels of MSI2 and MDR1.

The human MSI2 open reading frame (ORF) was cloned into the pcDNA3.1-Myc/His vector (Invitrogen-Thermo Fisher Scientific), and the cells were transfected into the plasmids using Lipofectamine^® (Invitrogen-Thermo Fisher Scientific). The MSI2 and scramble siRNAs were purchased from Bioneer Corp. (Daejeon, Korea), and the cells were transfected with siRNA using Lipofectamine^® RNAiMAX (Invitrogen-Thermo Fisher Scientific). To generate the MSI2 lentiviral plasmid, ORF of MSI2 was cloned into the pLentiHT vector. The lentiviral vectors expressing shRNA against MSI2 were purchased from Sigma-Aldrich. To generate MSI2 ORF and MSI2 shRNA lentiviral particles, the 293T cells were co-transfected with the pCMV-VSV-G plasmid (Addgene, Cambridge, MA, USA) and pCMV-dR 8.2 (Addgene), and the viral particles were harvested after 72 h. The cells were infected in the presence of polybrene (8 μg/ml). The cells were subsequently transduced and selected using 1 μg/ml puromycin (Amresco, Solon, OH, USA).

To analyze apoptosis, the cells were washed with phosphate-buffered saline (PBS; Thermo Fisher Scientific) and resuspended in 1X binding buffer (100 mM HEPES, pH 7.4, 140 mM NaOH, 2.5 mM CaCl₂). The cells were incubated with Annexin V-FITC and PI staining solution (FITC Annexin V apoptosis detection kit; BD Biosciences, Franklin Lakes, NJ, USA) for 15 min at RT in the dark. The quantity of apoptotic cells was determined using a BD FACSCalibur (BD Biosciences), and the results were analyzed using the CellQuest program (BD Biosciences).

The expression level of MSI2 in ovarian cancer tissue samples was analyzed using ovarian cancer tissue microarrays (58 cases) (Super BioChips, Seoul, Korea). The tissue array was deparaffinized using xylene (Thermo Fisher Scientific) and rehydrated using graded ethanol (100, 95, 90, 80 and 70%). After the slide was washed with distilled water, H&E staining was performed using a Mayer's hamalum solution (Merck-Millipore, Billerica, MA, USA) and an Eosin Y-solution 0.5%, aqueous (Merck-Millipore). The stained slide was then dehydrated using graded ethanol (70, 90, 95 and 100%) and xylene (Thermo Fisher Scientific), and the staining was visualized under a microscope. For IHC analysis, the ovarian cancer tissues were first incubated with a normal goat serum blocking solution (2% goat serum) and then stained for MSI2 by incubating with the MSI2-antibody (Abcam) diluted 1:100 overnight at 4°C. The slide was washed with PBS supplemented with Tween-20 (0.05% PBST) and subsequently incubated with HRP-conjugated secondary antibody diluted 1:5,000 for 90 min at room temperature. After the slides were washed in PBST, staining was visualized using a DAB substrate (Thermo Fisher Scientific). MSI2-positive cells were counted by a pathologist (Dr Yi Kyeong Chun, Cheil General Hospital and Women's Healthcare Center, Dankook University, College of Medicine, Seoul, Korea). Staining intensity was scored as follows: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining.

The results of three experiments per group were statistically analyzed. The data are presented as the mean ± standard deviation (SD). Statistical analyses were conducted using a two-tailed Student's t-test analysis, chi-square test, or Fisher's exact test and Spearman's rank correlation coefficient analysis. P<0.05 was considered statistically significant.

To establish the clinical significance of MSI2, we examined MSI2 expression in ovarian cancer tissues. Using a tissue microarray comprising 58 human ovarian cancer tissues, we conducted an IHC analysis of MSI2 expression and analyzed the correlation between MSI2 expression and various clinicopathological parameters such as tumor stage and subtype. In addition to being the most common histological subtype, serous ovarian cancer is the most lethal type of ovarian cancer, followed by mucinous, endometrioid and clear cell carcinoma (21). Our IHC results indicated that MSI2 was expressed at significantly higher levels in serous ovarian carcinoma tissues (P<0.001) than in other types of ovarian carcinoma tissues (Fig. 1A). In addition, MSI2 expression levels in advanced ovarian cancer tissues (grade III and IV) were significantly greater than in grade I and II tissues (P<0.05; Fig. 1B and C). Together, these data suggested that MSI2 plays an important role in ovarian cancer growth and progression.

Previous studies have shown that MSI2 regulates cell growth in several types of cancers including colorectal and brain cancers (3,22). Therefore, we hypothesized that MSI2 affects the growth and proliferation in ovarian cancer cells. To address this hypothesis, we transfected MSI2 plasmids into ovarian cancer SKOV3 and A2780 cells and conducted a WST-1-based cell viability assay. As shown in Fig. 2A, cell viability significantly increased in cells overexpressing MSI2 than in the control cells. We further analyzed cell proliferation in the control and MSI2-transfected SKOV3 and A2780 cells using the BrdU incorporation assay. Consistent with the results of the cell viability assay, MSI2 overexpression significantly increased cell proliferation (Fig. 2B). We confirmed the results of the cell proliferation and viability assays using a cell growth assay. The control and MSI2-transfected cells were cultured for 5 days, and the relative cell numbers were evaluated daily using trypan blue staining. As expected, MSI2-overexpressing SKOV3 and A2780 cells exhibited a significant increase in growth rate compared with the control cells (Fig. 2C). Together, these results suggested that inhibiting MSI2 expression is a promising strategy for regulating ovarian cancer growth.

Approximately 70% of patients with ovarian cancer are diagnosed at an advanced stage, and a majority of patients who initially respond to treatment ultimately relapse (23), which is commonly due to the development of chemoresistance (24). We found that MSI2 expression levels were significantly greater in stage III and IV ovarian cancer tissues than in stage tumor I and II tissues (Fig. 1) and that MSI2 overexpression enhanced ovarian cancer cell growth (Fig. 2). Therefore, we sought to determine if MSI2 also plays a role in paclitaxel resistance. To this end, we evaluated the expression levels of MSI2 in IOSE ovarian epithelial cells and various paclitaxel-sensitive and -resistant ovarian cancer cell lines (HeyA8, SKOV3, SKOV3-TR and A2780). To evaluate MSI2 protein levels, the cultured cells were lysed and analyzed using SDS-PAGE and immunoblotting with an MSI2-specific antibody. As shown in Fig. 3A, MSI2 protein levels were the highest in the paclitaxel-resistant SKOV3-TR cell line compared to the other cell lines. Moderate MSI2 levels were observed in A2780 cells, and relatively lower levels were observed in the IOSE, HeyA8 and SKOV3 cells. Next, we investigated MSI2 mRNA levels using qRT-PCR and found that MSI2 mRNA was highly expressed in the SKOV3-TR cells (Fig. 3B). However, the overall pattern of MSI2 mRNA expression was not identical to that of MSI2 protein expression, suggesting that post-transcriptional regulation influences the pattern of MSI2 expression in ovarian cancer cells. To further analyze the relationship between MSI2 protein levels and paclitaxel sensitivity in ovarian cancer cells, we conducted a WST-1-based cytotoxicity assay in the paclitaxel-treated cells. As shown in Fig. 1C, the SKOV3-TR cells, which exhibited elevated MSI2 protein levels, exhibited the strongest paclitaxel resistance at all doses examined. The A2780 cells, which exhibited only moderate MSI2 protein levels, exhibited moderate paclitaxel resistance at low doses (<20 ng/ml); however, higher doses of paclitaxel were significantly cytotoxic. Ovarian cancer cells with low MSI2 protein levels (IOSE, HeyA8 and SKOV3) exhibited paclitaxel sensitivity even at low doses (Fig. 3C). These data suggested that paclitaxel sensitivity and resistance are regulated by MSI2 protein levels in ovarian cancer cells.

Based on the results described above, we hypothesized that MSI2 regulates paclitaxel cytotoxicity in ovarian cancer cells. To investigate this hypothesis, we generated stable MSI2 knockdown cells using an shRNA-expressing lentivirus. As shown in Fig. 4A, we observed a significant reduction in MSI2 mRNA and protein levels in shMSI2-expressing SKOV3-TR cells. In addition, paclitaxel exerted a significantly greater toxicity in the shMSI2-expressing SKOV3-TR cells than in the corresponding control cells (SKOV3-TR shScramble) (Fig. 4B), indicating MSI2 knockdown abolished paclitaxel resistance in the SKOV3-TR cells. To confirm these data, we evaluated apoptosis in the control and MSI2-knockdown SKOV3-TR cells treated with paclitaxel. Consistent with the results of the cytotoxicity assays, paclitaxel-induced apoptosis (both early and late apoptosis) increased in the MSI2-knockdown SKOV3-TR cells compared with that in the control SKOV3-TR cells (Fig. 4C). Previous reports have demonstrated that paclitaxel resistance is correlated with multidrug resistant 1 (MDR1) expression levels (25–27). To determine if shMSI2-induced paclitaxel sensitivity was associated with changes in MDR1 levels, we evaluated MDR1 levels in the cells transfected with MSI2-specific siRNA using qRT-PCR and immunoblotting assays. As shown in Fig. 4D, MDR1 mRNA and protein levels significantly decreased in the SKOV3-TR cells transfected with MSI2 siRNA in a dose-dependent manner. Together, these data suggested that MSI2 plays an important role in paclitaxel resistance in ovarian cancer cells.

Next, we examined if MSI2 overexpression can induce paclitaxel resistance in paclitaxel-sensitive SKOV3 cells. To this end, we generated stable MSI2-overexpressing SKOV3 (SKOV3-MSI2) cells using an MSI2-expressing lentivirus (Fig. 5A, right panel) and analyzed their cell viability after paclitaxel treatment. As shown in Fig. 5A, the paclitaxel-mediated inhibition of cell viability significantly decreased in the MSI2-overexpressing SKOV3 cells than in the control SKOV3 cells. To further evaluate the inhibitory effect of MSI2 on paclitaxel-induced apoptosis, we treated the control and MSI2-overexpressing SKOV3 cells with paclitaxel and analyzed apoptosis using Annexin V/PI staining and flow cytometry. As shown in Fig. 5B, paclitaxel-induced apoptosis significantly decreased in the MSI2-overexpressing SKOV3 cells than in the control SKOV3 cells. As siRNA directed against MSI2 inhibited MDR1 expression in the SKOV3-TR cells (Fig. 4D), we investigated the relationship between MSI2-mediated paclitaxel resistance and MDR1 expression in the SKOV3 cells. As expected, transient MSI2 overexpression upregulated MDR1 mRNA and protein levels in the SKOV3 cells in a dose-dependent manner (Fig. 5C). Finally, we sought to determine if the effect of MSI2 on paclitaxel resistance in the SKOV3-TR cells was similar to that in other ovarian cancer cells (HeyA8 and A2780). Therefore, we used the WST-1 assay to evaluate cell viability in the MSI2-overexpressing HeyA8 and A2780 cells treated with the indicated doses of paclitaxel for 48 h. Consistent with that observed in the SKOV3 cells, paclitaxel resistance increased in the MSI2-expressing HeyA8 and A2780 cells compared with that in the corresponding non-transfected control cells (Fig. 5D). Together, these data indicate that MSI2 is a novel contributor to paclitaxel resistance in ovarian cancer cells.