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MiR-199a-3p enhances breast cancer cell sensitivity to cisplatin by downregulating TFAM (TFAM)
Biomedicine & Pharmacotherapy 88 (2017) 507–514
Available online at
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Original article
MiR-199a-3p enhances breast cancer cell sensitivity to cisplatin by downregulating TFAM (TFAM) Xuelong Fana,b , Shangcheng Zhouc, Miao Zhengd , Xiyun Denga , Yinsha Yia , Tieniu Huangb,e,* a
Medicine College of Hunan Normal University, Changsha, Hunan, 410006, China School of Public Health, Central South University, Changsha, Hunan, 410078, China Public Health and Management School of Hubei Medical College, Shiyan, Hubei, 442000, China d Anbu Overseas Chinese Hospital of Chaoan County of Guangdong Province, Chaozhou, Guangdong, 515638, China e Xiangtan Medicine & Health Vocational College, Xiangtan, Hunan, 411102, China b c
A R T I C L E I N F O
Article history: Received 2 December 2016 Received in revised form 28 December 2016 Accepted 9 January 2017 Keywords: Chemotherapy resistance Cisplatin Breast cancer MicroRNAs Apoptosis TFAM
A B S T R A C T
Chemotherapy resistance is the major obstacle to the effective therapy of cancer. While the mechanism of chemotherapy resistance is still not fully understood. Increasing evidences demonstrated that microRNAs (miRNAs) may have a crucial function in chemotherapy resistance through modulating intracellular pathways. MiR-199a has been shown to be involved in multiple malignancy-related processes, although the precise mechanism is unclear at present. In this study, we found that the expression level of miR-199a-3p was lower in cisplatin (DDP) resistant breast cancer MDA-MB-231/DDP cells compared with parental DDP-sensitive cells. Inhibition of miR-199a-3p in MDA-MB-231 cells significantly attenuated DDP-induced apoptosis and anti-proliferative effects, while overexpression of miR-199a-3p in MDA-MB-231/DDP cells increased the sensitivity to DDP. Moreover, expression levels of mitochondrial transcription factor A (TFAM) were modulated by miR-199a-3p. The luciferase reporter assay indicated that TFAM may be the target gene of miR-199a. Knocking down of TFAM could partially reverse DDP resistance in MDA-MB-231 cells induced by miR-199a-3p inhibition, while TFAM overexpression could partially restore miR-199a-3p-induced chemo-sensitivity of MDA-MB-231/DDP cells to DDP. These results show that miR-199a-3p is able to attenuate cisplatin resistance in breast cancer cells through inhibiting TFAM expression. © 2017 Published by Elsevier Masson SAS.
1. Introduction As the second leading cause of cancer death among women, breast cancer is lacking in obvious early symptoms. Many patients are diagnosed with the disease at late stages [1,2]. Cytotoxic chemotherapy has contributed to dramatic improvements of survival rate of breast cancer patients. While other factors may contribute to the overall poor prognosis of breast cancer, chemotherapy resistance is the major obstacle to the effective therapy of breast cancer. Cisplatin (DDP) is a widely used chemotherapeutic agent in breast cancer and other solid tumors [3]. Clinically relevant levels of resistance can develop quickly after
* Corresponding author at: School of Public Health, Central South University, No. 238 Shangmayuanling Road, Changsha, Hunan, 410078, China and Xiangtan Medicine & Health Vocational College, Shuangyong Middle Road, Xiangtan, Hunan,411102 China. E-mail address: [email protected] (T. Huang). http://dx.doi.org/10.1016/j.biopha.2017.01.058 0753-3322/© 2017 Published by Elsevier Masson SAS.
DDP treatment. The process of DDP resistance is multifactorial, including changes in drug accumulation or drug-target interaction, increased DNA-repair, or alteration of apoptosis signal pathway [4]. However, the mechanism for DDP resistance is not fully characterized. MiRNAs (miRNAs) are a large family of highly conserved small non-coding RNAs which regulate diverse biological process by modulating gene expression at the posttranscriptional level [5]. Numerous studies have reported that miRNAs are involved in tumorigenesis, tumor progression and response to therapy [6,7]. There is a significant association between miRNA expression profiling and clinical pathological characteristics [8]. Some miRNAs could even be used as biomarkers for the diagnosis and prognosis of cancer patients [9]. For instance, miR-199a has been shown to be involved in multiple malignancy-related processes, including cell cycle process, apoptosis and invasion [10,11]. MiR199a is found to be expressed at different levels in various human cancers. For example, it was expressed at a low level in
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hepatocellular carcinoma [12], but highly expressed in ovarian and gastric cancer [13,14]. In addition, in different breast cancer cell lines, miR-199a shows dual roles in autophagy, which inhibits radiation-induced autophagy in MCF-7 cells, but upregulates autophagy in MDA-MB-231 cells [15]. Hence, expression of miR199a-3p and its role in breast cancer cell need to be further studied. In this study, we first confirmed that a breast cancer cell line resistant to DDP MDA-MB-231/DDP exhibited a significantly lower expression level of miR-199a-3p compared with its parental cell line MDA-MB-231. Furthermore, we found that miR-199a-3p could regulate the expression of mitochondrial transcription factor A (TFAM), a crucial factor in drug resistance and tumor progression, by repressing TFAM 30 UTR. This interaction consequently regulates the resistance of breast cancer cell to DDP.
vector (Yrbio, China), designated pYr-TFAM-3U and PYr-TFAM3Umt. The primers for the wild-type 30 UTR were: forward primer: 50 -aaa ctc gag gtc tca ata cct gaa gct-30 ; reverse primer: 50 -aaa gcg gcc gc cct gcc tcc ata ata taa-30 , containing XhoI and NotI restriction sites. The primers for the mutant 30 UTR were: forward: 50 -tat ttt gtg ttt agg aga tga cag gat cag agt aat cca ag-30 ; reverse primer: 50 ctt gga tta ctc tga tcc tgt cat ctc cta aac aca aaa ta-30 . HEK293 cells (ATCC, USA) were placed into a 96 well plate and co-transfected with 100 ng pYr-TFAM-3U or pYr-TFAM-3Umt and miRNA mimics (50 nM), using Lipofectamine 2000. Luciferase activity was determined by the Dual-Luciferase reporter assay kit (Promega, USA) following the manufacturer’s instructions. Each experiment was repeated at least three times in triplicate. 2.5. Cell viability assay
2. Materials and methods 2.1. Cell lines and cell culture Human breast cancer cell line MDA-MB-231 (ATCC, USA) and its DDP resistant variant MDA-MB-231/DDP (YRbio, China) were cultured in DMEM medium (GIBCO, USA) supplemented with 10% fetal calf serum (GIBCO, USA), 100 units/ml penicillin and 100 mg/ ml streptomycin in a humidified incubator with 5% CO2 at 37 C. 2.2. Transfection of miR-199a-3p mimic and inhibitor The miR-199a-3p mimics and inhibitors were purchased from GenePharm, China. Cells were seeded at 3 105 per well in 6-well plates and cultured for 24 h. Then the cells were transfected with the mimics or inhibitor of miR-199a-3p or negative control (NC) RNA, at a final concentration of 50 nM, using Lipofectamine 2000 (Invitrogen, USA) and serum-free Opti-MEM medium (GIBCO, USA). After 6 h, the medium was replaced with DMEM containing 10% FBS. Total RNAs and proteins were extracted after 48 h of transfection. 2.3. Knockdown or overexpression of TFAM Specific siRNA of TFAM (si-TFAM, sense:aag uug ucc aaa gaa acc utt; antisense: agg uuu cuu ugg aca acu utt) and negative control (siRNA, sense: 50 -uuc ucc gaa cgu guc acg utt-30 ; antisense: 50 -acg uga cac guu cgg aga att-30 ) were purchased from GenePharma, Co., Ltd., Shanghai, China. Full length human TFAM cDNA generated by reverse transcription-polymerase chain reaction (using olig 50 -ccc aag ctt atg gcg ttt ctc cga agc-30 and 50 -cgc gga tcc tta aca ctc agc acc-30 ) with HindIII and BamH I sites at the 50 and 30 ends was cloned into the pcDNA expression vector. For TFAM knockdown treatment, MDA-MB-231/DDP cells (1.5 105) grown on six-well plates were transfected with 100 pmol si-TFAM or control siRNA using 8 ml siRNA-Mate transfection reagent (GenePharma, Co., Ltd., Shanghai, China). For TFAM overexpression treatment, MDA-MB231 cells (2 105) grown on six-well plates were transfected with 2 mg of TFAM overexpression plasmid or pCDNA using Lipofectamine 2000 (Invirtogen, USA) and serum-free Opti-MEM medium (GIBCO, USA).as described by the manufacturer. The cells were harvested after 48 h. Western blot analyses or other experiments were performed. 2.4. Construction of vector and luciferase reporter assay The 30 -UTR of TFAM gene (position 213–220) was predicted to be complementary to the sequence of miR-199a-3p according to an analysis of the miRNA target gene prediction database, TargetScan. The fragment sequence of wild-type and mutant TFAM 30 -UTR was amplified by PCR and cloned into the pYr-MirTarget luciferase
The cytotoxic effect of DDP was measured by MTT assay as previously described [16]. Briefly, the MDA-MB-231 or MDA-MB231/DDP was seeded in 96-well plate at 2 104 per well and cultured for 24 h, and transfected with the miR-199a-3p mimics or inhibitors for 24 h. The cells were then treated with different concentration of DDP (0, 5, 10, 20, 40, 60, 80,100 mM) for 48 h. The MTT (Sigma, USA) was added to a final concentration of 0.5 mg/ml, and the cells were incubated for 4 h at 37 C. The absorbance at 570 nm was measured in a microplate reader (Bio-rad, USA). Each experiment was repeated at least three times in triplicate. 2.6. Detection of mtDNA copy number [17] MtDNA copy number was measured using a real time quantitative polymerase chain reaction (PCR) using an Applied Biosystems 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). One primer pair specific for the mitochondrial DNA (ND1) and another primer pair specific for the nuclear DNA (18s) were designed for relative quantification for mtDNA copy number. The primer sequences for the mitochondrial ND1 gene were: forward primer 50 - ccc taa aac ccg cca cat ct-30 ; reverse primer 50 gag cga tgg tga gag cta agg t-30 . The primer pair used for the amplification of the nuclear gene 18s was as follows: forward primer, 50 -tag agg gac aag tgg cgt tc-30 ; reverse primer, 50 -cgc tga gcc agt cag tgt-30 . 2.7. Apoptosis assay Analysis of cell apoptosis was performed with the Annexin VFITC apoptosis detection kit (Keygen, China). 24 h after transfection, the MDA-MB-231 and MDA-MB-231/DDP cells were treated with DDP (20 mM) for 48 h. Then the cell samples were harvested with 0.25% trypsin without EDTA washed twice with ice-cold PBS and re-suspended in 500 ml binding buffer. Then the cells were incubated with 5 ml Annexin V-FITC and 5 ml propidium iodide (PI) for 15–20 min in the dark and detected by BD FascAria flow cytometer (BD, USA) with the excitation wavelength of 488 nm and emission wavelength of 530 nm. Each experiment was repeated three times in triplicate. 2.8. qRT-PCR analysis Total microRNAs were extracted by the miRNeasy Kit (Qiagen, German) according to the manufacturer’s instructions. RNA was converted to cDNA using the miScriptRT kit (Qiagen, German). QRT-PCR was performed using a miScript SYBR Green PCR kit (Qiagen, German) on the ABI 7900HT Real-time PCR System (Applied Biosystems, USA). The expression of miRNAs was normalized using the RNU6 as endogenous control. The primers of miR-199a and U6 were purchased from Yrbio Biotech (China).
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Total RNA was isolated with Trizol Reagent (Invitrogen, USA) according to the manufacturer’s instructions. RNA was converted to cDNA using the PrimeScript1 1 st Strand cDNA Synthesis Kit (Takara, Japan). QRT-PCR was performed using SYBRR Premix Ex TaqTM (TaKaRa, Japan) on ABI7500 Real-time PCR Systerm (Applied Biosysterm, USA). TFAM primers were: forward 50 -tcc aag aag cta agg gtg-30 ; and reverse 50 -ttc cca aga ctt cat ttc a-30 . b-actin primers were: 50 -ggg aaa tcg tgc tgc gtg ac- 30 ; and reverse 50 -ttg cca atg gtg atg acct g- 30 . All of the qRT-PCR assays were performed in triplicate and the change was calculated using the 2DDCt method. 2.9. Western blot analysis After transfection and DDP treatment, cells were harvested and homogenized with lysis buffer (Beyotime, China). The protein concentrations were measured using the BCA protein assay kit (Beyotime, China). Proteins (30 to 50 mg) were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane (Millipore, USA). After blocking with 5% non-fat dry milk, the membranes were incubated with primary antibodies specific to TFAM and b-actin (1:1000, Santa Cruz, USA). The membranes were further incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (1:5000, Santa Cruz, USA). The immunoblots were quantified by the ImageJ software.
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231/DDP cells (Fig. 1B, p < 0.01). Therefore, the effect of miR-199a3p on DDP resistance in MDA-MB-231 cells was further investigated. 3.2. Effect of miR-199a-3p on sensitivity to DDP in MDA-MB-231 and MDA-MB-231/DDP cells To investigate the effect of miR-199a-3p on regulating MDAMB-231 cells resistant to DDP, the MDA-MB-231 and MDA-MB231/DDP cells were respectively transfected with miR-199a-3p inhibitor or mimics and then treated with 20 mM DDP for 48 h. Transfection of miR-199a-3p inhibitor down-regulated miR-199a3p expression and attenuated DDP-induced apoptosis in MDA-MB231 cells compared with negative control (NC) transfection (Fig. 2A and C). On the contrary, MDA-MB-231/DDP cells transfected with miR-199a-3p mimics effectively increased the miR-199a-3p expression and enhanced the apoptosis to DDP compared with the cells transfected with NC (Fig. 2B and D). Moreover, transfection with miR-199a-3p inhibitor decreased the sensitivity of MDA-MB-231 to DDP. In contrast, transfection with miR-199a3p mimics significantly enhanced the sensitivity of MDA-MB-231/ DDP cells to DDP (Fig. 2E and F). These results indicated that miR199a-3p is involved in the DDP resistance of breast cancer cells. 3.3. Regulation of TFAM expression by miR-199a-3p
2.10. Statistical analysis The Student’s t-test was used for comparison between two groups. p < 0.05 was considered to indicate statistical significance. Test data from independent experiments were repeated three times. 3. Results 3.1. Expression levels of miR-199a-3p in MDA-MB-231 and MDA-MB231/DDP cells The cell viability of MDA-MB-231 and MDA-MB-231/DDP cells under cisplatin treatment for 48 h was shown in Fig. 1A. The IC50 of MDA-MB-231/DDP cells showed 4.42 fold higher than that of MDAMB-231 cells (22.1 vs. 97.6 mM, p < 0.01). To investigate the involvement of miR-199a-3p in DDP resistant breast cancer cells MDA-MB-231/DDP, the levels of miR-199a-3p were detected by qRT-PCR. Compared with MDA-MB-231 cells, the expression levels of miR-199a-3p were significantly down-regulated in MDA-MB-
TFAM has also been implicated as a key regulator involved in mtDNA transcription and replication [18]. Furthermore, it was reported that TFAM is involved in breast cancer development and drug resistance [19]. As shown in Fig. 3A, the expression of TFAM was significantly higher in MDA-MB-231/DDP cells compared with MDA-MB-231 cells. To determine whether miR-199a-3p is involved in the modulation of TFAM expression, the miR-199a-3p inhibitor and mimics were transfected into the MDA-MB-231 or MDA-MB-231/ DDP cells. The expression of TFAM was increased following miR199a-3p inhibitor transfection in MDA-MB-231 cells (Fig. 3B). In contrast, the reduced TFAM expression was observed in MDA-MB231/DDP cells transfected with miR-199a-3p mimics compared with the NC group (Fig. 3C). 3.4. TFAM may be a target gene of miR-199a-3p To identify whether TFAM is the target gene of miR-199a-3p, the miRNA target prediction public database (TargetScan, USA) was
Fig. 1. Expression levels of miR-199a-3p in MDA-MB-231 and MDA-MB-231/DDP cells. (A) Survival curves of MDA-MB-231 and MDA-MB-231/DDP cells. The cells were exposed to different concentration of cisplatin (DDP) (0, 5, 10, 20, 40, 60, 80, 100 mM) for 48 h. Cell viability was determined by MTT assay. (B) Expression level of miR-199a-3p was lower in MDA-MB-231/DDP cells compared with MDA-MB-231 cells (p < 0.01 vs.MDA-MB-231 cells).
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Fig. 2. MiR-199a-3p inhibits cisplatin resistance in MDA-MB-231/DDP cells. (A, B) After 48 h transfection of miR-199a-3p mimics or inhibitor, the expression of miR-199a-3p in MDA-MB-231 cells and MDA-MB-231/DDP cells was determined by qRT-PCR, respectively. (C) Inhibition of miR-199a-3p attenuated DDP-induced apoptosis in MDA-MB231 cells after 48 h 20 mM DDP treatment. Apoptosis rate was detected by flow cytometry with annexin V-FITC/PI staining. (D) Overexpression of miR-199a-3p enhanced the DDP-induced apoptosis in MDA-MB-231/DDP cells after 48 h 20 mM DDP treatment. Apoptosis rate was detected by flow cytometry with annexin V-FITC/PI staining. (E) After 48 h transfection of miR-199a-3p inhibitor, MDA-MB-231 cells decreased sensitivity to various doses of DDP treatment. Cell viability was assessed by MTT assay. (F) After 48 h transfection of miR-199a-3p mimics in MDA-MB-231/DDP cells, sensitivity to various doses of DDP treatment increased. Cell viability was assessed by MTT assays. *p < 0.05 and **p < 0.01 vs. NC mimics or inhibitor.
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Fig. 3. Regulation of TFAM by miR-199a-3p. (A) Expression of TFAM in MDA-MB-231 and MDA-MB-231/DDP cells was determined by western blot analysis. The densitometric analysis results were shown in the right panels. Expression of TFAM in MDA-MB-231 cells transfected with miR-199a-3p inhibitor (B) and in MDA-MB-231/DDP cells transfected with miR-199a mimics (C). The densitometric analysis results were shown in the right panels. *p < 0.05 and **p < 0.01 vs. NC mimics or inhibitor.
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used for analysis. It was found that the 30 -UTR mRNA of TFAM contained a highly conserved binding site for miR-199a-3p (Fig. 4A). To investigate the association between TFAM and miR199a-3p, the HEK293 cells were co-transfected with pYr-TFAM-3U or pYr-TFAM-3Umt and miR-199a mimics or miR-199a-3p inhibitor. The results showed that miR-199a-3p mimics dramatically reduced the luciferase activity of pYr-TFAM-3U, while there was no difference in the luciferase activity of pYr-TFAM-3Umt; miR-199a3p inhibitor increased the luciferase activity of pYr-TFAM-3U, while there was no difference in the luciferase activity of pYrTFAM-3Umt (Fig. 4B). These data suggested that TFAM may be a target gene of miR-199a-3p. 3.5. MiR-199a-3p/TFAM modulates cell proliferation and mtDNA copy number in MDA-MB-231 and MDA-MB-231/DDP cells To investigate the role of miR-199a-3p/TFAM in regulating the chemoresistance of MDA-MB-231, we performed a series of functional assays. MDA-MB-231 cells were co-transfected with NC inhibitor/miR-199a-3p inhibitor and siRNA/si-TFAM; MDA-MB231/DDP cells were co-transfected with NC mimics/miR-199a-3p mimics and pcDNA/TFAM. Transfection with miR-199a-3p inhibitor resulted in TFAM protein level and mtDNA copy number significantly up-regulated, while this promoting effect of miR199a-3p inhibitor could be partially restored by si-TFAM transfection in MDA-MB-231 cells (Fig. 5A and C). In MDA-MB-231/DDP cells, transfection with miR-199a-3p mimics led to a strong repression of TFAM protein level and mtDNA copy number, while this inhibitory effect of miR-199a-3p mimics could be partially restored by TFAM overexpression (Fig. 5B and D). To investigate the role of miR-199a-3p/TFAM in regulating breast cancer cells’
Fig. 4. TFAM may be a target gene of miR-199a-3p. (A) TFAM’s UTR mRNA includes a highly-conserved binding site for miR-199a-3p. (B) The HEK293 cells were cotransfected with pYr-TFAM-3U or pYr-TFAM-3Umt and miR-199a-3p mimics or inhibitor. After 24 h transfection, the luciferase activity decreased in pYr-TFAM-3U and miR-199a-3p mimics co-transfected cells, while increased in pYr-TFAM-3U and miR-199a-3p inhibitor co-transfected cells. The modification of luciferase activity by miR-199a-3p mimics and inhibitor was abolished when the potential target site was mutated. **p < 0.01.
chemo-resistance, the viability of cells under DDP treatment was monitored. Results showed that miR-199a-3p inhibitor reduced the chemo-sensitivity of MDA-MB-231 cells to DDP, while this effect could be partially restored by si-TFAM transfection (Fig. 5E). The chemoresistance of MDA-MB-231/DDP cells could be attenuated by miR-199a-3p mimics, while this effect could be partially restored by TFAM overexpression. These data indicated that miR-199a-3p/TFAM regulates the chemo-resistance of breast cancer cells to DDP. 4. Discussion MiRNAs play a crucial role in many biological processes, such as proliferation, apoptosis and differentiation, through modulating gene expression at the posttranscriptional level [5]. Aberrant miRNAs could affect the expression of target genes, which may modulate cell death signal pathway, drug target proteins and cell cycle-related proteins. This may lead to resistance of cancer cells to chemotherapy [20]. For instance, upregulation of miR-22 and miR205 induces resistance of breast cancer cells to DDP through modulating AKT signal pathway [21,22]. Upregulation of miR-127 is associated with increasing cell sensitivity to DDP [23]. In the present study, we first demonstrated that miR-199a-3p was expressed at a lower level in DDP resistant breast cancer cell line MDA-MB-231/DDP compared with the parental MDA-MB-231 cells. Furthermore, overexpression of miR-199a-3p in MDA-MB231/DDP cells inhibited TFAM expression, promoting the sensitivity of MDA-MB-231/DDP cells to DDP. Inhibition of miR-199a-3p in MDA-MB-231 cells led to promoting TFAM, while the sensitivity to cisplatin was decreased. These findings indicate that upregulation of miR-199a-3p may promote the sensitivity of breast cancer cells to DDP through downregulation of TFAM expression. Accumulating lines of evidence have confirmed that miR-199a is involved in cancer development by targeting oncogenes or tumor suppressors [11,24]. The expression of miR-199a was diversely deregulated in several types of cancer. For instance, miR-199a was downregulated in ovarian cancer, prostate cancer and bladder cancer, but it was upregulated in gastric cancer and cervical carcinoma [25,26]. Moreover, it has been demonstrated that miR-199a was the differential expressed between chemosensitive and chemoresistant cells which may participate in the cell chemoresistance. Downregulation of miR-199a is found in ovarian cancer cells and hepatoma cells resistant to DDP [27,28]. MiR-199a-3p is downregulated in hepatocellular carcinoma, restoring attenuated levels of miR-199a-3p in hepatocellular carcinoma cells, leading to increased sensitivity to doxorubicininduced apoptosis [29]. These studies indicated that miR-199a-3p may play a crucial role in chemotherapy resistance. Here we found that miR-199a-3p expression was significantly down-regulated in MDA-MB-231/DDP cells compared with the parental cells (Fig. 1B). Inhibition of miR-199a-3p in MDA-MB-231 cells reduced DDPinduced cell apoptosis and anti-proliferative effect; while overexpression of miR-199a-3p significantly promoted cisplatininduced apoptosis and anti-proliferative effect in MDA-MB-231/ DDP cells (Fig. 2). To further investigate the mechanism of miR-199a-3p in the DDP resistance, we predicted a possible target mRNA for miR199a-3p using computational algorithms (TargetScan). The intersection of algorithms and luciferase reporter assay indicated that TFAM was a target gene of miR-199a-3p (Fig. 4). In MDA-MB-231/ DDP cells, TFAM was highly expressed compared with MDA-MB231 cells, and could be negatively regulated by miR-199a-3p in both MDA-MB-231 and MDA-MB-231/DDP cells. As a regulator of mtDNA replication and transcription, TFAM is also known to be critical for maintenance of mitochondrial biogenesis and function [30]. Mitochondrial function and/or mtDNA content acceleration
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Fig. 5. MiR-199a-3p/TFAM modulates cell proliferation and mtDNA copy number in MDA-MB-231 and MDA-MB-231/DDP cells. (A) MDA-MB-231 cells were co-transfected with NC inhibitor/miR-199a-3p inhibitor and siRNA/si-TFAM. The protein levels of TFAM were determined using Western blot analysis. (B) MDA-MB-231/DDP cells were cotransfected with NC mimics/miR-199a-3p mimics and pcDNA/TFAM. The protein levels of TFAM were determined using Western blot analysis. (C, D) The mtDNA copy number of MDA-MB-231 and MDA-MB-231/DDP cells was determined in each of the indicated groups. (E, F) The cell viability of MDA-MB-231 and MDA-MB-231/DDP cells was determined in each of the indicated groups using MTT assay. *p < 0.05 and **p < 0.01 vs. NC mimics or inhibitor. ##p < 0.01 miR-199a inhibitor +siRNA vs. miR-199a inhibitor +si-TFAM or miR-199a mimics +pcDNA vs. miR-199a mimics +TFAM.
plays a key role in tumor formation and cancer pathogenesis [31,32]. Therefore, the modulation of TFAM has been involved in tumorigenesis and cancer progression [33]. Recently, it has been reported that TFAM could promote breast cancer cell proliferation and enhance DDP resistance in estrogen receptor positive breast cancer [34,35]. In the current study, we demonstrated that inhibition of miR-199a-3p in MDA-MB-231 cells led to increased TFAM expression, increased mtDNA copy numbers and reduced sensitivity of breast cancer cells to DDP. Overexpression of miR-
199a-3p in MDA-MB-231/DDP cells caused the opposite effects, i.e., reduced TFAM expression, reduced mtDNA copy number and enhanced sensitivity of breast cancer cells to DDP. Moreover, the effect of miR-199a-3p inhibition or overexpression could be partially restored by TFAM knockdown or overexpression, respectively (Fig. 5). The modulation of TFAM by miR-199a-3p contributes to the change of cell sensitivity to DDP. In summary, our finding showed that miR-199a-3p could attenuate the DDP resistance by blocking the expression of TFAM in
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cisplatin-resistant breast cancer cells. Moreover, we demonstrated that TFAM may be a target gene of miR-199a-3p and play an important role in resistance of breast cancer cells to DDP. Despite the growing evidence of cancer-specific miRNA patterns, the role of miRNA in cancer is tissue-specific and context-dependent, switching from repressors to activators in different microenvironment [36]. Therefore, future investigations using animal models and clinic patient samples are necessary to further support the function of miR-199a-3p in breast cancer resistance to DDP. Conflict of interest All authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Acknowledgements This study was funded by National Natural Science Foundation of China (No. 71273083 and 81472496) and Key Research Center for Humanities and Social Sciences in Hubei Province (Hubei University of Medicine, No. 2016YB002) References [1] J. Ferlay, H.R. Shin, F. Bray, D. Forman, C. Mathers, D.M. Parkin, Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008, Int. J. Cancer 127 (12) (2010) 2893–2917. [2] M. Kaufmann, G. von Minckwitz, H.D. Bear, A. Buzdar, P. McGale, H. Bonnefoi, M. Colleoni, C. Denkert, W. Eiermann, R. Jackesz, et al., Recommendations from an international expert panel on the use of neoadjuvant (primary) systemic treatment of operable breast cancer: new perspectives 2006, Ann. Oncol. 18 (12) (2007) 1927–1934. [3] D.P. Silver, A.L. Richardson, et al., Efficacy of neoadjuvant Cisplatin in triplenegative breast cancer, J. Clin. Oncol. 28 (7) (2010) 1145–1153. [4] A.-M. Florea, D. Büsselberg, Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects, Cancers 3 (1) (2011) 1351–1371. [5] N. Lynam-Lennon, S.G. Maher, J.V. Reynolds, The roles of microRNA in cancer and apoptosis, Biol. Rev. 84 (1) (2009) 55–71. [6] A. Fendler, M. Jung, C. Stephan, R.J. Honey, R.J. Stewart, K.T. Pace, A. Erbersdobler, S. Samaan, K. Jung, G.M. Yousef, miRNAs can predict prostate cancer biochemical relapse and are involved in tumor progression, Int. J. Oncol. 39 (5) (2011) 1183–1192. [7] M. Garofalo, C. Quintavalle, G. Romano, C.M. Croce, G. Condorelli, miR221/222 in cancer: their role in tumor progression and response to therapy, Curr. Mol. Med. 12 (1) (2012) 27–33. [8] Z. Lu, Y. Ye, D. Jiao, J. Qiao, S. Cui, Z. Liu, miR-155 and miR-31 are differentially expressed in breast cancer patients and are correlated with the estrogen receptor and progesterone receptor status, Oncol. Lett. 4 (5) (2012) 1027–1032. [9] Q. Wang, P. Li, A. Li, J. Wei, W. Hong, J. Wang, K. Xie, Plasma specific miRNAs as predictive biomarkers for diagnosis and prognosis of glioma, J. Exp. Clin. Cancer Res. 31 (2) (2012) 1–10. [10] H. He, J. Ge, J. Dong, L. Wang, Effect of miR-199a-5ptransfection on the growth of clear-cell renal-cell carcinoma 786-0 cells, J. Med. Postgrad. 26 (9) (2013) 921–924. [11] S.Q. Li, Z.H. Wang, X.G. Mi, L. Liu, Y. Tan, MiR-199a/b-3p suppresses migration and invasion of breast cancer cells by downregulating PAK4/MEK/ERK signaling pathway, IUBMB Life 67 (10) (2015) 768–777. [12] Y. Murakami, T. Yasuda, K. Saigo, T. Urashima, H. Toyoda, T. Okanoue, K. Shimotohno, Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues, Oncogene 25 (17) (2006) 2537–2545. [13] S.D. Li, J.R. Zhang, Y.Q. Wang, X.P. Wan, The role of microRNAs in ovarian cancer initiation and progression, J. Cell. Mol. Med. 14 (9) (2010) 2240–2249.
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Original article
MiR-199a-3p enhances breast cancer cell sensitivity to cisplatin by downregulating TFAM (TFAM) Xuelong Fana,b , Shangcheng Zhouc, Miao Zhengd , Xiyun Denga , Yinsha Yia , Tieniu Huangb,e,* a
Medicine College of Hunan Normal University, Changsha, Hunan, 410006, China School of Public Health, Central South University, Changsha, Hunan, 410078, China Public Health and Management School of Hubei Medical College, Shiyan, Hubei, 442000, China d Anbu Overseas Chinese Hospital of Chaoan County of Guangdong Province, Chaozhou, Guangdong, 515638, China e Xiangtan Medicine & Health Vocational College, Xiangtan, Hunan, 411102, China b c
A R T I C L E I N F O
Article history: Received 2 December 2016 Received in revised form 28 December 2016 Accepted 9 January 2017 Keywords: Chemotherapy resistance Cisplatin Breast cancer MicroRNAs Apoptosis TFAM
A B S T R A C T
Chemotherapy resistance is the major obstacle to the effective therapy of cancer. While the mechanism of chemotherapy resistance is still not fully understood. Increasing evidences demonstrated that microRNAs (miRNAs) may have a crucial function in chemotherapy resistance through modulating intracellular pathways. MiR-199a has been shown to be involved in multiple malignancy-related processes, although the precise mechanism is unclear at present. In this study, we found that the expression level of miR-199a-3p was lower in cisplatin (DDP) resistant breast cancer MDA-MB-231/DDP cells compared with parental DDP-sensitive cells. Inhibition of miR-199a-3p in MDA-MB-231 cells significantly attenuated DDP-induced apoptosis and anti-proliferative effects, while overexpression of miR-199a-3p in MDA-MB-231/DDP cells increased the sensitivity to DDP. Moreover, expression levels of mitochondrial transcription factor A (TFAM) were modulated by miR-199a-3p. The luciferase reporter assay indicated that TFAM may be the target gene of miR-199a. Knocking down of TFAM could partially reverse DDP resistance in MDA-MB-231 cells induced by miR-199a-3p inhibition, while TFAM overexpression could partially restore miR-199a-3p-induced chemo-sensitivity of MDA-MB-231/DDP cells to DDP. These results show that miR-199a-3p is able to attenuate cisplatin resistance in breast cancer cells through inhibiting TFAM expression. © 2017 Published by Elsevier Masson SAS.
1. Introduction As the second leading cause of cancer death among women, breast cancer is lacking in obvious early symptoms. Many patients are diagnosed with the disease at late stages [1,2]. Cytotoxic chemotherapy has contributed to dramatic improvements of survival rate of breast cancer patients. While other factors may contribute to the overall poor prognosis of breast cancer, chemotherapy resistance is the major obstacle to the effective therapy of breast cancer. Cisplatin (DDP) is a widely used chemotherapeutic agent in breast cancer and other solid tumors [3]. Clinically relevant levels of resistance can develop quickly after
* Corresponding author at: School of Public Health, Central South University, No. 238 Shangmayuanling Road, Changsha, Hunan, 410078, China and Xiangtan Medicine & Health Vocational College, Shuangyong Middle Road, Xiangtan, Hunan,411102 China. E-mail address: [email protected] (T. Huang). http://dx.doi.org/10.1016/j.biopha.2017.01.058 0753-3322/© 2017 Published by Elsevier Masson SAS.
DDP treatment. The process of DDP resistance is multifactorial, including changes in drug accumulation or drug-target interaction, increased DNA-repair, or alteration of apoptosis signal pathway [4]. However, the mechanism for DDP resistance is not fully characterized. MiRNAs (miRNAs) are a large family of highly conserved small non-coding RNAs which regulate diverse biological process by modulating gene expression at the posttranscriptional level [5]. Numerous studies have reported that miRNAs are involved in tumorigenesis, tumor progression and response to therapy [6,7]. There is a significant association between miRNA expression profiling and clinical pathological characteristics [8]. Some miRNAs could even be used as biomarkers for the diagnosis and prognosis of cancer patients [9]. For instance, miR-199a has been shown to be involved in multiple malignancy-related processes, including cell cycle process, apoptosis and invasion [10,11]. MiR199a is found to be expressed at different levels in various human cancers. For example, it was expressed at a low level in
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hepatocellular carcinoma [12], but highly expressed in ovarian and gastric cancer [13,14]. In addition, in different breast cancer cell lines, miR-199a shows dual roles in autophagy, which inhibits radiation-induced autophagy in MCF-7 cells, but upregulates autophagy in MDA-MB-231 cells [15]. Hence, expression of miR199a-3p and its role in breast cancer cell need to be further studied. In this study, we first confirmed that a breast cancer cell line resistant to DDP MDA-MB-231/DDP exhibited a significantly lower expression level of miR-199a-3p compared with its parental cell line MDA-MB-231. Furthermore, we found that miR-199a-3p could regulate the expression of mitochondrial transcription factor A (TFAM), a crucial factor in drug resistance and tumor progression, by repressing TFAM 30 UTR. This interaction consequently regulates the resistance of breast cancer cell to DDP.
vector (Yrbio, China), designated pYr-TFAM-3U and PYr-TFAM3Umt. The primers for the wild-type 30 UTR were: forward primer: 50 -aaa ctc gag gtc tca ata cct gaa gct-30 ; reverse primer: 50 -aaa gcg gcc gc cct gcc tcc ata ata taa-30 , containing XhoI and NotI restriction sites. The primers for the mutant 30 UTR were: forward: 50 -tat ttt gtg ttt agg aga tga cag gat cag agt aat cca ag-30 ; reverse primer: 50 ctt gga tta ctc tga tcc tgt cat ctc cta aac aca aaa ta-30 . HEK293 cells (ATCC, USA) were placed into a 96 well plate and co-transfected with 100 ng pYr-TFAM-3U or pYr-TFAM-3Umt and miRNA mimics (50 nM), using Lipofectamine 2000. Luciferase activity was determined by the Dual-Luciferase reporter assay kit (Promega, USA) following the manufacturer’s instructions. Each experiment was repeated at least three times in triplicate. 2.5. Cell viability assay
2. Materials and methods 2.1. Cell lines and cell culture Human breast cancer cell line MDA-MB-231 (ATCC, USA) and its DDP resistant variant MDA-MB-231/DDP (YRbio, China) were cultured in DMEM medium (GIBCO, USA) supplemented with 10% fetal calf serum (GIBCO, USA), 100 units/ml penicillin and 100 mg/ ml streptomycin in a humidified incubator with 5% CO2 at 37 C. 2.2. Transfection of miR-199a-3p mimic and inhibitor The miR-199a-3p mimics and inhibitors were purchased from GenePharm, China. Cells were seeded at 3 105 per well in 6-well plates and cultured for 24 h. Then the cells were transfected with the mimics or inhibitor of miR-199a-3p or negative control (NC) RNA, at a final concentration of 50 nM, using Lipofectamine 2000 (Invitrogen, USA) and serum-free Opti-MEM medium (GIBCO, USA). After 6 h, the medium was replaced with DMEM containing 10% FBS. Total RNAs and proteins were extracted after 48 h of transfection. 2.3. Knockdown or overexpression of TFAM Specific siRNA of TFAM (si-TFAM, sense:aag uug ucc aaa gaa acc utt; antisense: agg uuu cuu ugg aca acu utt) and negative control (siRNA, sense: 50 -uuc ucc gaa cgu guc acg utt-30 ; antisense: 50 -acg uga cac guu cgg aga att-30 ) were purchased from GenePharma, Co., Ltd., Shanghai, China. Full length human TFAM cDNA generated by reverse transcription-polymerase chain reaction (using olig 50 -ccc aag ctt atg gcg ttt ctc cga agc-30 and 50 -cgc gga tcc tta aca ctc agc acc-30 ) with HindIII and BamH I sites at the 50 and 30 ends was cloned into the pcDNA expression vector. For TFAM knockdown treatment, MDA-MB-231/DDP cells (1.5 105) grown on six-well plates were transfected with 100 pmol si-TFAM or control siRNA using 8 ml siRNA-Mate transfection reagent (GenePharma, Co., Ltd., Shanghai, China). For TFAM overexpression treatment, MDA-MB231 cells (2 105) grown on six-well plates were transfected with 2 mg of TFAM overexpression plasmid or pCDNA using Lipofectamine 2000 (Invirtogen, USA) and serum-free Opti-MEM medium (GIBCO, USA).as described by the manufacturer. The cells were harvested after 48 h. Western blot analyses or other experiments were performed. 2.4. Construction of vector and luciferase reporter assay The 30 -UTR of TFAM gene (position 213–220) was predicted to be complementary to the sequence of miR-199a-3p according to an analysis of the miRNA target gene prediction database, TargetScan. The fragment sequence of wild-type and mutant TFAM 30 -UTR was amplified by PCR and cloned into the pYr-MirTarget luciferase
The cytotoxic effect of DDP was measured by MTT assay as previously described [16]. Briefly, the MDA-MB-231 or MDA-MB231/DDP was seeded in 96-well plate at 2 104 per well and cultured for 24 h, and transfected with the miR-199a-3p mimics or inhibitors for 24 h. The cells were then treated with different concentration of DDP (0, 5, 10, 20, 40, 60, 80,100 mM) for 48 h. The MTT (Sigma, USA) was added to a final concentration of 0.5 mg/ml, and the cells were incubated for 4 h at 37 C. The absorbance at 570 nm was measured in a microplate reader (Bio-rad, USA). Each experiment was repeated at least three times in triplicate. 2.6. Detection of mtDNA copy number [17] MtDNA copy number was measured using a real time quantitative polymerase chain reaction (PCR) using an Applied Biosystems 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). One primer pair specific for the mitochondrial DNA (ND1) and another primer pair specific for the nuclear DNA (18s) were designed for relative quantification for mtDNA copy number. The primer sequences for the mitochondrial ND1 gene were: forward primer 50 - ccc taa aac ccg cca cat ct-30 ; reverse primer 50 gag cga tgg tga gag cta agg t-30 . The primer pair used for the amplification of the nuclear gene 18s was as follows: forward primer, 50 -tag agg gac aag tgg cgt tc-30 ; reverse primer, 50 -cgc tga gcc agt cag tgt-30 . 2.7. Apoptosis assay Analysis of cell apoptosis was performed with the Annexin VFITC apoptosis detection kit (Keygen, China). 24 h after transfection, the MDA-MB-231 and MDA-MB-231/DDP cells were treated with DDP (20 mM) for 48 h. Then the cell samples were harvested with 0.25% trypsin without EDTA washed twice with ice-cold PBS and re-suspended in 500 ml binding buffer. Then the cells were incubated with 5 ml Annexin V-FITC and 5 ml propidium iodide (PI) for 15–20 min in the dark and detected by BD FascAria flow cytometer (BD, USA) with the excitation wavelength of 488 nm and emission wavelength of 530 nm. Each experiment was repeated three times in triplicate. 2.8. qRT-PCR analysis Total microRNAs were extracted by the miRNeasy Kit (Qiagen, German) according to the manufacturer’s instructions. RNA was converted to cDNA using the miScriptRT kit (Qiagen, German). QRT-PCR was performed using a miScript SYBR Green PCR kit (Qiagen, German) on the ABI 7900HT Real-time PCR System (Applied Biosystems, USA). The expression of miRNAs was normalized using the RNU6 as endogenous control. The primers of miR-199a and U6 were purchased from Yrbio Biotech (China).
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Total RNA was isolated with Trizol Reagent (Invitrogen, USA) according to the manufacturer’s instructions. RNA was converted to cDNA using the PrimeScript1 1 st Strand cDNA Synthesis Kit (Takara, Japan). QRT-PCR was performed using SYBRR Premix Ex TaqTM (TaKaRa, Japan) on ABI7500 Real-time PCR Systerm (Applied Biosysterm, USA). TFAM primers were: forward 50 -tcc aag aag cta agg gtg-30 ; and reverse 50 -ttc cca aga ctt cat ttc a-30 . b-actin primers were: 50 -ggg aaa tcg tgc tgc gtg ac- 30 ; and reverse 50 -ttg cca atg gtg atg acct g- 30 . All of the qRT-PCR assays were performed in triplicate and the change was calculated using the 2DDCt method. 2.9. Western blot analysis After transfection and DDP treatment, cells were harvested and homogenized with lysis buffer (Beyotime, China). The protein concentrations were measured using the BCA protein assay kit (Beyotime, China). Proteins (30 to 50 mg) were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane (Millipore, USA). After blocking with 5% non-fat dry milk, the membranes were incubated with primary antibodies specific to TFAM and b-actin (1:1000, Santa Cruz, USA). The membranes were further incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (1:5000, Santa Cruz, USA). The immunoblots were quantified by the ImageJ software.
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231/DDP cells (Fig. 1B, p < 0.01). Therefore, the effect of miR-199a3p on DDP resistance in MDA-MB-231 cells was further investigated. 3.2. Effect of miR-199a-3p on sensitivity to DDP in MDA-MB-231 and MDA-MB-231/DDP cells To investigate the effect of miR-199a-3p on regulating MDAMB-231 cells resistant to DDP, the MDA-MB-231 and MDA-MB231/DDP cells were respectively transfected with miR-199a-3p inhibitor or mimics and then treated with 20 mM DDP for 48 h. Transfection of miR-199a-3p inhibitor down-regulated miR-199a3p expression and attenuated DDP-induced apoptosis in MDA-MB231 cells compared with negative control (NC) transfection (Fig. 2A and C). On the contrary, MDA-MB-231/DDP cells transfected with miR-199a-3p mimics effectively increased the miR-199a-3p expression and enhanced the apoptosis to DDP compared with the cells transfected with NC (Fig. 2B and D). Moreover, transfection with miR-199a-3p inhibitor decreased the sensitivity of MDA-MB-231 to DDP. In contrast, transfection with miR-199a3p mimics significantly enhanced the sensitivity of MDA-MB-231/ DDP cells to DDP (Fig. 2E and F). These results indicated that miR199a-3p is involved in the DDP resistance of breast cancer cells. 3.3. Regulation of TFAM expression by miR-199a-3p
2.10. Statistical analysis The Student’s t-test was used for comparison between two groups. p < 0.05 was considered to indicate statistical significance. Test data from independent experiments were repeated three times. 3. Results 3.1. Expression levels of miR-199a-3p in MDA-MB-231 and MDA-MB231/DDP cells The cell viability of MDA-MB-231 and MDA-MB-231/DDP cells under cisplatin treatment for 48 h was shown in Fig. 1A. The IC50 of MDA-MB-231/DDP cells showed 4.42 fold higher than that of MDAMB-231 cells (22.1 vs. 97.6 mM, p < 0.01). To investigate the involvement of miR-199a-3p in DDP resistant breast cancer cells MDA-MB-231/DDP, the levels of miR-199a-3p were detected by qRT-PCR. Compared with MDA-MB-231 cells, the expression levels of miR-199a-3p were significantly down-regulated in MDA-MB-
TFAM has also been implicated as a key regulator involved in mtDNA transcription and replication [18]. Furthermore, it was reported that TFAM is involved in breast cancer development and drug resistance [19]. As shown in Fig. 3A, the expression of TFAM was significantly higher in MDA-MB-231/DDP cells compared with MDA-MB-231 cells. To determine whether miR-199a-3p is involved in the modulation of TFAM expression, the miR-199a-3p inhibitor and mimics were transfected into the MDA-MB-231 or MDA-MB-231/ DDP cells. The expression of TFAM was increased following miR199a-3p inhibitor transfection in MDA-MB-231 cells (Fig. 3B). In contrast, the reduced TFAM expression was observed in MDA-MB231/DDP cells transfected with miR-199a-3p mimics compared with the NC group (Fig. 3C). 3.4. TFAM may be a target gene of miR-199a-3p To identify whether TFAM is the target gene of miR-199a-3p, the miRNA target prediction public database (TargetScan, USA) was
Fig. 1. Expression levels of miR-199a-3p in MDA-MB-231 and MDA-MB-231/DDP cells. (A) Survival curves of MDA-MB-231 and MDA-MB-231/DDP cells. The cells were exposed to different concentration of cisplatin (DDP) (0, 5, 10, 20, 40, 60, 80, 100 mM) for 48 h. Cell viability was determined by MTT assay. (B) Expression level of miR-199a-3p was lower in MDA-MB-231/DDP cells compared with MDA-MB-231 cells (p < 0.01 vs.MDA-MB-231 cells).
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Fig. 2. MiR-199a-3p inhibits cisplatin resistance in MDA-MB-231/DDP cells. (A, B) After 48 h transfection of miR-199a-3p mimics or inhibitor, the expression of miR-199a-3p in MDA-MB-231 cells and MDA-MB-231/DDP cells was determined by qRT-PCR, respectively. (C) Inhibition of miR-199a-3p attenuated DDP-induced apoptosis in MDA-MB231 cells after 48 h 20 mM DDP treatment. Apoptosis rate was detected by flow cytometry with annexin V-FITC/PI staining. (D) Overexpression of miR-199a-3p enhanced the DDP-induced apoptosis in MDA-MB-231/DDP cells after 48 h 20 mM DDP treatment. Apoptosis rate was detected by flow cytometry with annexin V-FITC/PI staining. (E) After 48 h transfection of miR-199a-3p inhibitor, MDA-MB-231 cells decreased sensitivity to various doses of DDP treatment. Cell viability was assessed by MTT assay. (F) After 48 h transfection of miR-199a-3p mimics in MDA-MB-231/DDP cells, sensitivity to various doses of DDP treatment increased. Cell viability was assessed by MTT assays. *p < 0.05 and **p < 0.01 vs. NC mimics or inhibitor.
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Fig. 3. Regulation of TFAM by miR-199a-3p. (A) Expression of TFAM in MDA-MB-231 and MDA-MB-231/DDP cells was determined by western blot analysis. The densitometric analysis results were shown in the right panels. Expression of TFAM in MDA-MB-231 cells transfected with miR-199a-3p inhibitor (B) and in MDA-MB-231/DDP cells transfected with miR-199a mimics (C). The densitometric analysis results were shown in the right panels. *p < 0.05 and **p < 0.01 vs. NC mimics or inhibitor.
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used for analysis. It was found that the 30 -UTR mRNA of TFAM contained a highly conserved binding site for miR-199a-3p (Fig. 4A). To investigate the association between TFAM and miR199a-3p, the HEK293 cells were co-transfected with pYr-TFAM-3U or pYr-TFAM-3Umt and miR-199a mimics or miR-199a-3p inhibitor. The results showed that miR-199a-3p mimics dramatically reduced the luciferase activity of pYr-TFAM-3U, while there was no difference in the luciferase activity of pYr-TFAM-3Umt; miR-199a3p inhibitor increased the luciferase activity of pYr-TFAM-3U, while there was no difference in the luciferase activity of pYrTFAM-3Umt (Fig. 4B). These data suggested that TFAM may be a target gene of miR-199a-3p. 3.5. MiR-199a-3p/TFAM modulates cell proliferation and mtDNA copy number in MDA-MB-231 and MDA-MB-231/DDP cells To investigate the role of miR-199a-3p/TFAM in regulating the chemoresistance of MDA-MB-231, we performed a series of functional assays. MDA-MB-231 cells were co-transfected with NC inhibitor/miR-199a-3p inhibitor and siRNA/si-TFAM; MDA-MB231/DDP cells were co-transfected with NC mimics/miR-199a-3p mimics and pcDNA/TFAM. Transfection with miR-199a-3p inhibitor resulted in TFAM protein level and mtDNA copy number significantly up-regulated, while this promoting effect of miR199a-3p inhibitor could be partially restored by si-TFAM transfection in MDA-MB-231 cells (Fig. 5A and C). In MDA-MB-231/DDP cells, transfection with miR-199a-3p mimics led to a strong repression of TFAM protein level and mtDNA copy number, while this inhibitory effect of miR-199a-3p mimics could be partially restored by TFAM overexpression (Fig. 5B and D). To investigate the role of miR-199a-3p/TFAM in regulating breast cancer cells’
Fig. 4. TFAM may be a target gene of miR-199a-3p. (A) TFAM’s UTR mRNA includes a highly-conserved binding site for miR-199a-3p. (B) The HEK293 cells were cotransfected with pYr-TFAM-3U or pYr-TFAM-3Umt and miR-199a-3p mimics or inhibitor. After 24 h transfection, the luciferase activity decreased in pYr-TFAM-3U and miR-199a-3p mimics co-transfected cells, while increased in pYr-TFAM-3U and miR-199a-3p inhibitor co-transfected cells. The modification of luciferase activity by miR-199a-3p mimics and inhibitor was abolished when the potential target site was mutated. **p < 0.01.
chemo-resistance, the viability of cells under DDP treatment was monitored. Results showed that miR-199a-3p inhibitor reduced the chemo-sensitivity of MDA-MB-231 cells to DDP, while this effect could be partially restored by si-TFAM transfection (Fig. 5E). The chemoresistance of MDA-MB-231/DDP cells could be attenuated by miR-199a-3p mimics, while this effect could be partially restored by TFAM overexpression. These data indicated that miR-199a-3p/TFAM regulates the chemo-resistance of breast cancer cells to DDP. 4. Discussion MiRNAs play a crucial role in many biological processes, such as proliferation, apoptosis and differentiation, through modulating gene expression at the posttranscriptional level [5]. Aberrant miRNAs could affect the expression of target genes, which may modulate cell death signal pathway, drug target proteins and cell cycle-related proteins. This may lead to resistance of cancer cells to chemotherapy [20]. For instance, upregulation of miR-22 and miR205 induces resistance of breast cancer cells to DDP through modulating AKT signal pathway [21,22]. Upregulation of miR-127 is associated with increasing cell sensitivity to DDP [23]. In the present study, we first demonstrated that miR-199a-3p was expressed at a lower level in DDP resistant breast cancer cell line MDA-MB-231/DDP compared with the parental MDA-MB-231 cells. Furthermore, overexpression of miR-199a-3p in MDA-MB231/DDP cells inhibited TFAM expression, promoting the sensitivity of MDA-MB-231/DDP cells to DDP. Inhibition of miR-199a-3p in MDA-MB-231 cells led to promoting TFAM, while the sensitivity to cisplatin was decreased. These findings indicate that upregulation of miR-199a-3p may promote the sensitivity of breast cancer cells to DDP through downregulation of TFAM expression. Accumulating lines of evidence have confirmed that miR-199a is involved in cancer development by targeting oncogenes or tumor suppressors [11,24]. The expression of miR-199a was diversely deregulated in several types of cancer. For instance, miR-199a was downregulated in ovarian cancer, prostate cancer and bladder cancer, but it was upregulated in gastric cancer and cervical carcinoma [25,26]. Moreover, it has been demonstrated that miR-199a was the differential expressed between chemosensitive and chemoresistant cells which may participate in the cell chemoresistance. Downregulation of miR-199a is found in ovarian cancer cells and hepatoma cells resistant to DDP [27,28]. MiR-199a-3p is downregulated in hepatocellular carcinoma, restoring attenuated levels of miR-199a-3p in hepatocellular carcinoma cells, leading to increased sensitivity to doxorubicininduced apoptosis [29]. These studies indicated that miR-199a-3p may play a crucial role in chemotherapy resistance. Here we found that miR-199a-3p expression was significantly down-regulated in MDA-MB-231/DDP cells compared with the parental cells (Fig. 1B). Inhibition of miR-199a-3p in MDA-MB-231 cells reduced DDPinduced cell apoptosis and anti-proliferative effect; while overexpression of miR-199a-3p significantly promoted cisplatininduced apoptosis and anti-proliferative effect in MDA-MB-231/ DDP cells (Fig. 2). To further investigate the mechanism of miR-199a-3p in the DDP resistance, we predicted a possible target mRNA for miR199a-3p using computational algorithms (TargetScan). The intersection of algorithms and luciferase reporter assay indicated that TFAM was a target gene of miR-199a-3p (Fig. 4). In MDA-MB-231/ DDP cells, TFAM was highly expressed compared with MDA-MB231 cells, and could be negatively regulated by miR-199a-3p in both MDA-MB-231 and MDA-MB-231/DDP cells. As a regulator of mtDNA replication and transcription, TFAM is also known to be critical for maintenance of mitochondrial biogenesis and function [30]. Mitochondrial function and/or mtDNA content acceleration
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Fig. 5. MiR-199a-3p/TFAM modulates cell proliferation and mtDNA copy number in MDA-MB-231 and MDA-MB-231/DDP cells. (A) MDA-MB-231 cells were co-transfected with NC inhibitor/miR-199a-3p inhibitor and siRNA/si-TFAM. The protein levels of TFAM were determined using Western blot analysis. (B) MDA-MB-231/DDP cells were cotransfected with NC mimics/miR-199a-3p mimics and pcDNA/TFAM. The protein levels of TFAM were determined using Western blot analysis. (C, D) The mtDNA copy number of MDA-MB-231 and MDA-MB-231/DDP cells was determined in each of the indicated groups. (E, F) The cell viability of MDA-MB-231 and MDA-MB-231/DDP cells was determined in each of the indicated groups using MTT assay. *p < 0.05 and **p < 0.01 vs. NC mimics or inhibitor. ##p < 0.01 miR-199a inhibitor +siRNA vs. miR-199a inhibitor +si-TFAM or miR-199a mimics +pcDNA vs. miR-199a mimics +TFAM.
plays a key role in tumor formation and cancer pathogenesis [31,32]. Therefore, the modulation of TFAM has been involved in tumorigenesis and cancer progression [33]. Recently, it has been reported that TFAM could promote breast cancer cell proliferation and enhance DDP resistance in estrogen receptor positive breast cancer [34,35]. In the current study, we demonstrated that inhibition of miR-199a-3p in MDA-MB-231 cells led to increased TFAM expression, increased mtDNA copy numbers and reduced sensitivity of breast cancer cells to DDP. Overexpression of miR-
199a-3p in MDA-MB-231/DDP cells caused the opposite effects, i.e., reduced TFAM expression, reduced mtDNA copy number and enhanced sensitivity of breast cancer cells to DDP. Moreover, the effect of miR-199a-3p inhibition or overexpression could be partially restored by TFAM knockdown or overexpression, respectively (Fig. 5). The modulation of TFAM by miR-199a-3p contributes to the change of cell sensitivity to DDP. In summary, our finding showed that miR-199a-3p could attenuate the DDP resistance by blocking the expression of TFAM in
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cisplatin-resistant breast cancer cells. Moreover, we demonstrated that TFAM may be a target gene of miR-199a-3p and play an important role in resistance of breast cancer cells to DDP. Despite the growing evidence of cancer-specific miRNA patterns, the role of miRNA in cancer is tissue-specific and context-dependent, switching from repressors to activators in different microenvironment [36]. Therefore, future investigations using animal models and clinic patient samples are necessary to further support the function of miR-199a-3p in breast cancer resistance to DDP. Conflict of interest All authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Acknowledgements This study was funded by National Natural Science Foundation of China (No. 71273083 and 81472496) and Key Research Center for Humanities and Social Sciences in Hubei Province (Hubei University of Medicine, No. 2016YB002) References [1] J. Ferlay, H.R. Shin, F. Bray, D. Forman, C. Mathers, D.M. Parkin, Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008, Int. J. Cancer 127 (12) (2010) 2893–2917. [2] M. Kaufmann, G. von Minckwitz, H.D. Bear, A. Buzdar, P. McGale, H. Bonnefoi, M. Colleoni, C. Denkert, W. Eiermann, R. Jackesz, et al., Recommendations from an international expert panel on the use of neoadjuvant (primary) systemic treatment of operable breast cancer: new perspectives 2006, Ann. Oncol. 18 (12) (2007) 1927–1934. [3] D.P. Silver, A.L. Richardson, et al., Efficacy of neoadjuvant Cisplatin in triplenegative breast cancer, J. Clin. Oncol. 28 (7) (2010) 1145–1153. [4] A.-M. Florea, D. Büsselberg, Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects, Cancers 3 (1) (2011) 1351–1371. [5] N. Lynam-Lennon, S.G. Maher, J.V. Reynolds, The roles of microRNA in cancer and apoptosis, Biol. Rev. 84 (1) (2009) 55–71. [6] A. Fendler, M. Jung, C. Stephan, R.J. Honey, R.J. Stewart, K.T. Pace, A. Erbersdobler, S. Samaan, K. Jung, G.M. Yousef, miRNAs can predict prostate cancer biochemical relapse and are involved in tumor progression, Int. J. Oncol. 39 (5) (2011) 1183–1192. [7] M. Garofalo, C. Quintavalle, G. Romano, C.M. Croce, G. Condorelli, miR221/222 in cancer: their role in tumor progression and response to therapy, Curr. Mol. Med. 12 (1) (2012) 27–33. [8] Z. Lu, Y. Ye, D. Jiao, J. Qiao, S. Cui, Z. Liu, miR-155 and miR-31 are differentially expressed in breast cancer patients and are correlated with the estrogen receptor and progesterone receptor status, Oncol. Lett. 4 (5) (2012) 1027–1032. [9] Q. Wang, P. Li, A. Li, J. Wei, W. Hong, J. Wang, K. Xie, Plasma specific miRNAs as predictive biomarkers for diagnosis and prognosis of glioma, J. Exp. Clin. Cancer Res. 31 (2) (2012) 1–10. [10] H. He, J. Ge, J. Dong, L. Wang, Effect of miR-199a-5ptransfection on the growth of clear-cell renal-cell carcinoma 786-0 cells, J. Med. Postgrad. 26 (9) (2013) 921–924. [11] S.Q. Li, Z.H. Wang, X.G. Mi, L. Liu, Y. Tan, MiR-199a/b-3p suppresses migration and invasion of breast cancer cells by downregulating PAK4/MEK/ERK signaling pathway, IUBMB Life 67 (10) (2015) 768–777. [12] Y. Murakami, T. Yasuda, K. Saigo, T. Urashima, H. Toyoda, T. Okanoue, K. Shimotohno, Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues, Oncogene 25 (17) (2006) 2537–2545. [13] S.D. Li, J.R. Zhang, Y.Q. Wang, X.P. Wan, The role of microRNAs in ovarian cancer initiation and progression, J. Cell. Mol. Med. 14 (9) (2010) 2240–2249.
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