Lung cancer is one of the most common malignant cancers leading the most cancer-related deaths around the world . In China, lung cancer causes about 23% of deaths annually among men, and the overall five-year survival of lung cancer patients remains under 17% . Besides surgical excision, platinum-based chemotherapy such as cisplatin (DDP) is one of the most effective ways concerning various cancer treatments, including lung cancer . However, the long-term and continuous infusion of DDP frequently results in the drug resistance, causing treatment interruption or failure . Therefore, it is urging to discover the underlying mechanisms of DDP resistance in lung cancer cells in order to improve chemotherapy efficiency.
MicroRNAs (miRNAs) are small, non-coding regulatory RNAs containing 21-25 nucleotides, functioning as critical regulators of post-transcription gene expression [5,6]. Accumulating evidences proved that dysregulation of miRNAs was involved in regulating cancer cell development and drug resistance in various tumors, such as lung cancer , breast cancer , gastric cancer  and et al. Previous studies reported that up-regulation of miR-1236-3p could significantly inhibit lung cancer cell proliferation, migration and invasion . In consequence, miR-1236-3p may function as a tumor suppressor and participate in modulating the tumor development process in lung cancer. However, whether or not miR-1236-3p is related to drug resistance in lung cancer still remains to be explored.
In the current study, the underlying mechanism of miR-1236-3p in DDP-resistant lung cancer cells was illustrated. The results suggested that up-regulation of miR-1236-3p could reverse DDP resistance in lung cancer cells through targeting TPT1 and inhibition of the Pim-3 signaling pathway.
2 Materials and methods
30 pairs of lung cancer tumor tissues (Table. 1) and non-tumor adjacent tissues were obtained from Weifang Traditional Chinese Hospital between July 2014 and April 2016 from patients who had undergone surgical resection. All the patients were pathologically diagnosed with lung cancer without receiving any radiotherapy or chemotherapy. After resection, all of the tissues were preserved in liquid nitrogen at -80°. This project was approved by the Ethical Committee of Weifang Traditional Chinese Hospital and the written consent was obtained from each patient or relative.
2.2 Cell culture
Human normal lung epithelial cells (BESA-2B) and lung cancer cell line (A549) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). DDP resistant A549 sub-line A549/DDP was purchased from the Cancer Hospital of Peking Union Medical College, Chinese Academy of Medical Sciences. All the cell lines were cultivated in Dulbecco’s Modified Eagle’s medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing penicillin-streptomycin. All the cell lines were cultivated in a humid atmosphere with 5% CO2 at 37°.
2.3 Dual-luciferase reporter assay
TPT1 3’UTR was amplified from cDNA of 293 cells and inserted into pGL-3 (Promega, USA). The 293 cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cotransfected with the wild-type 3’UTR of TPT1 containing the two putative miR-1236-3p binding sites (Site 1: 473-480 and Site 2: 1085-1091) and mutant TPT1 3’UTR (TPT1-3’UTR) with either NC mimics or miR-1236-3p mimics via Lipofectamine®2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA). After transfection, the cells were cultivated at 37°, 5% CO2 for 4 h. Then, the luciferase activities were confirmed using a dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
2.4 Cell transfection
The NC mimics and miR-1236-3p mimics were purchased from Sigma-Aldrich (Shanghai, China). According to the project, A549/DDP cells were divided into three different groups in order to measure the transfection efficiency of miR-1236-3p: i) the control group (un-treated cells); ii) the negative control (NC) group (to eliminate non-sequence specific effects) and iii) the mimics group (cells were transfected with miR-1236-3p mimics). The A549/DDP cells were seeded into six-well plates, with the density of 4×105 cells/well, then the miR-1236-3p or the NC mimics were transfected into cells with Lipofectamine®2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer’s protocol.
The pcDNA3.1 plasmids were purchased from Gene-Chem (Shanghai, China). The pcDNA3.1-TPT1 plasmids were constructed by inserting the TPT1 CDS expression into the pcDNA3.1 vector. According to the project, A549/DDP cells were divided into three different groups in order to confirm the expression of TPT1 gene in different groups: i) the control group (un-treated cells); ii) pcDNA3.1 group (cells transfected with blank plasmid) and iii) pcD-NA3.1-TPT1 (cells transfected with pcDNA3.1-TPT1 plasmids). The A549/DDP cells were seeded into six-well plates, with the density of 4×105 cells/well, then the pcDNA3.1 or the pcDNA3.1-TPT1 were transfected into cells using Lipofectamine®2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the instructions.
2.5 RNA extraction and PR-qPCR assay were conducted
Total RNA from tissues or cells were isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. Meanwhile, miRNAs were isolated from tissues or cells via the miRNeasy Mini Kit (Qiagen, Valencia, CA, USA). Afterward, TaqMAnTM MicroRNA Reverse Transcription Kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was applied to synthesize cDNA. The RNA quantity was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE, USA). Then, 2 μl RNA were reverse transcribed into cDNA using TaqManTM Reverse Transcription Reagents (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Subsequently, RT-qPCR (Real-time Quantitative polymerase chain reaction) analysis was performed as followed. Briefly, 2 μl-cDNA, 10 μl-SYBR Green RT-qPCR Master Mix (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1 μl-primers and 7 μl ddH2O were mixed together during the whole PCR reaction process. The PCR reaction conditions were described as followed: i) initially denatured at 94° for 3min; ii) denatured at 94° for 30s, annealed at 55° for 30s, extended at 72° for 30s, this procedure was repeated 30 cycles and iii) finally extended at 72° for 10min. In data processing, the relative expression level of miR-1236-3p was normalized to U6 small nuclear RNA (Thermo Fisher Scientific, Inc., Waltham, MA, USA), while the expression levels of TPT1 and related proteins were normalized to β-actin (R&D Systems China, Co. Ltd, Shanghai, China) according to the 2-ΔΔCt method. The primer sequences were described as followed: miR-1236-3p forward, 5′-CCAATCAGCCTCT-TCCCCTT-3′ and reverse, 5′-TATGGTTGTTCACGACTCCT-TCAC-3′; U6 forward, 5′-ATTGGAACGATACAGAGAAGATT-3′ and reverse, 5′-GGAACGCTTCACGAATTTG-3′; TPT1 forward, 5’- TATTGGACTACCGTGAGG-3’ and reverse, 5’-CAAGCAGAAGCCAGTTATG-3’; β-actin forward, 5’-AGAT-GTGGATCAGCAAGCAG-3’ and reverse, 5’-GCGCAAGTTAG-GTTTTGTCA-3’.
2.6 The IC50 of DDP determined by MTT assay
The cytotoxic activity of DDP was determined by the colorimetric MTT assay. In brief, cells with the density of 4×103 cells/ml were seeded on a 96-well plate with 100 μl culture medium per well. Cells were incubated at 37° in a humid atmosphere with 5% CO2 for 24 h and treated with cisplatin subsequently. Refer to previous study and experiment , a concentration of 20 μg/ml-DDP was prepared and added to the 96-well plate. After incubating for 24 h, 20 μl-3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT, 5 mg/ml) was added to the cells in the dark and incubated for 4 h at 37°, 5% CO2, covered with aluminum foil. Afterward, 100 μl DMSO (Solarbio®, Beijing, China) was added to each well to dissolve the formazan crystals formed, and the 490/570 nm double-wave-length measurement optical density was measured via a microplate reader (Bio-Tek Instruments, Winooski, VT, USA). The potency of cell growth inhibition for the test agent was expressed as the half maximal (50%) inhibitory concentration, IC50. All the procedures were conducted in triplicate.
2.7 The proliferation rate of A549/DDP cells confirmed by MTT assay
According to the experimental design, all the following experiments were divided into four different groups: i) the control group (un-treated cells); ii) the negative control (NC) group (to eliminate non-sequence specific effects); iii) the miR-1236-3p mimics group (cells were transfected with miR-1236-3p mimics), and iv) the miR-1236-3p mimics+pcDNA3.1-TPT1 group (cells were transfected with miR-1236-3p mimics and pcDNA3.1-TPT1).
Cells from different groups were seeded on a 96-well plated, with the density of 5×103 cells/well. After incubation for 24 h, 20 μl-3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml) was added to the cells in the dark and incubated for 4 h at 37°, 5% CO2, covered with aluminum foil. Afterward, 100 μl DMSO (Solarbio®, Beijing, China) was added to each well to dissolve the formazan crystals formed, and the absorbance was read at 490 nm using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA). All the procedures were performed in triplicate.
2.8 Apoptosis rate of A549/DDP cells confirmed by a flow cytometry
The apoptosis rate of cultured A549/DDP cells was determined using Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; BD Pharmigen, San Diego, CA, USA). In brief, cells from different groups with the density of 3×105cells/well were seeded in a six-well plate and cultured for 24 h. After being washed with phosphate-buffered saline (PBS; Solarbio®, Beijing, China) three times, cells were stained with eBioscienceTM flow cytometry staining buffer (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 15 min in the dark. The fluorescence was assessed by CytoFLEX flow cytometry (Beckman Coulter, Inc., CA, USA).
2.9 Effect of miR-1236-3p on the expression levels of TPT1, Pim-3, BAD and Bcl-XL
After washed with phosphate-buffered saline (PBS; Solar-bio®, Beijing, China), cells from different groups were lysed by the RIPA buffer (Solarbio®, Beijing, China) supplemented with a protease inhibitor cocktail (MedChem Express, Shanghai, China). All the protein was quantified using a Bradford protein assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Equal amounts (20 μg) of the cell lysates were separated by 8% SDS-PAGE (Detai-Bio, Nanjing, China) and transferred onto polyvinylidene difluoride membranes (PVDF; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Then, the blots were blocked in 50 ml 5% bovine serum albumin (BSA; Haoran Biological Technology Co., Ltd, Shanghai, China) for 1 h. Later on, the primary antibodies were added on the membrane at 37° overnight: mouse anti-TPT1 (1:1000; A5442; ABclonal, Wuhan, China), rabbit anti-Pim-3 (1:500; ab71321; Abcam, Cambridge, MA, USA); rabbit anti-phospho-BAD (1:5000; ab129192; Abcam, Cambridge, MA, USA); rabbit anti-Bcl-XL (1:1000; ab32370; Abcam, Cambridge, MA, USA) and mouse anti-β-actin (1:1000; ab8226; Abcam, Cambridge, MA, USA). After washed with phosphate-buffered saline (PBS; Solarbio®, Beijing, China) three times, the secondary antibodies IgG H&L (HRP; 1:1000; ab7090; Abcam, Cambridge, MA, USA) and IgG H&L (Cy3® ;1:2000; ab97035; Abcam, Cambridge, MA, USA). Subsequently, the membranes were washed four times with phosphate-buffered saline (PBS; Solarbio®, Beijing, China), containing 0.1% Tween 20 (Sigma-Aldrich, Shanghai, China). The results were visualized using the X-ray films after adding 200 μl chemiluminescent system (Thermo Fisher Scientific, Inc., Waltham, MA, USA). A molecular Imager Chem-iDox XRS system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to quantify the relative expression levels of TPT1 and related-proteins. β-actin functioned as the reference protein.
2.10 Statistical analysis
SPSS 17.0 statistical software (IBM Corporation, Armonk, NY, USA) was used to analyze the data. The data were presented as the mean ± deviation. Student’s t-test was used to determine differences between two groups, while one-way analysis of variation (ANOVA) followed by NewmanKeuls analysis was applied to verify differences among three or four groups. A P value <0.05 was considered to be statistically significant.
3.1 The lower expression level of miR-1236-3p in tumor and cancer cells
As shown in Fig. 1A the expression level of miR-1236-3p was significantly lower in the lung cancer tumor tissues as compared with non-tumor adjacent tissues (P<0.01). Meanwhile, Fig. 1B further demonstrated that the expression level of miR-1236-3p was remarkably restrained in the A549 lung cancer cell line, compared to the human normal lung epithelial cells BEAS-2B (P<0.01). Collectively, these results demonstrated that the expression level of miR-1236-3p was lower in lung cancer tumor tissues and cell lines than in non-tumor adjacent tissues and normal cells, respectively.
3.2 The IC50 of DDP and miR-1236-3p expression level in lung cancer cells
To detect the half maximal inhibitory concentration (IC50) of DDP for chemotherapy agent, two lung cancer cell lines were exposed to 20 μg/ml DDP for 24 h. After that, the MTT data revealed that the IC50 of DDP in A549 cells was lower than that in A549/DDP cells (Fig. 2A; P<0.01). Subsequently, the expression level of miR-1236-3p in two cell line was observed via RT-qPCR assay. As the results presented, the expression level of miR-1236-3p was markedly decreased in the A549/DDP cells compared with the A549 cells (Fig. 2B; P<0.01). Hence, our data suggested that DDP-resistant cell line A549/DDP was presented with a down-regulated expression level of miR-1236-3p.
3.3 miR-1236-3p regulates the DDP drug resistance in lung cancer cells
After the transfection of cells with NC mimics or miR-1236-3p mimics, the transfection efficiency of miR-1236-3p was detected by RT-qPCR assay. As shown in Fig. 3A the expression level of miR-1236-3p was prominently increased in the miR-1236-3p mimics group as compared to NC mimics group (P<0.01). Afterward, the IC50 of DDP in different groups was measured by MTT assay. The data in Fig. 3B demonstrated that the IC50 value of DDP was significantly decreased in the miR-1236-3p mimics group in contrast with NC mimics group (P<0.01).
3.4 TPT1 gene is the direct target of miR-1236-3p
Combining the online bioinformatics tool TargetScan, TPT1 was predicted to be a possible target gene of miR-1236-3p. In order to verify this prediction, a dual-luciferase reporter assay was conducted subsequently. As shown in Fig. 4A and B the 3’-UTR of the gene TPT1 were proved to contain the binding sequences for the miR-1236-3p in both 473-480 and 1085-1091 positions, suggesting that TPT1 may be a downstream target gene of miR-1236-3p. Meanwhile, as shown in Fig. 4C and D the luciferase activities in the wild-type TPT1 3’UTR (TPT1-3’UTR) plasmid-transfected cells were significantly restrained by the transfection of miR-1236-3p-mimics, whereas there were no variation in the mutant-type TPT1 3’UTR (TPT1-3’UTR) plasmid-transfected cells (P<0.01).
3.5 The expression level of TPT1 gene is confirmed after pcDNA3.1-TPT1 transfection
Since TPT1 was proved to be the direct target gene of miR-1236-3p, cells were transfected with pcDNA3.1 or pcD-NA3.1-TPT1 in order to confirm the expression level of TPT1 in different groups prior to the identification of the effects of TPT1 overexpression on miR-1236-3p mimics induced changes. The RT-qPCR assay results illustrated that the mRNA expression level of TPT1 was higher in the pcD-NA3.1-TPT1 group, in contrast with the pcDNA3.1 group (Fig. 5; P<0.01). Moreover, the protein expression of TPT1 by western blot assay presented the same trend of variation as well (Fig. 5B and C; P<0.01).
3.6 Up-regulation of miR-1236-3p markedly decreases the proliferation rate of A549/DDP cells by targeting TPT1
For the sake of further studying the effect of miR-1236-3p on lung cancer cells, A549/DDP cells were transfected with NC mimics, miR-1236-3p mimics, and miR-1236-3p mimics plus pcDNA3.1-TPT1. Analysis of the cell proliferation rate of A549/DDP cells demonstrated that miR-1236-3p mimics may restrain the proliferation rate (P<0.01) and this phenomenon could be reversed by pcDNA3.1-TPT1 (Fig. 6; P<0.01).
3.7 Up-regulation of miR-1236-3p markedly increases the apoptosis rate of A549/DDP cells by targeting TPT1
In order to confirm the effect of miR-1236-3p on lung cancer cells, a flow cytometry method was performed accordingly. The Fig. 7A-D represented the apoptosis rate among four different groups respectively, while the Fig. 7E was the corresponding quantified results of the flow cytometry assay. As the data exhibited, the apoptosis rate in cells transfected with miR-1236-3p mimics was significantly promoted (P<0.01), whereas the variation could be reversed by pcDNA3.1-TPT1 (P<0.05).
3.8 Effect of miR-1236-3p on the expression levels of TPT1, Pim-3, BAD and Bcl-XL
As to confirm whether miR-1236-3p and TPT1 participated in the regulation of apoptosis, the expression levels of Pim-3, BAD and Bcl-XL in different groups were measured by western blot assay. As shown in Fig. 8A and B the expression levels of TPT1, Pim-3, p-BAD and Bcl-XL were prominently decreased in the miR-1236-3p mimics group (P<0.01), whereas the variation was reversed by pcD-NA3.1-TPT1 (P<0.01).
According to previous studies, miR-1236-3p was reported to be engaged in regulating the tumor development process. For instance, miR-1236-3p could inhibit invasion and migration in gastric cancer , bladder cancer , ovarian carcinoma  and lung adenocarcinoma . Consistent with previous studies, our data demonstrated that miR-1236-3p was down-regulated in lung cancer tumor tissues and A549 cell lines, suggesting that miR-1236-3p functioned as a tumor suppressor in lung cancer. Furthermore, according to the IC50 value determined by MTT assay, the DDP-resistant cell line A549/DDP was presented with the down-regulated expression level of miR-1236-3p, indicating that miR-1236-3p was involved in regulating DDP resistance in lung cancer.
As the results exhibited, TPT1 was proved to be the direct target gene of miR-1236-3p by a dual-luciferase reporter assay. Recently, TPT1 was associated with cancer progression and was highly expressed in various tumors . As known, knockdown of TPT1 was proved to inhibit
cell proliferation or metastasis in colon cancer  and prostate cancer . Moreover, the TPT1 gene was reported to be up-regulated in A549 lung cancer cells as well . Thus, we suggested that TPT1 exerted the function as an oncogene taking part in modulating tumor development and evolution, however, whether TPT1 was involved in modulating drug resistance remained uncertain. Accordingly, our study illustrated the function that TPT1 exerted concerning the DDP resistance in lung cancer cells. As stated in MTT and flow cytometry, up-regulated miR-1236-3p significantly inhibited A549/DDP cell proliferation but promoted apoptosis by down-regulating the expression level of TPT1. That is to say, miR-1236-3p could reverse DDP resistance in lung cancer cells by restraining the expression level of TPT1. Collectively, our study firstly discovered the potential mechanism of miR-1236-3p and its target gene TPT1 in regards to the DDP resistance in lung cancer, which may contribute to the clinical chemotherapy treatment.
In order to further illustrate the mechanism of how miR-1236-3p mediated the DDP resistance in lung cancer cell by targeting TPT1 gene, the expression levels of related proteins were measured by western blot assay. Pim-3 is a member of the proto-oncogene Pim family , which could prevent apoptosis and promote cell proliferation . In addition, TPT1 was considered to be able to interact with Pim-3 and enhance Pim-3 protein stability . According to our results, miR-1236-3p down-regulated the expression levels of TPT1 and Pim-3, which were corresponded to previous studies. Meanwhile, a previous study pointed out that Pim-3 could inactivate BAD in human pancreas and colon carcinoma by phosphorylating BAD (p-BAD), that is Pim-3 may enhance the expression level of p-BAD [22,23]. BAD is known as a member of Bcl-2 family, binding to the Bcl-2/Bcl-XL complex and promoting apoptosis [24,25]. However, p-BAD dissociates from this complex, leading to the release of Bcl-2 and Bcl-XL, exerting the function of suppressing cell apoptosis [26,27]. Bcl-XL, a member of the Bcl-2 family as well, was found to be highly increased in A549/DDP cells . Meanwhile, Bcl-XL was also reported to be up-regulated in A549 cell lines, inhibiting lung cancer cell apoptosis as well . That is, TPT1, Pim-3, p-BAD and Bcl-XL functioned as oncogenes suppressing cell apoptosis in lung cancer cells. Another study had been reported that downregulation of TPT1 could restrain the expression levels of Pim3, p-BAD and Bcl-XL, promoting cell apoptosis . Results of western blot exhibited that miR-1236-3p may inhibit the expression levels of TPT1, Pim-3, p-BAD and Bcl-XL, the known tumor promoter genes in lung cancer, by targeting TPT1.
To sum up, up-regulating of miR-1236-3p could reverse DDP resistance in lung cancer cells by targeting TPT1 through Pim-3 signaling pathway.
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About the article
Published Online: 2019-01-16
Conflicts of interest: The authors have no conflicts of interest to declare.