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MiR-451a attenuates doxorubicin resistance in lung cancer via suppressing epithelialmesenchymal transition (EMT) through targeting c-Myc
Biomedicine & Pharmacotherapy 125 (2020) 109962
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MiR-451a attenuates doxorubicin resistance in lung cancer via suppressing epithelialmesenchymal transition (EMT) through targeting c-Myc
T
Li Taoa,b,1, Wang Shu-Lingb,1, Hao Jing-Boc, Zhang Yingb, Hu Ronga,d, Liu Xiang-Quna, Cui Wen-Jieb, Zhou Lin-Fua,* a
Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu 210029, China Department of Respiratory Medicine, The Municipal Hospital Affiliated to Xuzhou Medical University, Xuzhou, Jiangsu 221002, China c Department of Geriatrics, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221000, China d Department of Respiratory and Critical Care Medicine, The First People’s Hospital of Lianyungang, Lianyungang, Jiangsu 222006, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Chemoresistance Lung cancer miR-451a EMT c-Myc
Chemoresistance is still a major obstacle for lung cancer treatment. Increasing studies have demonstrated that microRNAs (miRNAs) are essential meditators of chemoresistance during cancer progression. MiR-451a is reported to be a tumor suppressor during cancer development. However, its effects on lung cancer and drug resistance in lung cancer are still unclear. In the study, the results showed that miR-451a exhibited a significant role in suppressing the drug resistance in lung cancer cells when treated with doxorubicin (DOX) through alleviating epithelialmesenchymal transition (EMT), as evidenced by the markedly reduced expression of N-cadherin and Vimentin, while the enhanced expression of E-cadherin. In addition, miR-451a over-expression markedly promoted the sensitivity of lung cancer cells to DOX treatments, and also disrupted the EMT of lung cancer cells. Mechanistically, miR-451a was found to directly target c-Myc to affect the EMT and drug resistance in lung cancer cells in response to DOX incubation. Furthermore, c-Myc knockdown markedly elevated the sensitivity of lung cancer cells to DOX, whereas over-expressing c-Myc markedly reversed the anti-tumor role of DOX, which was slightly diminished by miR-451a mimic. The in vivo experiments confirmed that miR-451a promoted the sensitivity of lung cancer cells-derived tumors to DOX treatment by reducing c-Myc. Therefore, our results revealed a new insight into DOX resistance of lung cancer cells and miR-451a could be considered as a potential therapeutic target to overcome drug resistance in lung cancer.
1. Introduction Lung cancer is a major cause of tumor-associated mortality among human worldwide with a 5-year survival rate of < 15 % [1,2]. As reported, the non-small-cell lung cancer (NSCLC) is consisted of a largely heterogeneous group of malignancies and accounts for about 85 % of all diagnosed lung cancers [3]. Accumulating studies have demonstrated that surgical resection is effective for early-stage non-metastatic lung tumors. Chemotherapy alone or in combination with radiation is defined as the frontline therapeutic strategy for the treatment of advanced lung cancer [4,5]. Nevertheless, because of the inherent or acquired drug resistance, the efficiency of chemotherapy has been ephemeral and significantly limited, leading to poor survival rate consequently [6,7]. For instance, doxorubicin (DOX) has been widely used for lung cancer treatment. However, DOX resistance limits its clinical outcomes
[8]. A better understanding of the molecular mechanisms revealing chemoresistance, including DOX resistance, is important for achieving better survival rates for patients with lung cancer. MicroRNAs (miRNAs) are small non-coding RNAs, and negatively modulate gene expression through binding to the 3′-untranslated region (3′-UTR) of their targeting mRNAs, resulting in translational repression and/or mRNA degradation [9,10]. Abnormal expression of miRNA has also been reported during lung cancer progression, which is tightly associated with the cell proliferation, invasion, migration and chemoresistance [11,12]. As reported, miR-451a acted as a tumor suppressor, and was down-regulated in gastric cancer, papillary thyroid carcinoma and prostate cancer [13–15]. Recently, miR-451a was also reported to suppress NSCLC progression through repressing migration and invasion by targeting activating transcription factor 2 (ATF2) [16]. In acute myeloid leukemia, miR-451a abolished treatment resistance
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Corresponding author. E-mail address: [email protected] (Z. Lin-Fu). 1 The first two authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2020.109962 Received 8 January 2020; Received in revised form 19 January 2020; Accepted 23 January 2020 0753-3322/ © 2020 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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2.5. Western blot analysis
[17]. Although miR-451a is involved in chemoresistance during tumor growth, its physiological effects on DOX resistance of lung cancer are still unclear. In this study, our results demonstrated that miR-451a over-expression attenuated DOX resistance in lung cancer cells through targeting cMyc. In addition, EMT was also inhibited by miR-451a in lung cancer cells when treated with DOX, which was also by directly targeting cMyc. Our study provided functional and mechanistic links between miR-451a and DOX resistance, and thus demonstrated a promising therapeutic target for lung cancer treatment.
Total proteins from the cells or tumor tissues were extracted with a protein lysis buffer (Beyotime, Nanjing, China) with PhosStop phosphatase inhibitor cocktail and protease inhibitor cocktail (Roche Diagnostics, USA). Lysates were denatured prior to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, UK). Then, the membranes were blocked using 5 % nonfat milk at room temperature for 1.5 h, followed by incubation with primary antibodies at 4 °C overnight. Next, the membranes were further incubated with secondary antibodies (Beyotime) for 1 h. The immunoreactive signals were finally visualized with the enhanced chemiluminescence reagents (ECL, Pierce, USA). The primary antibodies used in the study were shown as followings: anti-Vimentin (ab92547, Abcam, USA), anti-Ecadherin (PA5-85088, ThermoFisher Scientific, USA), anti-N-cadherin (MA5-32088, ThermoFisher Scientific), anti-c-Myc (ab32072, Abcam) and anti-GAPDH (ab8245, Abcam).
2. Materials and methods 2.1. Cells and culture Human lung cancer cells lines, including A549, PC9, H1299 and H460 were obtained from the American Type Culture Collection (ATCC, USA). All cells were cultured in RPMI 1640 medium (Gibco, USA) or Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10 % fetal bovine serum (FBS, Hyclone, USA) containing 100 units/mL penicillin and 100 mg/mL streptomycin. Cell lines were grown at 37 °C in a humidified atmosphere with 5 % CO2 and 95 % air. For the A549/ADR and PC9/ADR cells with drug resistance, the resistant variants were originated through growing initial A549 and PC9 cells with raising concentrations DOX (Sigma-Aldrich, USA). DOX was added to cells every 3 days. A549/ADR and PC9/ADR cells were 35 times as much resistant to the DOX cytotoxicity when compared to the initial A549 and PC9 cells. DOX at a final concentration of 1 mg/l was subjected to cells for maintaining the drug resistance until 1 week prior to the experiments.
2.6. Real-time quantitative PCR (RT-qPCR) analysis Total RNA from the cultured cells was extracted using the RNAeasy Mini Kit (Qiagen, USA) according to the manufacturer’s protocols, and reverse-transcribed using the High Capacity Reverse Transcription Kit (Applied Biosystems, USA). Then, qPCR analysis was performed in duplicate using GoTaq qPCR Master Mix (Promega, USA) on the ABI7900HT Real-Time PCR system (Applied Biosystems). All results were analyzed using the 2–ΔΔCt method [18]. The miRNA expression levels were normalized to U6. The probes for U6 and miR-451a were purchased from Generay Biotech (Shanghai, China). The sequence of primers used for qPCR were listed as followings: miR-451a forward: 5′ACA CTC CAG CTG GGA AAC CGT TAC CAT TAC-3′; reverse, 5′-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG CTT ACA G-3′; U6 forward: 5′-GCT TCG GCA GCA CAT ATA CTA A-3′; reverse, 5′-AAC GCT TCA CGA ATT TGC GT-3′.
2.2. Cell viability analysis Cells after transfection and/or DOX treatments were seeded into a 96-well plate at 1000 cells per well, and cultured at 37 °C at 5 % CO2 for 4 h. For cell viability calculation, the cells were incubated with 10 μL of CCK-8 (Dojindo, Japan) for 2 h at 37 °C according to the manufacturer’s protocols. Finally, the density was measured at a wavelength of 450 nm with a microplate reader.
2.7. Xenograft model All animal experiments were performed following the standards regarding the use of laboratory animals and all experiments were approved by the Institutional Animal Care and Usage Committee of the First Affiliated Hospital, Nanjing Medical University (Nanjing, China). The male BALB/c nude mice (4–6 weeks old) were purchased from the Animal Center of Nanjing Medical University (Nanjing). To produce a xenograft tumor model, a total of 1 × 107 the A549 cells were injected into the right dorsal flanks of nude mice. After tumor volume reached to 50 cm3, the mice were randomly divided in to 4 groups: 1) PBS group, 2) DOX group (2 mg/kg body weight, every 2 days), miR-451a agomir (2 nmol each mouse, every 3 days), or DOX combined with miR-451a agomir. MiR-451a agomir was chemically modified from miR-451a mimic. Tumor size was measured using Vernier calipers every 3 days for 28 days. The tumor volume (mm3) was calculated as length × width2 × 0.5. After 4 weeks, the tumors were isolated, weighed and frozen or paraffin embedded for immunohistochemical calculation.
2.3. Transfection in vitro miR-451a mimics, miR-451a inhibitor, and their corresponding negative controls (NC) oligonucleotide were synthesized and purchased from RiboBio Co. Ltd. (Guangzhou, China). Small interfering RNA against c-Myc (si-c-Myc) and its nonspecific NC siRNA were purchased from GenePharma Co., Ltd. (Shanghai, China). The plasmids for c-Myc overexpression were purchased from GeneChem Co. Ltd. (Shanghai). All transfection were performed using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s recommendations. 2.4. Immunofluorescence staining After treatments, all cells were fixed with 4 % paraformaldehyde for 10 min, followed by blocking with 10 % BSA (Solarbio, Beijing, China) for 30 min at room temperature. Then, the cells were incubated with primary antibodies (N-cadhrin or E-cadherin, at 1:100 dilutions; Abcam, USA) at 4 °C overnight. Next, the cells were incubated with an Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) secondary antibodies (at 1:50 dilutions; Abcam) at room temperature for 1 h. The nucleus was stained with 4′6diamidino-2-phenylindole (DAPI, Solarbio). Finally, the fluorescent signals were detected with an inverted fluorescence microscope (Nikon Co., Ltd., Japan).
2.8. Immunohistochemical staining Tumor tissues were fixed with 4 % formalin and embedded in paraffin. Tissue sections (4 μm thickness) were deparaffinized in xylene, rehydrated in a serial of graded ethanol, and antigens were retrieved by boiling. Endogenous peroxidase activity was blocked using 10 % goat serum (Solarbio) for 30 min, followed by incubation with primary antibodies (KI-67 and c-Myc, Abcam) at 4 °C overnight. Then, all sections were treated with a secondary antibody (Solarbio) for 1 h at room temperature. The immunocomplexes were visualized with a 3,3′-diaminobenzidine solution (DAB, Solarbio). Finally, the sections were 2
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Fig. 1. MiR-451a over-expression sensitized lung cancer cells to DOX treatment. (A) DOX (0, 0.1, 0.25, 0.5, 1, 2.5, 5, 7.5, 10, 20, 50 and 100 μM) was treated to lung cancer cells (A549/ADR, A549, PC9/ADR, PC9, H1299 and H460) with or without drug resistance for 24 h. Then, all cells were collected for cell viability calculation using CCK8 analysis. (B) RT-qPCR analysis for miR-451a in lung cancer cells with or without drug resistance. (C) Lung cancer cells were transfected with miR-451a mimic for 24 h, followed by transfection efficacy measurement using RT-qPCR analysis. ***p < 0.001 vs the Ctrl group. (D) A549/ADR, (E) A549, (F) PC9/ADR, (G) PC9, (H) H1299 and (I) H460 cells were transfected with miR-451a mimic for 24 h, and then were subjected to DOX (0, 0.1, 0.25, 0.5, 1 and 2.5 μM) for another 24 h. Then, all cells were collected for cell viability calculation using CCK8. *p < 0.05 vs the corresponding Ctrl groups. (J) Lung cancer cells were transfected with miR451a inhibitor for 24 h, followed by transfection efficacy determination by RT-qPCR analysis. (K) A549/ADR, (L) A549, (M) PC9/ADR, (N) PC9, (O) H1299 and (P) H460 cells were transfected with miR-451a inhibitor for 24 h, and then were treated with DOX (0, 0.1, 0.25, 0.5, 1 and 2.5 μM) for another 24 h. Next, all cells were harvested for cell viability assessment with CCK8. *p < 0.05 vs the corresponding Ctrl groups. Data were expressed as mean ± SEM (n = 4 in each group).
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Fig. 2. MiR-451a over-expression suppressed EMT in DOX-treated lung cancer cells. A549 and PC9 cells were treated with DOX (2.5 μM) for 24 h. (A) Then, all cells were collected for western blot analysis of N-cadherin, Vimentin and E-cadherin, and for (B) RT-qPCR analysis of miR-451a in cells. A549 and PC9 cells were transfected with miR-451a inhibitor for 24 h, and then were incubated with DOX (2.5 μM) for another 24 h. **p < 0.01 vs the Ctrl group. All cells were collected for (C) western blot analysis of N-cadherin, Vimentin and E-cadherin, as well as (D) immunofluorescence staining for N-cadherin and E-cadherin. Data were expressed as mean ± SEM (n = 4 in each group).
PC9, H1299 and H460 cells displayed the lowest cell viability (Fig. 1A). Then, the expression of miR-451a was measured in lung cancer cells with or without drug resistance. As shown in Fig. 1B, miR-451a expression levels displayed an opposite trend relative to that of the cell proliferation, showing a positive relationship between miR-451a and DOX sensitivity in lung cancer cells. In order to further explore the effects of miR-451a on drug resistance, we over-expressed miR-451a in lung cancer cells by transfection with miR-451a mimic. Transfection efficacy was confirmed using RT-qPCR (Fig. 1C). CCK8 analysis showed that miR-451a over-expression significantly promoted the suppressive effects of DOX on lung cancer cells with or without drug resistance (Fig. 1D-I). In contrast, reducing miR-451a by its inhibitor (Fig. 1J) markedly elevated the DOX resistance in lung cancer cells (Fig. 1K-P). Together, these findings demonstrated a significant role of miR-451a in suppressing the DOX resistance in lung cancer cells.
stained with hematoxylin (Sigma), and observed under a light microscope (Olympus, Japan). 2.9. Statistical analysis All statistical analysis were performed using GraphPad prism v.6.0 (GraphPad Software, La Jolla, USA). Two tailed Student’s t-test was used to compare the difference between groups. The values are presented as means ± SEM. P values < 0.05 were considered statistical difference. 3. Results 3.1. MiR-451a over-expression sensitized lung cancer cells to DOX treatment CCK8 analysis showed that A549/ADR and PC9/ADR exhibited the highest viability in response to DOX treatments, whereas the A549, 4
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Fig. 3. MiR-451a negatively regulated c-Myc to control the proliferation and EMT in lung cancer cells. (A) TargetScan-predicted binding sequences of miR-451a in the 3′-UTR of c-Myc. A549 and PC9 cells were transfected with (B) miR-451a mimic or (C) miR-451a inhibitor for 24 h. Then, all cells were collected for western blot analysis of c-Myc. (D) Western blotting analysis of c-Myc in lung cancer cells with or without drug resistance. (E) A549 and A549/ADR cells, (F) PC9 and PC9/ADR cells were transfected with si-c-Myc for 24 h, followed by transfection efficacy measurement using western blot analysis. A549, A549/ADR, PC9 and PC9/ADR cells were co-transfected with miR-451a inhibitor and si-c-Myc for 24 h, followed by DOX (0, 0.1, 0.25, 0.5, 1 and 2.5 μM) incubation for another 24 h. Then, (G) the cell viability was assessed using CCK8 analysis, and (H) western blot analysis was used to determine N-cadherin and E-cadherin expression. *p < 0.05 vs the corresponding NC groups; +p < 0.05 vs the corresponding si-c-Myc + miR-451a inhibitor group. (I) A549, A549/ADR, PC9 and PC9/ADR cells were transfected with cMyc plasmid for 24 h to over-express the cellular c-Myc. Western blot analysis was used for the transfection efficiency determination. A549, A549/ADR, PC9 and PC9/ADR cells were transfected with c-Myc plasmid for 24 h, and then were treated with DOX for another 24 h. Next, all cells were subjected to (J) western blot analysis of N-cadherin and E-cadherin, and (K) CCK8 analysis of cell viability. Data were expressed as mean ± SEM (n = 4 in each group).
time, miR-451a expression levels were induced by DOX (Fig. 2B). Then, we found that the role of DOX in regulating the EMT was reversed by the transfection of miR-451a inhibitor through western blot and immunofluorescence staining (Fig. 2C and D). MiR-451a over-expression inhibited EMT in DOX-treated lung cancer cells
3.2. MiR-451a over-expression suppressed EMT in DOX-treated lung cancer cells In this part, we investigated whether EMT, a critical factor during the progression of drug resistance, was implicated in DOX sensitivity regulated by miR-451a in lung cancer cells. As shown in Fig. 2A, Ncadherin and Vimentin expression levels in A549 and PC9 cells were reduced by DOX treatment, while E-cadherin was up-regulated, demonstrating the reduction of EMT in lung cancer cells. At the same 5
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Fig. 4. MiR-451a over-expression promoted the anti-tumor role of DOX in vivo. (A) Representative images of tumor samples from each group of mice. (B) Tumor volume and (C) tumor weight were measured. (D) Immunohistochemical staining of KI-67 and c-Myc in tumor sections. (E) Quantification of KI-67 and c-Myc expression following the immunohistochemical analysis. (F) Western blotting analysis of c-Myc, N-cadherin an E-cadherin in tumor samples. Data were expressed as mean ± SEM (n = 8 in each group). **p < 0.01 vs the Ctrl group.
or without drug resistance (Fig. 3E and F). CCK8 assays indicated that si-c-Myc markedly promoted the inhibitory role of DOX in reducing cell viability in A549 and A549/ADR cells, while these effects were significantly abrogated by miR-451a inhibitor. Similar trends of cell viability were observed in PC9 and PC9/ADR cells transfected with si-cMyc and/or miR-451a inhibitor following DOX treatments (Fig. 3G). Ncadherin expression levels suppressed by DOX were further reduced by si-c-Myc, while being rescued in lung cancer cells with or without drug resistance when transfected with miR-451a inhibitor; however, an opposite result was observed in the expression change of E-cadherin (Fig. 3H). Subsequently, we over-expressed c-Myc in lung cancer cells (Fig. 3I). Western blot analysis indicated that DOX-reduced N-cadherin and -increased E-cadherin were reversed by c-Myc over-expression,
3.3. MiR-451a negatively regulated c-Myc to control the proliferation and EMT in lung cancer cells To further explore the molecular mechanism revealing the chemoresistance regulated by miR-451a in lung cancer cells with or without drug resistance, a miRNA target prediction website was applied to find the target gene of miR-451a. Here, c-Myc was identified as a miR-451a target gene containing the binding sequence as shown in Fig. 3A. Western blot results showed that miR-451a over-expression reduced c-Myc in A549 and PC9 cells, while miR-451a inhibition promoted c-Myc (Fig. 3B and C). Then, we found that A549/ADR and PC9/ ADR cells had higher c-Myc expression than that of the A549 and PC9 cells (Fig. 3D). Then, c-Myc was knocked down in lung cancer cells with 6
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The in vivo experiments were performed to further investigate the effects of miR-451a on DOX resistance. As displayed in Fig. 4A-C, miR451a agomir combined with DOX markedly reduced the tumor volume and weight. Immunohistochemical staining demonstrated significant decreases in KI-67 and c-Myc in tumor sections from mice co-treated with miR-451a agomir and DOX (Fig. 4D and E). Western blot results showed that DOX and miR-451a agomir co-treatments markedly reduced c-Myc and N-cadherin expression levels in tumor samples, while enhanced E-cadherin protein expression levels (Fig. 4F). Together, these results demonstrated that miR-451a over-expression promoted the drug sensitivity in the treatment of lung cancer.
while miR-451a knockdown led to the increases in c-Myc. The increase of c-Myc was demonstrated to be associated with drug resistance and the outcome of patients with lung cancer [35]. Suppressing c-Myc expression markedly sensitized lung cancer cells to cisplatin treatment [36]. Therefore, c-Myc is a critical molecule that regulates chemoresistance in lung cancer cells. In this study, we confirmed that miR-451a could also directly target c-Myc at the 3′-UTR and thereafter reduce cMyc expression in lung cancer cells. Moreover, we also found that both miR-451a over-expression and c-Myc knockdown could markedly inhibit EMT and suppress DOX resistance in lung cancer cells. According to all these results, we supposed that c-Myc was a functional target gene of miR-451a, thus modulating DOX resistance in lung caner, which were confirmed by the in vivo experiments. In summary, our findings identified an essential molecular mechanism through which miR-451a regulated DOX resistance in lung cancer. Promoting miR-451a enhanced the sensitivity of lung cancer cells to DOX treatment by repressing EMT through targeting c-Myc. We therefore established new therapeutic strategy to overcome chemoresistance in lung cancer. However, if other genes participate in the improved DOX sensitivity still require further exploration.
4. Discussion
Declaration of Competing Interest
which were, however, abolished by miR-451a mimic (Fig. 3J). Finally in this part, CCK8 results showed that DOX-reduced cell viability in lung cancer cells was rescued by c-Myc over-expression; consistently, these effects were reversed by miR-451a mimic (Fig. 3K). Findings in this regard suggested that miR-451a negatively regulated c-Myc to mediate the cell viability and EMT in lung cancer cells. 3.4. MiR-451a over-expression promoted the anti-tumor role of DOX in vivo
The authors declare that they have no competing interests.
Despite the use of DOX has significantly improved the survival rate of patients with lung cancer, the drug resistance limits its wide application and results in the treatment failure [2–4,19]. Increasing evidences have suggested that miRNAs suppress gene expression through restraining transcription or eliciting the degradation of the targeted genes [9,10]. The abnormal expression of miRNA is commonly observed during lung cancer progression, which is also involved in chemoresistance [11,12,20]. Nevertheless, the regulatory role of miRNAs is pretty complicated, and their effects on drug resistance of lung cancer are still unclear. MiR-451a is suggested to function as a tumor suppressor in different types of cancers, such as prostate cancer, gastric cancer and melanoma [13,15,21]. For instance, miR-451a repressed the migration and invasion to inhibit melanoma progression [21]. In addition, miR-451a has also been reported to reduce the resistance of cancer cells to chemotherapy [17]. Accordingly, miR-451a suppressed cell proliferation and promoted the tamoxifen sensitive in breast cancer through macrophage migration inhibitory factor [22]. Therefore, miR-451a might be a promising target for tumor treatment. In this study, we further confirmed that miR-451a was implicated in DOX resistance of lung cancer cells. Increasing studies have suggested that EMT is a biological process through which epithelial cells are transformed into mesenchymal-phenotype cells. EMT plays an essential role in regulating tumor pathogenesis [23,24]. EMT activation is frequently detected during the progression of various types of tumors, including lung cancer [25,26]. Abnormal EMT promotes the cancer cells have highly malignant properties, which include invasion, migration, as well as distant metastasis [27]. Furthermore, a large number of studies have reported that tumor cells undergoing EMT exhibit cancer stem cell characteristics, enhancing the chemoresistance development consequently [28,29]. Targeting EMT could overcome chemoresistance and subsequently repress tumor progression [30]. In lung cancer, there are studies have indicated that EMT is closely associated with chemoresistance [31,32]. In the present study, we showed the EMT suppressive mechanism by miR-451a in lung cancer cells. We for the first time demonstrated that miR-451a mediated EMT to suppress DOX resistance in lung cancer cells, as evidenced by the reduced N-cadherin and the enhanced Ecadherin expression levels, which are critical markers for EMT progression [33,34]. The bioinformatics assay here identified c-Myc as a taregt of miR451a in lung cancer cells. Results in the study indicated that miR-451a over-expression resulted in the reduction of c-Myc in lung cancer cells,
Acknowledgments This work was supported by (1) The National Key Research and Development Program of China (2018YFC1313600); (2) Major International (Regional) Joint Research Project (81820108001); (3) National Natural Science Foundation of China (81670029); and (4) Xuzhou Administration of Science & Technology (KC16SH034). References [1] M.G. Oser, M.J. Niederst, L.V. Sequist, et al., Transformation from non-small-cell lung cancer to small-cell lung cancer: molecular drivers and cells of origin, Lancet Oncol. 16 (4) (2015) e165–e172. [2] F.R. Hirsch, G.V. Scagliotti, J.L. Mulshine, et al., Lung cancer: current therapies and new targeted treatments, Lancet 389 (10066) (2017) 299–311. [3] C. Swanton, R. Govindan, Clinical implications of genomic discoveries in lung cancer, N. Engl. J. Med. 374 (19) (2016) 1864–1873. [4] S. Gettinger, N.A. Rizvi, L.Q. Chow, et al., Nivolumab monotherapy for first-line treatment of advanced non–small-cell lung cancer, J. Clin. Oncol. 34 (25) (2016) 2980. [5] C. Zappa, S.A. Mousa, Non-small cell lung cancer: current treatment and future advances, Transl. Lung Cancer Res. 5 (3) (2016) 288. [6] B. Tu, J. Zhu, S. Liu, et al., Mesenchymal stem cells promote osteosarcoma cell survival and drug resistance through activation of STAT3, Oncotarget 7 (30) (2016) 48296. [7] J.A. Moscow, K.H. Cowan, B.I. Sikic, Drug resistance and its clinical circumvention, Holland-Frei Cancer Medicine, (2016), pp. 1–7. [8] L. Lv, X. An, H. Li, et al., Effect of miR-155 knockdown on the reversal of doxorubicin resistance in human lung cancer A549/dox cells, Oncol. Lett. 11 (2) (2016) 1161–1166. [9] Y. Peng, C.M. Croce, The role of MicroRNAs in human cancer, Signal Transduct. Target. Ther. 1 (2016) 15004. [10] A. Ramassone, S. Pagotto, A. Veronese, et al., Epigenetics and microRNAs in cancer, Int. J. Mol. Sci. 19 (2) (2018) 459. [11] K. Inamura, Y. Ishikawa, MicroRNA in lung cancer: novel biomarkers and potential tools for treatment, J. Clin. Med. 5 (3) (2016) 36. [12] P. Ren, F. Gong, Y. Zhang, et al., MicroRNA-92a promotes growth, metastasis, and chemoresistance in non-small cell lung cancer cells by targeting PTEN, Tumor Biol. 37 (3) (2016) 3215–3225. [13] I. Riquelme, O. Tapia, P. Leal, et al., miR-101-2, miR-125b-2 and miR-451a act as potential tumor suppressors in gastric cancer through regulation of the PI3K/AKT/ mTOR pathway, Cell. Oncol. 39 (1) (2016) 23–33. [14] E. Minna, P. Romeo, M. Dugo, et al., Correction: miR-451a is underexpressed and targets AKT/mTOR pathway in papillary thyroid carcinoma, Oncotarget 9 (15) (2018) 12534. [15] C. Chang, J. Liu, W. He, et al., A regulatory circuit HP1γ/miR-451a/c-Myc promotes prostate cancer progression, Oncogene 37 (4) (2018) 415. [16] Y.Y. Shen, J.Y. Cui, J. Yuan, et al., MiR-451a suppressed cell migration and invasion in non-small cell lung cancer through targeting ATF2, Eur. Rev. Med. Pharmacol. Sci. 22 (2018) 5554–5561.
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[17] R.H.E. Krakowsky, A.A. Wurm, D. Gerloff, et al., miR-451a abrogates treatment resistance in FLT3-ITD-positive acute myeloid leukemia, Blood Cancer J. 8 (3) (2018) 36. [18] H. El Hadi, I. Abdellaoui-Maane, D. Kottwitz, et al., Development and evaluation of a novel RT-qPCR based test for the quantification of HER2 gene expression in breast cancer, Gene 605 (2017) 114–122. [19] D.H. Shin, Y.J. Choi, J.W. Park, SIRT1 and AMPK mediate hypoxia-induced resistance of non-small cell lung cancers to cisplatin and doxorubicin, Cancer Res. 74 (1) (2014) 298–308. [20] S. Naidu, M. Garofalo, microRNAs: an emerging paradigm in lung cancer chemoresistance, Front. Med. 2 (2015) 77. [21] S. Babapoor, E. Fleming, R. Wu, et al., A novel miR-451a isomiR, associated with amelanotypic phenotype, acts as a tumor suppressor in melanoma by retarding cell migration and invasion, PLoS One 9 (9) (2014) e107502. [22] Z. Liu, T. Miao, T. Feng, et al., miR-451a inhibited cell proliferation and enhanced tamoxifen sensitive in breast cancer via macrophage migration inhibitory factor, Biomed Res. Int. 2015 (2015). [23] Q.C. Zhu, R.Y. Gao, W. Wu, et al., Epithelial-mesenchymal transition and its role in the pathogenesis of colorectal cancer, Asian Pacific J. Cancer Prev. 14 (5) (2013) 2689–2698. [24] J.E. Larsen, V. Nathan, J.K. Osborne, et al., ZEB1 drives epithelial-to-mesenchymal transition in lung cancer, J. Clin. Invest. 126 (9) (2016) 3219–3235. [25] X. Zheng, J.L. Carstens, J. Kim, et al., Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer, Nature 527 (7579) (2015) 525. [26] Y. Tang, G. Shu, X. Yuan, et al., FOXA2 functions as a suppressor of tumor metastasis by inhibition of epithelial-to-mesenchymal transition in human lung cancers,
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
MiR-451a attenuates doxorubicin resistance in lung cancer via suppressing epithelialmesenchymal transition (EMT) through targeting c-Myc
T
Li Taoa,b,1, Wang Shu-Lingb,1, Hao Jing-Boc, Zhang Yingb, Hu Ronga,d, Liu Xiang-Quna, Cui Wen-Jieb, Zhou Lin-Fua,* a
Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu 210029, China Department of Respiratory Medicine, The Municipal Hospital Affiliated to Xuzhou Medical University, Xuzhou, Jiangsu 221002, China c Department of Geriatrics, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221000, China d Department of Respiratory and Critical Care Medicine, The First People’s Hospital of Lianyungang, Lianyungang, Jiangsu 222006, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Chemoresistance Lung cancer miR-451a EMT c-Myc
Chemoresistance is still a major obstacle for lung cancer treatment. Increasing studies have demonstrated that microRNAs (miRNAs) are essential meditators of chemoresistance during cancer progression. MiR-451a is reported to be a tumor suppressor during cancer development. However, its effects on lung cancer and drug resistance in lung cancer are still unclear. In the study, the results showed that miR-451a exhibited a significant role in suppressing the drug resistance in lung cancer cells when treated with doxorubicin (DOX) through alleviating epithelialmesenchymal transition (EMT), as evidenced by the markedly reduced expression of N-cadherin and Vimentin, while the enhanced expression of E-cadherin. In addition, miR-451a over-expression markedly promoted the sensitivity of lung cancer cells to DOX treatments, and also disrupted the EMT of lung cancer cells. Mechanistically, miR-451a was found to directly target c-Myc to affect the EMT and drug resistance in lung cancer cells in response to DOX incubation. Furthermore, c-Myc knockdown markedly elevated the sensitivity of lung cancer cells to DOX, whereas over-expressing c-Myc markedly reversed the anti-tumor role of DOX, which was slightly diminished by miR-451a mimic. The in vivo experiments confirmed that miR-451a promoted the sensitivity of lung cancer cells-derived tumors to DOX treatment by reducing c-Myc. Therefore, our results revealed a new insight into DOX resistance of lung cancer cells and miR-451a could be considered as a potential therapeutic target to overcome drug resistance in lung cancer.
1. Introduction Lung cancer is a major cause of tumor-associated mortality among human worldwide with a 5-year survival rate of < 15 % [1,2]. As reported, the non-small-cell lung cancer (NSCLC) is consisted of a largely heterogeneous group of malignancies and accounts for about 85 % of all diagnosed lung cancers [3]. Accumulating studies have demonstrated that surgical resection is effective for early-stage non-metastatic lung tumors. Chemotherapy alone or in combination with radiation is defined as the frontline therapeutic strategy for the treatment of advanced lung cancer [4,5]. Nevertheless, because of the inherent or acquired drug resistance, the efficiency of chemotherapy has been ephemeral and significantly limited, leading to poor survival rate consequently [6,7]. For instance, doxorubicin (DOX) has been widely used for lung cancer treatment. However, DOX resistance limits its clinical outcomes
[8]. A better understanding of the molecular mechanisms revealing chemoresistance, including DOX resistance, is important for achieving better survival rates for patients with lung cancer. MicroRNAs (miRNAs) are small non-coding RNAs, and negatively modulate gene expression through binding to the 3′-untranslated region (3′-UTR) of their targeting mRNAs, resulting in translational repression and/or mRNA degradation [9,10]. Abnormal expression of miRNA has also been reported during lung cancer progression, which is tightly associated with the cell proliferation, invasion, migration and chemoresistance [11,12]. As reported, miR-451a acted as a tumor suppressor, and was down-regulated in gastric cancer, papillary thyroid carcinoma and prostate cancer [13–15]. Recently, miR-451a was also reported to suppress NSCLC progression through repressing migration and invasion by targeting activating transcription factor 2 (ATF2) [16]. In acute myeloid leukemia, miR-451a abolished treatment resistance
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Corresponding author. E-mail address: [email protected] (Z. Lin-Fu). 1 The first two authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2020.109962 Received 8 January 2020; Received in revised form 19 January 2020; Accepted 23 January 2020 0753-3322/ © 2020 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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2.5. Western blot analysis
[17]. Although miR-451a is involved in chemoresistance during tumor growth, its physiological effects on DOX resistance of lung cancer are still unclear. In this study, our results demonstrated that miR-451a over-expression attenuated DOX resistance in lung cancer cells through targeting cMyc. In addition, EMT was also inhibited by miR-451a in lung cancer cells when treated with DOX, which was also by directly targeting cMyc. Our study provided functional and mechanistic links between miR-451a and DOX resistance, and thus demonstrated a promising therapeutic target for lung cancer treatment.
Total proteins from the cells or tumor tissues were extracted with a protein lysis buffer (Beyotime, Nanjing, China) with PhosStop phosphatase inhibitor cocktail and protease inhibitor cocktail (Roche Diagnostics, USA). Lysates were denatured prior to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, UK). Then, the membranes were blocked using 5 % nonfat milk at room temperature for 1.5 h, followed by incubation with primary antibodies at 4 °C overnight. Next, the membranes were further incubated with secondary antibodies (Beyotime) for 1 h. The immunoreactive signals were finally visualized with the enhanced chemiluminescence reagents (ECL, Pierce, USA). The primary antibodies used in the study were shown as followings: anti-Vimentin (ab92547, Abcam, USA), anti-Ecadherin (PA5-85088, ThermoFisher Scientific, USA), anti-N-cadherin (MA5-32088, ThermoFisher Scientific), anti-c-Myc (ab32072, Abcam) and anti-GAPDH (ab8245, Abcam).
2. Materials and methods 2.1. Cells and culture Human lung cancer cells lines, including A549, PC9, H1299 and H460 were obtained from the American Type Culture Collection (ATCC, USA). All cells were cultured in RPMI 1640 medium (Gibco, USA) or Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10 % fetal bovine serum (FBS, Hyclone, USA) containing 100 units/mL penicillin and 100 mg/mL streptomycin. Cell lines were grown at 37 °C in a humidified atmosphere with 5 % CO2 and 95 % air. For the A549/ADR and PC9/ADR cells with drug resistance, the resistant variants were originated through growing initial A549 and PC9 cells with raising concentrations DOX (Sigma-Aldrich, USA). DOX was added to cells every 3 days. A549/ADR and PC9/ADR cells were 35 times as much resistant to the DOX cytotoxicity when compared to the initial A549 and PC9 cells. DOX at a final concentration of 1 mg/l was subjected to cells for maintaining the drug resistance until 1 week prior to the experiments.
2.6. Real-time quantitative PCR (RT-qPCR) analysis Total RNA from the cultured cells was extracted using the RNAeasy Mini Kit (Qiagen, USA) according to the manufacturer’s protocols, and reverse-transcribed using the High Capacity Reverse Transcription Kit (Applied Biosystems, USA). Then, qPCR analysis was performed in duplicate using GoTaq qPCR Master Mix (Promega, USA) on the ABI7900HT Real-Time PCR system (Applied Biosystems). All results were analyzed using the 2–ΔΔCt method [18]. The miRNA expression levels were normalized to U6. The probes for U6 and miR-451a were purchased from Generay Biotech (Shanghai, China). The sequence of primers used for qPCR were listed as followings: miR-451a forward: 5′ACA CTC CAG CTG GGA AAC CGT TAC CAT TAC-3′; reverse, 5′-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG CTT ACA G-3′; U6 forward: 5′-GCT TCG GCA GCA CAT ATA CTA A-3′; reverse, 5′-AAC GCT TCA CGA ATT TGC GT-3′.
2.2. Cell viability analysis Cells after transfection and/or DOX treatments were seeded into a 96-well plate at 1000 cells per well, and cultured at 37 °C at 5 % CO2 for 4 h. For cell viability calculation, the cells were incubated with 10 μL of CCK-8 (Dojindo, Japan) for 2 h at 37 °C according to the manufacturer’s protocols. Finally, the density was measured at a wavelength of 450 nm with a microplate reader.
2.7. Xenograft model All animal experiments were performed following the standards regarding the use of laboratory animals and all experiments were approved by the Institutional Animal Care and Usage Committee of the First Affiliated Hospital, Nanjing Medical University (Nanjing, China). The male BALB/c nude mice (4–6 weeks old) were purchased from the Animal Center of Nanjing Medical University (Nanjing). To produce a xenograft tumor model, a total of 1 × 107 the A549 cells were injected into the right dorsal flanks of nude mice. After tumor volume reached to 50 cm3, the mice were randomly divided in to 4 groups: 1) PBS group, 2) DOX group (2 mg/kg body weight, every 2 days), miR-451a agomir (2 nmol each mouse, every 3 days), or DOX combined with miR-451a agomir. MiR-451a agomir was chemically modified from miR-451a mimic. Tumor size was measured using Vernier calipers every 3 days for 28 days. The tumor volume (mm3) was calculated as length × width2 × 0.5. After 4 weeks, the tumors were isolated, weighed and frozen or paraffin embedded for immunohistochemical calculation.
2.3. Transfection in vitro miR-451a mimics, miR-451a inhibitor, and their corresponding negative controls (NC) oligonucleotide were synthesized and purchased from RiboBio Co. Ltd. (Guangzhou, China). Small interfering RNA against c-Myc (si-c-Myc) and its nonspecific NC siRNA were purchased from GenePharma Co., Ltd. (Shanghai, China). The plasmids for c-Myc overexpression were purchased from GeneChem Co. Ltd. (Shanghai). All transfection were performed using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s recommendations. 2.4. Immunofluorescence staining After treatments, all cells were fixed with 4 % paraformaldehyde for 10 min, followed by blocking with 10 % BSA (Solarbio, Beijing, China) for 30 min at room temperature. Then, the cells were incubated with primary antibodies (N-cadhrin or E-cadherin, at 1:100 dilutions; Abcam, USA) at 4 °C overnight. Next, the cells were incubated with an Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) secondary antibodies (at 1:50 dilutions; Abcam) at room temperature for 1 h. The nucleus was stained with 4′6diamidino-2-phenylindole (DAPI, Solarbio). Finally, the fluorescent signals were detected with an inverted fluorescence microscope (Nikon Co., Ltd., Japan).
2.8. Immunohistochemical staining Tumor tissues were fixed with 4 % formalin and embedded in paraffin. Tissue sections (4 μm thickness) were deparaffinized in xylene, rehydrated in a serial of graded ethanol, and antigens were retrieved by boiling. Endogenous peroxidase activity was blocked using 10 % goat serum (Solarbio) for 30 min, followed by incubation with primary antibodies (KI-67 and c-Myc, Abcam) at 4 °C overnight. Then, all sections were treated with a secondary antibody (Solarbio) for 1 h at room temperature. The immunocomplexes were visualized with a 3,3′-diaminobenzidine solution (DAB, Solarbio). Finally, the sections were 2
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Fig. 1. MiR-451a over-expression sensitized lung cancer cells to DOX treatment. (A) DOX (0, 0.1, 0.25, 0.5, 1, 2.5, 5, 7.5, 10, 20, 50 and 100 μM) was treated to lung cancer cells (A549/ADR, A549, PC9/ADR, PC9, H1299 and H460) with or without drug resistance for 24 h. Then, all cells were collected for cell viability calculation using CCK8 analysis. (B) RT-qPCR analysis for miR-451a in lung cancer cells with or without drug resistance. (C) Lung cancer cells were transfected with miR-451a mimic for 24 h, followed by transfection efficacy measurement using RT-qPCR analysis. ***p < 0.001 vs the Ctrl group. (D) A549/ADR, (E) A549, (F) PC9/ADR, (G) PC9, (H) H1299 and (I) H460 cells were transfected with miR-451a mimic for 24 h, and then were subjected to DOX (0, 0.1, 0.25, 0.5, 1 and 2.5 μM) for another 24 h. Then, all cells were collected for cell viability calculation using CCK8. *p < 0.05 vs the corresponding Ctrl groups. (J) Lung cancer cells were transfected with miR451a inhibitor for 24 h, followed by transfection efficacy determination by RT-qPCR analysis. (K) A549/ADR, (L) A549, (M) PC9/ADR, (N) PC9, (O) H1299 and (P) H460 cells were transfected with miR-451a inhibitor for 24 h, and then were treated with DOX (0, 0.1, 0.25, 0.5, 1 and 2.5 μM) for another 24 h. Next, all cells were harvested for cell viability assessment with CCK8. *p < 0.05 vs the corresponding Ctrl groups. Data were expressed as mean ± SEM (n = 4 in each group).
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Fig. 2. MiR-451a over-expression suppressed EMT in DOX-treated lung cancer cells. A549 and PC9 cells were treated with DOX (2.5 μM) for 24 h. (A) Then, all cells were collected for western blot analysis of N-cadherin, Vimentin and E-cadherin, and for (B) RT-qPCR analysis of miR-451a in cells. A549 and PC9 cells were transfected with miR-451a inhibitor for 24 h, and then were incubated with DOX (2.5 μM) for another 24 h. **p < 0.01 vs the Ctrl group. All cells were collected for (C) western blot analysis of N-cadherin, Vimentin and E-cadherin, as well as (D) immunofluorescence staining for N-cadherin and E-cadherin. Data were expressed as mean ± SEM (n = 4 in each group).
PC9, H1299 and H460 cells displayed the lowest cell viability (Fig. 1A). Then, the expression of miR-451a was measured in lung cancer cells with or without drug resistance. As shown in Fig. 1B, miR-451a expression levels displayed an opposite trend relative to that of the cell proliferation, showing a positive relationship between miR-451a and DOX sensitivity in lung cancer cells. In order to further explore the effects of miR-451a on drug resistance, we over-expressed miR-451a in lung cancer cells by transfection with miR-451a mimic. Transfection efficacy was confirmed using RT-qPCR (Fig. 1C). CCK8 analysis showed that miR-451a over-expression significantly promoted the suppressive effects of DOX on lung cancer cells with or without drug resistance (Fig. 1D-I). In contrast, reducing miR-451a by its inhibitor (Fig. 1J) markedly elevated the DOX resistance in lung cancer cells (Fig. 1K-P). Together, these findings demonstrated a significant role of miR-451a in suppressing the DOX resistance in lung cancer cells.
stained with hematoxylin (Sigma), and observed under a light microscope (Olympus, Japan). 2.9. Statistical analysis All statistical analysis were performed using GraphPad prism v.6.0 (GraphPad Software, La Jolla, USA). Two tailed Student’s t-test was used to compare the difference between groups. The values are presented as means ± SEM. P values < 0.05 were considered statistical difference. 3. Results 3.1. MiR-451a over-expression sensitized lung cancer cells to DOX treatment CCK8 analysis showed that A549/ADR and PC9/ADR exhibited the highest viability in response to DOX treatments, whereas the A549, 4
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Fig. 3. MiR-451a negatively regulated c-Myc to control the proliferation and EMT in lung cancer cells. (A) TargetScan-predicted binding sequences of miR-451a in the 3′-UTR of c-Myc. A549 and PC9 cells were transfected with (B) miR-451a mimic or (C) miR-451a inhibitor for 24 h. Then, all cells were collected for western blot analysis of c-Myc. (D) Western blotting analysis of c-Myc in lung cancer cells with or without drug resistance. (E) A549 and A549/ADR cells, (F) PC9 and PC9/ADR cells were transfected with si-c-Myc for 24 h, followed by transfection efficacy measurement using western blot analysis. A549, A549/ADR, PC9 and PC9/ADR cells were co-transfected with miR-451a inhibitor and si-c-Myc for 24 h, followed by DOX (0, 0.1, 0.25, 0.5, 1 and 2.5 μM) incubation for another 24 h. Then, (G) the cell viability was assessed using CCK8 analysis, and (H) western blot analysis was used to determine N-cadherin and E-cadherin expression. *p < 0.05 vs the corresponding NC groups; +p < 0.05 vs the corresponding si-c-Myc + miR-451a inhibitor group. (I) A549, A549/ADR, PC9 and PC9/ADR cells were transfected with cMyc plasmid for 24 h to over-express the cellular c-Myc. Western blot analysis was used for the transfection efficiency determination. A549, A549/ADR, PC9 and PC9/ADR cells were transfected with c-Myc plasmid for 24 h, and then were treated with DOX for another 24 h. Next, all cells were subjected to (J) western blot analysis of N-cadherin and E-cadherin, and (K) CCK8 analysis of cell viability. Data were expressed as mean ± SEM (n = 4 in each group).
time, miR-451a expression levels were induced by DOX (Fig. 2B). Then, we found that the role of DOX in regulating the EMT was reversed by the transfection of miR-451a inhibitor through western blot and immunofluorescence staining (Fig. 2C and D). MiR-451a over-expression inhibited EMT in DOX-treated lung cancer cells
3.2. MiR-451a over-expression suppressed EMT in DOX-treated lung cancer cells In this part, we investigated whether EMT, a critical factor during the progression of drug resistance, was implicated in DOX sensitivity regulated by miR-451a in lung cancer cells. As shown in Fig. 2A, Ncadherin and Vimentin expression levels in A549 and PC9 cells were reduced by DOX treatment, while E-cadherin was up-regulated, demonstrating the reduction of EMT in lung cancer cells. At the same 5
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Fig. 4. MiR-451a over-expression promoted the anti-tumor role of DOX in vivo. (A) Representative images of tumor samples from each group of mice. (B) Tumor volume and (C) tumor weight were measured. (D) Immunohistochemical staining of KI-67 and c-Myc in tumor sections. (E) Quantification of KI-67 and c-Myc expression following the immunohistochemical analysis. (F) Western blotting analysis of c-Myc, N-cadherin an E-cadherin in tumor samples. Data were expressed as mean ± SEM (n = 8 in each group). **p < 0.01 vs the Ctrl group.
or without drug resistance (Fig. 3E and F). CCK8 assays indicated that si-c-Myc markedly promoted the inhibitory role of DOX in reducing cell viability in A549 and A549/ADR cells, while these effects were significantly abrogated by miR-451a inhibitor. Similar trends of cell viability were observed in PC9 and PC9/ADR cells transfected with si-cMyc and/or miR-451a inhibitor following DOX treatments (Fig. 3G). Ncadherin expression levels suppressed by DOX were further reduced by si-c-Myc, while being rescued in lung cancer cells with or without drug resistance when transfected with miR-451a inhibitor; however, an opposite result was observed in the expression change of E-cadherin (Fig. 3H). Subsequently, we over-expressed c-Myc in lung cancer cells (Fig. 3I). Western blot analysis indicated that DOX-reduced N-cadherin and -increased E-cadherin were reversed by c-Myc over-expression,
3.3. MiR-451a negatively regulated c-Myc to control the proliferation and EMT in lung cancer cells To further explore the molecular mechanism revealing the chemoresistance regulated by miR-451a in lung cancer cells with or without drug resistance, a miRNA target prediction website was applied to find the target gene of miR-451a. Here, c-Myc was identified as a miR-451a target gene containing the binding sequence as shown in Fig. 3A. Western blot results showed that miR-451a over-expression reduced c-Myc in A549 and PC9 cells, while miR-451a inhibition promoted c-Myc (Fig. 3B and C). Then, we found that A549/ADR and PC9/ ADR cells had higher c-Myc expression than that of the A549 and PC9 cells (Fig. 3D). Then, c-Myc was knocked down in lung cancer cells with 6
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The in vivo experiments were performed to further investigate the effects of miR-451a on DOX resistance. As displayed in Fig. 4A-C, miR451a agomir combined with DOX markedly reduced the tumor volume and weight. Immunohistochemical staining demonstrated significant decreases in KI-67 and c-Myc in tumor sections from mice co-treated with miR-451a agomir and DOX (Fig. 4D and E). Western blot results showed that DOX and miR-451a agomir co-treatments markedly reduced c-Myc and N-cadherin expression levels in tumor samples, while enhanced E-cadherin protein expression levels (Fig. 4F). Together, these results demonstrated that miR-451a over-expression promoted the drug sensitivity in the treatment of lung cancer.
while miR-451a knockdown led to the increases in c-Myc. The increase of c-Myc was demonstrated to be associated with drug resistance and the outcome of patients with lung cancer [35]. Suppressing c-Myc expression markedly sensitized lung cancer cells to cisplatin treatment [36]. Therefore, c-Myc is a critical molecule that regulates chemoresistance in lung cancer cells. In this study, we confirmed that miR-451a could also directly target c-Myc at the 3′-UTR and thereafter reduce cMyc expression in lung cancer cells. Moreover, we also found that both miR-451a over-expression and c-Myc knockdown could markedly inhibit EMT and suppress DOX resistance in lung cancer cells. According to all these results, we supposed that c-Myc was a functional target gene of miR-451a, thus modulating DOX resistance in lung caner, which were confirmed by the in vivo experiments. In summary, our findings identified an essential molecular mechanism through which miR-451a regulated DOX resistance in lung cancer. Promoting miR-451a enhanced the sensitivity of lung cancer cells to DOX treatment by repressing EMT through targeting c-Myc. We therefore established new therapeutic strategy to overcome chemoresistance in lung cancer. However, if other genes participate in the improved DOX sensitivity still require further exploration.
4. Discussion
Declaration of Competing Interest
which were, however, abolished by miR-451a mimic (Fig. 3J). Finally in this part, CCK8 results showed that DOX-reduced cell viability in lung cancer cells was rescued by c-Myc over-expression; consistently, these effects were reversed by miR-451a mimic (Fig. 3K). Findings in this regard suggested that miR-451a negatively regulated c-Myc to mediate the cell viability and EMT in lung cancer cells. 3.4. MiR-451a over-expression promoted the anti-tumor role of DOX in vivo
The authors declare that they have no competing interests.
Despite the use of DOX has significantly improved the survival rate of patients with lung cancer, the drug resistance limits its wide application and results in the treatment failure [2–4,19]. Increasing evidences have suggested that miRNAs suppress gene expression through restraining transcription or eliciting the degradation of the targeted genes [9,10]. The abnormal expression of miRNA is commonly observed during lung cancer progression, which is also involved in chemoresistance [11,12,20]. Nevertheless, the regulatory role of miRNAs is pretty complicated, and their effects on drug resistance of lung cancer are still unclear. MiR-451a is suggested to function as a tumor suppressor in different types of cancers, such as prostate cancer, gastric cancer and melanoma [13,15,21]. For instance, miR-451a repressed the migration and invasion to inhibit melanoma progression [21]. In addition, miR-451a has also been reported to reduce the resistance of cancer cells to chemotherapy [17]. Accordingly, miR-451a suppressed cell proliferation and promoted the tamoxifen sensitive in breast cancer through macrophage migration inhibitory factor [22]. Therefore, miR-451a might be a promising target for tumor treatment. In this study, we further confirmed that miR-451a was implicated in DOX resistance of lung cancer cells. Increasing studies have suggested that EMT is a biological process through which epithelial cells are transformed into mesenchymal-phenotype cells. EMT plays an essential role in regulating tumor pathogenesis [23,24]. EMT activation is frequently detected during the progression of various types of tumors, including lung cancer [25,26]. Abnormal EMT promotes the cancer cells have highly malignant properties, which include invasion, migration, as well as distant metastasis [27]. Furthermore, a large number of studies have reported that tumor cells undergoing EMT exhibit cancer stem cell characteristics, enhancing the chemoresistance development consequently [28,29]. Targeting EMT could overcome chemoresistance and subsequently repress tumor progression [30]. In lung cancer, there are studies have indicated that EMT is closely associated with chemoresistance [31,32]. In the present study, we showed the EMT suppressive mechanism by miR-451a in lung cancer cells. We for the first time demonstrated that miR-451a mediated EMT to suppress DOX resistance in lung cancer cells, as evidenced by the reduced N-cadherin and the enhanced Ecadherin expression levels, which are critical markers for EMT progression [33,34]. The bioinformatics assay here identified c-Myc as a taregt of miR451a in lung cancer cells. Results in the study indicated that miR-451a over-expression resulted in the reduction of c-Myc in lung cancer cells,
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