Biomedicine & Pharmacotherapy 68 (2014) 7–12
Available online at
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Original article
MicroRNA-137 down-regulates KIT and inhibits small cell lung cancer cell proliferation Peipei Li a,b,1, Lijie Ma a,1, Yongjuan Zhang c, Fuyun Ji d, Faguang Jin a,* a
Department of Respiration, Tangdu Hospital, Fourth Military Medical University, 710038 Xi’an, China Department of Respiration, Harrison International Peace Hospital, 053000 Hengshui, China c Department of Physiology, Guangdong Medical College, 523808 Dongguan, China d Institute of Human Respiratory Disease, Xinqiao Hospital, Third Military Medical University, 400037 Chongqing, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 November 2013 Accepted 10 December 2013
MiR-137 expression was examined in parental and drug-resistant cell lines, H446 and H446/CDDP, of small lung cancer (SCLC), and the results showed there was fewer miR-137 expressed in H446/CDDP cells followed by KIT expression emergence. In order to confirm physiological function of these abnormal expressions, H446 and H446/CDDP cells were transfected with miR-137 inhibitor and miR-137 mimics, respectively, after that, miR-137 and KIT expression in two cell lines and drug sensitivity of these cells were evaluated. Results indicated that sensitivity of H446 cells to cisplatin significantly decreased after transfected with miR-137 inhibitor, while miR-137 mimics transfection increased drug sensitivity of H446/CDDP cells and deregulated KIT expression. Our data provided combined evidence that miR-137 was closely related to MDR of SCLC, and interfering of miR-137 expression may attenuate drug resistant of H446/CDDP cells to cisplatin partially through KIT expression regulation. Besides, it has also been proved that KIT might be only one of the downstream molecules of miR-137 that related to SCLC MDR. ß 2014 Published by Elsevier Masson SAS.
Keywords: MiR-137 KIT SCLC MDR
Abbreviations: CDDP, cisplatin RPMI, Roswell Park Memorial Institute PBS, phosphate buffered saline PVDF, Polyvinylidene Fluoride ECL, electrochemiluminescence SYBR, Synergy Brands CCK, Cell Counting Kit FITC, fluorescein isothiocyanate
1. Introduction Small cell lung cancer (SCLC) is an aggressive malignant tumor accounting for approximately 15–20% of all primary lung cancer, and its highly metastasizing characteristics result in poor prognosis and high mortality. In addition, 5-year survival rate of the SCLC is less than 15% [1]. Chemotherapy is the primary therapeutic approach for SCLC, and it is also sensitive to chemotherapy initially. Subsequently, the intrinsic or acquired multi-drug resistance (MDR) emerges and increases the rate of relapse and leads to poor prognosis [2]. Therefore, MDR has become the main obstacle for SCLC treatment in the world, and how to reverse MDR phenotype has become a central issue in improving the prognosis of SCLC. Previous studies have demonstrated that the MDR mechanism included multiple aspects, such as over expression of drug efflux pumps like P-glycoprotein (Pgp), multi-drug resistance associated protein 1 (MRP-1), lung * Corresponding author. Tel.: +86 29 84777425; fax: +86 29 84777425. E-mail address: [email protected] (F. Jin). 1 These authors contributed to the article equally. 0753-3322/$ – see front matter ß 2014 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.biopha.2013.12.002
resistance protein (LRP) in drug-resistant cells, the change of microenvironment in the cells, the enhanced function of DNA synthesis and repair, and the dysfunction of cell apoptosis pathway etc. [3]. All of the above indicated that the abnormal regulation of gene and protein expression played a critical role in the MDR process. MicroRNAs (miRNAs) are a cluster of short single-stranded, non-coding, endogenous RNA molecules, approximately 22 nucleotides that negatively regulate gene expression via binding to the 30 untranslated region (30 UTR) of mRNA, leading to repressing post-transcripional translation or the degradation of the target mRNA [4]. Extensive researches have indicated that aberrantly expressed miRNAs could affect cell biological activities, and dysregulation of miRNAs may be the mechanism of gene expression in cancer [5,6]. Besides, some miRNAs were related to the responses to chemotherapeutic treatment [7]. And miR-137, as one of the focused miRNAs, has been proved to participate in inhibiting epithelial mesenchymal [8], modulating the MDR of breast cancer cell [9], and suppressing lung cancer cell proliferation [10]. Though, further researches are needed to explore the role of miR-137 in the chemoresistance of small cell lung cancer cells.
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KIT, also named as CD117, is a transmembrane protein receptor encoded by the c-kit proto-oncogene [11]. Abnormal KIT expression is closely related to tumors, such as multiple myeloma [12], small cell lung cancer (SCLC) [13], and gastrointestinal stromal tumors [14,15], suggesting there is a complementary mechanism involving KIT expression in occurrence and development of tumors. The regulation of KIT by miRNA-221/miRNA-222 in erythroleukemic cells [16] and its regulation by miRNA-494 in gastrointestinal stromal tumor cells [17] have been explored, but no miRNAs targeting KIT have been identified in SCLC. In the present research, we used parental cells (H446) and drug resistant cell lines (H446/CDDP) to explore the possible mechanism of SCLC drug resistance, and proposed that miRNA-137 expression was involved in SCLC MDR probably through KIT pathway by showing that: miR-137 expression was negatively correlated to the expression of KIT; exogenous miR-137 suppressed KIT expression and led to higher sensitivity of H446/CDDP cells to cisplatin.
of the PCR bands using a Scion image analysis system (Beta 4.02, Scion Corporation). The results were expressed as the ratio to bactin mRNA level in the same RNA samples. 2.3. Western Blot analysis for KIT The cells were harvested and washed twice with PBS 72 h after transfection, then, the total protein was extracted using protein extraction reagent (Pierce Biotechnology, USA) and protease inhibitor (Roche Applied Science). After quantification, 20 mm protein was separated on 8% SDS-PAGE gels, transferred to PVDF membranes (Millipore, Bedford, MA) and blocked by 5% non-fat dry milk in TBS. After that, the membranes were incubated with rabbit monoclonal antibody to KIT (1:1000, Cell Signaling Technology #3074) and b-actin (1:1000, Abcam, USA) overnight at 4 8C. The membrane was incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Abcam, USA) for one hour at room temperature, and finally was detected with ECL method (Millipore, USA). 2.4. miRNA mimics and transfection
And, our results suggested that miR-137 was involved in the MDR of SCLC cells and KIT was one of its downstream molecules. 2. Materials and methods 2.1. Cell lines and culture The small cell lung cancer line H446 was purchased from the American type culture collection (ATCC, USA), and its multi-drugresistant cell line H446/CDDP was kindly gifted by Dr. Guisheng Qian (Institute of Human Respiratory Disease, Xinqiao Hospital, The Third Military Medical University). These cells were maintained in RPMI-1640 medium with 10% fetal calf serum (Gibico BRL, Grand Island, USA) in a humidified atmosphere containing 5% CO2 at 37 8C. To maintain the MDR phenotype, cisplatin (with final concentration of 0.5mg/ml) was added to the culture medium for H446/CDDP cells. 2.2. Determination of KIT mRNA expression in H446 and H446/CDDP cells The expression level of KIT mRNA was detected by reverse transcription-PCR (RT-PCR) following the manufacturer’s instructions (Takara, Japan). A total of 1 mg of RNA was incubated with reverse transcriptase mixture at 50 8C for 30 min, followed by amplification with the specific primers (synthesized by Invitrogen) listed as follows: KIT forward 50 -GAAATATCCTCCTTACTCATGGTCG-30 , reverse 50 -CGAGCGTTTCCTTTAACCACA-30 ; and bactin forward 50 -CTCCTCCACCTTTGACGCTG-30 , reverse 50 TCCTCTTGTGCTCTTGCTGG-30 . Thirty-five amplification cycles consisting of 30 s denaturation at 95 8C, 30 s of annealing (at the temperatures listed in Table 1), and 1 min of extension at 72 8C were used. After amplification, RT-PCR products were separated on 1% agarose gels, and the bands were visualized by ethidium bromide staining. Quantitation was obtained by density scanning Table 1 The sequence of miR-137 mimics, miR-137 inhibitor, negative control miRNA mimics and negative control miRNA inhibitor. has-miR-137 mimics has-miR137 negative control mimics has-miR-137 inhibitor has-miR137 negative control inhibitor
50 -UUAUUGCUUAAGAAUACGCGUAG-30 50 -ACGCGUAUUCUUAAGCAAUAAUU-30 50 -UUCUCCGAACGUGUCACGUTT-30 50 -ACGUGACACGUUCGGAGAATT-30 50 -CUACGCGUAUUCUUAAGCAAUAA-30 50 -CAGUACUUUUGUGUAGUACAA-30
The miR-137 mimics, miR-137 inhibitor, negative control miRNA mimics and negative control miRNA inhibitor (sequences showed in Table 1) were synthesized by Shanghai GenePharma Company (Shanghai, China) and transfected into H446 and H446/ CDDP cells by using lipofectamine 2000 (Invitrogen, Long Island, USA). 2.5. Real-time quantification of miR-137 after transfection The total RNA was extracted from the H446 and H446/CDDP cells using trizol (Invitrogen, Carlsbad, CA), and the concentration of the total RNA was quantitated by measuring the absorbance at 260 nm. Real-time PCR was carried out with SYBR green (Foster City, CA) detection using stem-loop reverse transcription followed by real-time polymerase chain reaction. The stem-loop primers were obtained from RiboBio Co. Ltd (Guangzhou, China) and the fold change of miR-137 in H446/CDDP cells was compared to that DD of H446 calculated using the 2 CT method. What was more; RTPCR was performed in triplicate. 2.6. In vitro drug sensitivity assay H446 and H446/CDDP cells were plated in 96-well plates (8000 cells/well). After adhesion, the H4446 cells were transfected with miR-137 inhibitor and the H446/CDDP cells were transfected with miR-137 mimics, both of the two kind cells were exposed to cisplatin with various concentrations (0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 mg/ml) for 48 h. Following the incubation, 10 ml of CCK-8 and 90 ml of 10% RPMI-1640 were added to each well. After incubation for 2 h at 37 8C, the absorbance of each well was detected with a microplate reader at a wavelength of 450 nm 2 h after incubation at 37 8C. Half maximal (50%) inhibitory concentration (IC50) of cisplatin was estimated by the relative survival curve. The experiments were conducted in triplicate. 2.7. Cell apoptosis assay H446/CDDP and H446 cells were seeded in 6-well plates (2 105/well) and transfected with miR-137 mimics or miR-137 mimics negative control. Twenty-four hours later, cisplatin with final concentration of 2 mg/ml was added to the medium. Fortyeight hours after incubation, the cells were trypainized and washed twice with PBS and stained with 5 ml annexin V-FITC for 5 min and kept in a dark place, and then stained with 10 ml
P. Li et al. / Biomedicine & Pharmacotherapy 68 (2014) 7–12
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Fig. 1. KIT expression in H446 and H446/CDDP cell lines. There was no KIT mRNA (A) and protein (B) expressed in H446 cells while both KIT mRNA and protein expression were found in H446/CDDP cells.
propidiumiodide (PI). Flow cytometry was carried out following the manufacturer’s instructions to detect the apoptosis of the cells by determining the relative amount of annexin V-FITC-positive, PI-negative cells.
accordance with its gene, which further confirmed that KIT only expressed in multi-drug-resistant cells.
2.8. Statistical analysis
MiR-137 expression was taken as ‘‘1’’ in H446 cells. Its expression in the two kind of cells was examined before transfection and it was confirmed that miR-137 expression in drug resistant cells was much lower than that in H446 (Fig. 2A) (P < 0.05). Besides, its expression after transfection was also determined in both H446 and H446/CDDP cells. The results showed that MiR-137 expression in H446 cells was inhibited after being transfected with miR-137 inhibitor (Fig. 2B) (**P < 0.05), while miR-137 mimics transfection significantly up-regulates miR-137 expression in H446/CDDP cells (Fig. 2C) (**P < 0.05). These results proved that the transfection was acceptable.
Each experiment was repeated at least three times. The numerical data were expressed as means standard deviation. The difference between means was analyzed with one-way ANOVA using SPSS 16.0 software. A statistical difference was accepted as significant if P < 0.05. 3. Results
3.2. miR-137 expression before and after transfection
3.1. KIT expression in H446 and H446/CDDP cell lines 3.3. KIT expression before and after transfection KIT expression was determined by RT-PCR and Western Blot. The results showed that KIT mRNA (Fig. 1A) expression was pretty higher in H446/CDDP cells than that in H446 cells where there was no KIT gene expressing. KIT protein expression (Fig. 1B) was in
In order to determine whether KIT expression was regulated by miR-137, we further evaluated the expression of KIT by Western Blot before and after transfection (Fig. 3). Results showed that there was
Fig. 2. miR-137 expression before and after transfection. Real-time PCR was carried out to examine miR-137 expression before and after transfection, and it was confirmed that there was fewer miR-137 expressed in H446/CDDP cells compared to that of H446 (P < 0.05) (A); besides, miR-137 inhibitor would significantly inhibit miR-137 expression in H446 cells (**P < 0.05) (B), while miR-137 mimics markedly increased miR-137 expression in H446/CDDP cells (**P < 0.05) (C).
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Fig. 3. KIT expression before and after transfection. In order to further explore the relationship between miR-137 and KIT, KIT protein expression was checked by Western Blot. There was no KIT expressed in H446 cells transfected with miR-137 inhibitor or not. While expression of KIT in H446/CDDP cells decreased after transfected with miR-137 mimics.
factors that hinder the outcomes. MDR mechanisms of SCLC is much complex, which can be roughly divided into endogenous and acquired drug resistance, both of which are caused by a variety of mechanisms involving multiple genes. MicroRNAs (miRNA), a kind of important gene regulation factors, participate in numerous physiological processes including the development of diseases. Available evidence indicates that miRNAs expression may regulate the occurrence, development and invasion of lung cancer, besides, there are also researches indicating that miRNAs play a certain role in MDR of cancers, such as gastrointestinal cancer [18] and ovarian cancer [19]. What is more, several pathways involving miRNAs in MDR have already been descripted precisely [20–22]. MiR-137, located in chromosome 1 p21. 3, is closely related to the prognosis of multiple tumors, such as colorectal cancer [23], gastric cancer [24], oral squamous cell carcinoma [25], breast cancer [26] and melanoma [27]. Evidence showed that upregulated paxillin (PXN) expression following low level of miR137 expression was involved in the invasion and poor prognosis of colorectal cancer [28], besides, miR-137 has been found to regulate the expression of Cdc42 and to block cancer cell cycle at G1 phase so as to inhibit invasion [8]. There are also some researches indicating that miR-137 may regulate the MDR of breast cancer cells by interfering with the expression of P-gP and MDR of lung cancer cells by Cdc42 and Cdk6, respectively [9,10]. Although it has been demonstrated that abnormal miR-137 expression was involved in many tumor pathological and physiological processes, there is no research available to explain the modulating role and mechanism of miR-137 in SCLC MDR. We wished the present study could explain how miR-137 modulated drug sensitivity of SCLC cells. We found that miR-137 expression was closely related to the MDR of SCLC for its deregulated expression in multi-drug-resistant cells and the positive results of gain-of-function and loss-offunction experiments. Besides, our results also showed that the sensitivity of H446/CDDP cells to cisplatin markedly increased after being transfected with miR-137 mimics, while the sensitivities of the H446 cells significantly reduced when transfected with
no KIT expressed in H446 cells whether transfected with miR-137 inhibitor or not, while miR-137 mimics transfection significantly suppressed KIT expression in H446/CDDP cells (*P < 0.05). 3.4. Drug sensitivity comparison before and after transfection In order to confirm the relation between miR-137 expression and drug sensitivity of the two cell lines, IC50 of the cells before and after transfection was examined (Fig. 4). Results showed that H446 cells were sensitive to cisplatin and inhibition of miR-137 expression could cause the sensitivity to decrease (**P < 0.05). But the upregulated miR-137 expression by miR-137 mimics sharply increased the sensitivity of H446/CDDP cells to cisplatin (##P < 0.05). 3.5. Association between miR-137 and cisplatin-induced apoptosis H446 and H446/CDDP cells apoptosis were assessed before and after transfection. The apoptosis rate of H446/CDDP cells was 5.36% 48 h after 0.2 m/ml cisplatin exposure, and the up-regulation of miR-137 expression by miR-137 mimics significantly increased H446/CDDP cell apoptosis to 16.41%. On the other hand, miR-137 inhibitor transfection led to a marked decrease of H446 cell death from 27.68% to 8.49% (Fig. 5). 4. Discussion Chemotherapy becomes the chief treatment for SCLC patients, and multi-drug resistance (MDR) is one of the most important
Fig. 4. Drug sensitivity comparison before and after transfection. In order further confirm the effects of transfection, drug sensitivity of H446 and H446/CDDP cells was evaluated and IC50 of cells in each group was calculated. Results showed that drug sensitivity of H446 cells significantly decreased after transfected with miR137 inhibitor (**P < 0.05) and miR-137 transfection would markedly increase drug sensitivity of H446/CDDP cells (##P < 0.05).
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Fig. 5. Cells apoptosis analysis before and after transfection. In order to evaluate impact of miR-137 expression on cell apoptosis, apoptosis rate was evaluated 48 h after treatment. Apoptosis rate of H446 cells was 27.68%, and dropped to 8.49% after transfected with miR-137 inhibitor (*P < 0.05). On the other hand, apoptosis rate of H446/ CDDP cells was 5.36%, and increased to 16.41% after miR-137 mimics transfection (*P < 0.05).
miR-137 inhibitor. In order to further confirm that miR-137 took part in SCLC MDR, cell apoptosis was evaluated and the results turned out that apoptosis of H446/CDDP cells transfected with miR-137 mimics significantly increased when stimulated by cisplatin, while apoptosis of H446 cells transfected with miR137 inhibitor decreased. All these results confirmed that miR-137 regulated SCLC MDR. In order to explore the mechanisms of how miR-137 modulated SCLC MDR, the target genes of miR-137 have been predicted by using the software on line (Target scan 6.2 http://www.targetscan.org/; http://www.microrna.org/microrna/home.do; and http:// mirdb.org/miRDB/). The sequence alignment of miR-137 indicates that KIT is one of the potential targets of miR-137. Since KIT is a multi-drug resistance gene in GIST, we hypothesized that miR-137 expression might sensitize H446/CDDP cells to cisplatin by repressing KIT expression. We detected KIT expressions in H446 and H446/CDDP cells on both gene and protein levels. Both gene and protein examination indicated that there was no KIT expressed in H446 cells, while the expression of KIT in H446/CDDP cells was very high. KIT, encoded by proto-oncogene c-kit, is a type I transmembrane glycoprotein belonging to the type III transmembrane tyrosine kinase receptor family, and its abnormal expression would be closely related to multiple kinds of tumorgeneous [4– 7,9,8]. It has been believed that KIT expression was a characteristic marker of gastrointestinal stromal tumors, and the total positive rate could reach 85% to 98% [29,30]. Besides, KIT was involved in the process of gastrointestinal stromal tumor MDR. It has been confirmed, in the present research, that KIT gene and protein would express in H446/CDDP cells but not in H446 cells, which indicated that KIT was an acquired MDR gene for SCLC cells. Then, the KIT protein expression was examined in parental and drug resistant cells after being transfected with miR-137 inhibitor and miR-137
mimics. The results showed that no KIT expression was stimulated in H446 cells when transfected with miR-137 inhibitor, while the expression of KIT in H446/CDDP cells was suppressed after miR137 mimics transfection. Notably, apoptosis rate of H446 cells transfected with miR-137 inhibitor decreased, indicating that target genes of miR-137 were far more than KIT, and there perhaps were other MDR related genes in H446/CDDP which called for further research to reveal SCLC MDR mechanisms. 5. Conclusion The present study demonstrated that miR-137 was closely related to MDR of SCLC, and interference of miR-137 expression may attenuate drug resistant of H446/CDDP cells to cisplatin, partially through KIT expression regulation. Besides, it has also been proved that KIT might be only one of the downstream molecules of miR-137 that related to SCLC MDR. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements The study was supported by the National Natural Science Foundation of China (No. 81071933). References [1] Lee H, Jin GY, Han YM, Chung GH, Lee YC, Kwon KS, et al. Comparison of survival rate in primary non-small-cell lung cancer among elderly patients treated with radiofrequency ablation, surgery, or chemotherapy. Cardiovasc Intervent Radiol 2012;35:343–50.
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[2] Zhu GY. [To improve the survival rate of patients suffering from locally advanced non-small cell lung cancer with combination of radiotherapy and chemotherapy]. Zhonghua Jie He He Hu Xi Za Zhi 2007;30:88–90. [3] Sharom FJ. ABC multidrug transporters: structure, function and role in chemoresistance. Pharmacogenomics 2008;9:105–27. [4] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281–97. [5] Blower PE, Chung JH, Verducci JS, Lin S, Park JK, Dai Z, et al. MicroRNAs modulate the chemosensitivity of tumor cells. Mol Cancer Ther 2008;7:1–9. [6] Nasser MW, Datta J, Nuovo G, Kutay H, Motiwala T, Majumder S, et al. Downregulation of micro-RNA-1 (miR-1) in lung cancer. Suppression of tumorigenic property of lung cancer cells and their sensitization to doxorubicin-induced apoptosis by miR-1. J Biol Chem 2008;283:33394–405. [7] Eitan R, Kushnir M, Lithwick-Yanai G, David MB, Hoshen M, Glezerman M, et al. Tumor microRNA expression patterns associated with resistance to platinum based chemotherapy and survival in ovarian cancer patients. Gynecol Oncol 2009;114:253–9. [8] Deng Y, Deng H, Bi F, Liu J, Bemis LT, Norris D, et al. MicroRNA-137 targets carboxyl-terminal binding protein 1 in melanoma cell lines. Int J Biol Sci 2011;7:133–7. [9] Zhu X, Li Y, Shen H, Li H, Long L, Hui L, et al. miR-137 restoration sensitizes multidrug-resistant MCF-7/ADM cells to anticancer agents by targeting YB-1. Acta Biochim Biophys Sin (Shanghai) 2013;45:80–6. [10] Zhu X, Li Y, Shen H, Li H, Long L, Hui L, et al. miR-137 inhibits the proliferation of lung cancer cells by targeting Cdc42 and Cdk6. FEBS Lett 2013;587:73–81. [11] Ali S, Ali S. Role of c-kit/SCF in cause and treatment of gastrointestinal stromal tumors (GIST). Gene 2007;401:38–45. [12] Montero JC, Lopez-Perez R, Miguel JFS, Pandiella A. Expression of c-Kit isoforms in multiple myeloma: differences in signaling and drug sensitivity. Haematologica 2008;93:851–9. [13] Yoshida C, Tsuji AB, Sudo H, Sugyo A, Kikuchi T, Koizumi M, et al. Therapeutic efficacy of c-kit-targeted radioimmunotherapy using 90Y-labeled anti-c-kit antibodies in a mouse model of small cell lung cancer. PLoS One 2013;8:e59248. [14] Edris B, Willingham S, Weiskopf K, Volkmer AK, Volkmer JP, Muhlenberg T, et al. Use of a KIT-specific monoclonal antibody to bypass imatinib resistance in gastrointestinal stromal tumors. Oncoimmunology 2013;2:e24452. [15] Lv A, Li Z, Tian X, Guan X, Zhao M, Dong B, et al. SKP2 high expression, KIT exon 11 deletions, and gastrointestinal bleeding as predictors of poor prognosis in primary gastrointestinal stromal tumors. PLoS One 2013;8:e62951. [16] Felli N, Fontana L, Pelosi E, Botta R, Bonci D, Facchiano F, et al. MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation. Proc Natl Acad Sci USA 2005;102:18081–6.
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Available online at
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Original article
MicroRNA-137 down-regulates KIT and inhibits small cell lung cancer cell proliferation Peipei Li a,b,1, Lijie Ma a,1, Yongjuan Zhang c, Fuyun Ji d, Faguang Jin a,* a
Department of Respiration, Tangdu Hospital, Fourth Military Medical University, 710038 Xi’an, China Department of Respiration, Harrison International Peace Hospital, 053000 Hengshui, China c Department of Physiology, Guangdong Medical College, 523808 Dongguan, China d Institute of Human Respiratory Disease, Xinqiao Hospital, Third Military Medical University, 400037 Chongqing, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 November 2013 Accepted 10 December 2013
MiR-137 expression was examined in parental and drug-resistant cell lines, H446 and H446/CDDP, of small lung cancer (SCLC), and the results showed there was fewer miR-137 expressed in H446/CDDP cells followed by KIT expression emergence. In order to confirm physiological function of these abnormal expressions, H446 and H446/CDDP cells were transfected with miR-137 inhibitor and miR-137 mimics, respectively, after that, miR-137 and KIT expression in two cell lines and drug sensitivity of these cells were evaluated. Results indicated that sensitivity of H446 cells to cisplatin significantly decreased after transfected with miR-137 inhibitor, while miR-137 mimics transfection increased drug sensitivity of H446/CDDP cells and deregulated KIT expression. Our data provided combined evidence that miR-137 was closely related to MDR of SCLC, and interfering of miR-137 expression may attenuate drug resistant of H446/CDDP cells to cisplatin partially through KIT expression regulation. Besides, it has also been proved that KIT might be only one of the downstream molecules of miR-137 that related to SCLC MDR. ß 2014 Published by Elsevier Masson SAS.
Keywords: MiR-137 KIT SCLC MDR
Abbreviations: CDDP, cisplatin RPMI, Roswell Park Memorial Institute PBS, phosphate buffered saline PVDF, Polyvinylidene Fluoride ECL, electrochemiluminescence SYBR, Synergy Brands CCK, Cell Counting Kit FITC, fluorescein isothiocyanate
1. Introduction Small cell lung cancer (SCLC) is an aggressive malignant tumor accounting for approximately 15–20% of all primary lung cancer, and its highly metastasizing characteristics result in poor prognosis and high mortality. In addition, 5-year survival rate of the SCLC is less than 15% [1]. Chemotherapy is the primary therapeutic approach for SCLC, and it is also sensitive to chemotherapy initially. Subsequently, the intrinsic or acquired multi-drug resistance (MDR) emerges and increases the rate of relapse and leads to poor prognosis [2]. Therefore, MDR has become the main obstacle for SCLC treatment in the world, and how to reverse MDR phenotype has become a central issue in improving the prognosis of SCLC. Previous studies have demonstrated that the MDR mechanism included multiple aspects, such as over expression of drug efflux pumps like P-glycoprotein (Pgp), multi-drug resistance associated protein 1 (MRP-1), lung * Corresponding author. Tel.: +86 29 84777425; fax: +86 29 84777425. E-mail address: [email protected] (F. Jin). 1 These authors contributed to the article equally. 0753-3322/$ – see front matter ß 2014 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.biopha.2013.12.002
resistance protein (LRP) in drug-resistant cells, the change of microenvironment in the cells, the enhanced function of DNA synthesis and repair, and the dysfunction of cell apoptosis pathway etc. [3]. All of the above indicated that the abnormal regulation of gene and protein expression played a critical role in the MDR process. MicroRNAs (miRNAs) are a cluster of short single-stranded, non-coding, endogenous RNA molecules, approximately 22 nucleotides that negatively regulate gene expression via binding to the 30 untranslated region (30 UTR) of mRNA, leading to repressing post-transcripional translation or the degradation of the target mRNA [4]. Extensive researches have indicated that aberrantly expressed miRNAs could affect cell biological activities, and dysregulation of miRNAs may be the mechanism of gene expression in cancer [5,6]. Besides, some miRNAs were related to the responses to chemotherapeutic treatment [7]. And miR-137, as one of the focused miRNAs, has been proved to participate in inhibiting epithelial mesenchymal [8], modulating the MDR of breast cancer cell [9], and suppressing lung cancer cell proliferation [10]. Though, further researches are needed to explore the role of miR-137 in the chemoresistance of small cell lung cancer cells.
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KIT, also named as CD117, is a transmembrane protein receptor encoded by the c-kit proto-oncogene [11]. Abnormal KIT expression is closely related to tumors, such as multiple myeloma [12], small cell lung cancer (SCLC) [13], and gastrointestinal stromal tumors [14,15], suggesting there is a complementary mechanism involving KIT expression in occurrence and development of tumors. The regulation of KIT by miRNA-221/miRNA-222 in erythroleukemic cells [16] and its regulation by miRNA-494 in gastrointestinal stromal tumor cells [17] have been explored, but no miRNAs targeting KIT have been identified in SCLC. In the present research, we used parental cells (H446) and drug resistant cell lines (H446/CDDP) to explore the possible mechanism of SCLC drug resistance, and proposed that miRNA-137 expression was involved in SCLC MDR probably through KIT pathway by showing that: miR-137 expression was negatively correlated to the expression of KIT; exogenous miR-137 suppressed KIT expression and led to higher sensitivity of H446/CDDP cells to cisplatin.
of the PCR bands using a Scion image analysis system (Beta 4.02, Scion Corporation). The results were expressed as the ratio to bactin mRNA level in the same RNA samples. 2.3. Western Blot analysis for KIT The cells were harvested and washed twice with PBS 72 h after transfection, then, the total protein was extracted using protein extraction reagent (Pierce Biotechnology, USA) and protease inhibitor (Roche Applied Science). After quantification, 20 mm protein was separated on 8% SDS-PAGE gels, transferred to PVDF membranes (Millipore, Bedford, MA) and blocked by 5% non-fat dry milk in TBS. After that, the membranes were incubated with rabbit monoclonal antibody to KIT (1:1000, Cell Signaling Technology #3074) and b-actin (1:1000, Abcam, USA) overnight at 4 8C. The membrane was incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Abcam, USA) for one hour at room temperature, and finally was detected with ECL method (Millipore, USA). 2.4. miRNA mimics and transfection
And, our results suggested that miR-137 was involved in the MDR of SCLC cells and KIT was one of its downstream molecules. 2. Materials and methods 2.1. Cell lines and culture The small cell lung cancer line H446 was purchased from the American type culture collection (ATCC, USA), and its multi-drugresistant cell line H446/CDDP was kindly gifted by Dr. Guisheng Qian (Institute of Human Respiratory Disease, Xinqiao Hospital, The Third Military Medical University). These cells were maintained in RPMI-1640 medium with 10% fetal calf serum (Gibico BRL, Grand Island, USA) in a humidified atmosphere containing 5% CO2 at 37 8C. To maintain the MDR phenotype, cisplatin (with final concentration of 0.5mg/ml) was added to the culture medium for H446/CDDP cells. 2.2. Determination of KIT mRNA expression in H446 and H446/CDDP cells The expression level of KIT mRNA was detected by reverse transcription-PCR (RT-PCR) following the manufacturer’s instructions (Takara, Japan). A total of 1 mg of RNA was incubated with reverse transcriptase mixture at 50 8C for 30 min, followed by amplification with the specific primers (synthesized by Invitrogen) listed as follows: KIT forward 50 -GAAATATCCTCCTTACTCATGGTCG-30 , reverse 50 -CGAGCGTTTCCTTTAACCACA-30 ; and bactin forward 50 -CTCCTCCACCTTTGACGCTG-30 , reverse 50 TCCTCTTGTGCTCTTGCTGG-30 . Thirty-five amplification cycles consisting of 30 s denaturation at 95 8C, 30 s of annealing (at the temperatures listed in Table 1), and 1 min of extension at 72 8C were used. After amplification, RT-PCR products were separated on 1% agarose gels, and the bands were visualized by ethidium bromide staining. Quantitation was obtained by density scanning Table 1 The sequence of miR-137 mimics, miR-137 inhibitor, negative control miRNA mimics and negative control miRNA inhibitor. has-miR-137 mimics has-miR137 negative control mimics has-miR-137 inhibitor has-miR137 negative control inhibitor
50 -UUAUUGCUUAAGAAUACGCGUAG-30 50 -ACGCGUAUUCUUAAGCAAUAAUU-30 50 -UUCUCCGAACGUGUCACGUTT-30 50 -ACGUGACACGUUCGGAGAATT-30 50 -CUACGCGUAUUCUUAAGCAAUAA-30 50 -CAGUACUUUUGUGUAGUACAA-30
The miR-137 mimics, miR-137 inhibitor, negative control miRNA mimics and negative control miRNA inhibitor (sequences showed in Table 1) were synthesized by Shanghai GenePharma Company (Shanghai, China) and transfected into H446 and H446/ CDDP cells by using lipofectamine 2000 (Invitrogen, Long Island, USA). 2.5. Real-time quantification of miR-137 after transfection The total RNA was extracted from the H446 and H446/CDDP cells using trizol (Invitrogen, Carlsbad, CA), and the concentration of the total RNA was quantitated by measuring the absorbance at 260 nm. Real-time PCR was carried out with SYBR green (Foster City, CA) detection using stem-loop reverse transcription followed by real-time polymerase chain reaction. The stem-loop primers were obtained from RiboBio Co. Ltd (Guangzhou, China) and the fold change of miR-137 in H446/CDDP cells was compared to that DD of H446 calculated using the 2 CT method. What was more; RTPCR was performed in triplicate. 2.6. In vitro drug sensitivity assay H446 and H446/CDDP cells were plated in 96-well plates (8000 cells/well). After adhesion, the H4446 cells were transfected with miR-137 inhibitor and the H446/CDDP cells were transfected with miR-137 mimics, both of the two kind cells were exposed to cisplatin with various concentrations (0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 mg/ml) for 48 h. Following the incubation, 10 ml of CCK-8 and 90 ml of 10% RPMI-1640 were added to each well. After incubation for 2 h at 37 8C, the absorbance of each well was detected with a microplate reader at a wavelength of 450 nm 2 h after incubation at 37 8C. Half maximal (50%) inhibitory concentration (IC50) of cisplatin was estimated by the relative survival curve. The experiments were conducted in triplicate. 2.7. Cell apoptosis assay H446/CDDP and H446 cells were seeded in 6-well plates (2 105/well) and transfected with miR-137 mimics or miR-137 mimics negative control. Twenty-four hours later, cisplatin with final concentration of 2 mg/ml was added to the medium. Fortyeight hours after incubation, the cells were trypainized and washed twice with PBS and stained with 5 ml annexin V-FITC for 5 min and kept in a dark place, and then stained with 10 ml
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Fig. 1. KIT expression in H446 and H446/CDDP cell lines. There was no KIT mRNA (A) and protein (B) expressed in H446 cells while both KIT mRNA and protein expression were found in H446/CDDP cells.
propidiumiodide (PI). Flow cytometry was carried out following the manufacturer’s instructions to detect the apoptosis of the cells by determining the relative amount of annexin V-FITC-positive, PI-negative cells.
accordance with its gene, which further confirmed that KIT only expressed in multi-drug-resistant cells.
2.8. Statistical analysis
MiR-137 expression was taken as ‘‘1’’ in H446 cells. Its expression in the two kind of cells was examined before transfection and it was confirmed that miR-137 expression in drug resistant cells was much lower than that in H446 (Fig. 2A) (P < 0.05). Besides, its expression after transfection was also determined in both H446 and H446/CDDP cells. The results showed that MiR-137 expression in H446 cells was inhibited after being transfected with miR-137 inhibitor (Fig. 2B) (**P < 0.05), while miR-137 mimics transfection significantly up-regulates miR-137 expression in H446/CDDP cells (Fig. 2C) (**P < 0.05). These results proved that the transfection was acceptable.
Each experiment was repeated at least three times. The numerical data were expressed as means standard deviation. The difference between means was analyzed with one-way ANOVA using SPSS 16.0 software. A statistical difference was accepted as significant if P < 0.05. 3. Results
3.2. miR-137 expression before and after transfection
3.1. KIT expression in H446 and H446/CDDP cell lines 3.3. KIT expression before and after transfection KIT expression was determined by RT-PCR and Western Blot. The results showed that KIT mRNA (Fig. 1A) expression was pretty higher in H446/CDDP cells than that in H446 cells where there was no KIT gene expressing. KIT protein expression (Fig. 1B) was in
In order to determine whether KIT expression was regulated by miR-137, we further evaluated the expression of KIT by Western Blot before and after transfection (Fig. 3). Results showed that there was
Fig. 2. miR-137 expression before and after transfection. Real-time PCR was carried out to examine miR-137 expression before and after transfection, and it was confirmed that there was fewer miR-137 expressed in H446/CDDP cells compared to that of H446 (P < 0.05) (A); besides, miR-137 inhibitor would significantly inhibit miR-137 expression in H446 cells (**P < 0.05) (B), while miR-137 mimics markedly increased miR-137 expression in H446/CDDP cells (**P < 0.05) (C).
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Fig. 3. KIT expression before and after transfection. In order to further explore the relationship between miR-137 and KIT, KIT protein expression was checked by Western Blot. There was no KIT expressed in H446 cells transfected with miR-137 inhibitor or not. While expression of KIT in H446/CDDP cells decreased after transfected with miR-137 mimics.
factors that hinder the outcomes. MDR mechanisms of SCLC is much complex, which can be roughly divided into endogenous and acquired drug resistance, both of which are caused by a variety of mechanisms involving multiple genes. MicroRNAs (miRNA), a kind of important gene regulation factors, participate in numerous physiological processes including the development of diseases. Available evidence indicates that miRNAs expression may regulate the occurrence, development and invasion of lung cancer, besides, there are also researches indicating that miRNAs play a certain role in MDR of cancers, such as gastrointestinal cancer [18] and ovarian cancer [19]. What is more, several pathways involving miRNAs in MDR have already been descripted precisely [20–22]. MiR-137, located in chromosome 1 p21. 3, is closely related to the prognosis of multiple tumors, such as colorectal cancer [23], gastric cancer [24], oral squamous cell carcinoma [25], breast cancer [26] and melanoma [27]. Evidence showed that upregulated paxillin (PXN) expression following low level of miR137 expression was involved in the invasion and poor prognosis of colorectal cancer [28], besides, miR-137 has been found to regulate the expression of Cdc42 and to block cancer cell cycle at G1 phase so as to inhibit invasion [8]. There are also some researches indicating that miR-137 may regulate the MDR of breast cancer cells by interfering with the expression of P-gP and MDR of lung cancer cells by Cdc42 and Cdk6, respectively [9,10]. Although it has been demonstrated that abnormal miR-137 expression was involved in many tumor pathological and physiological processes, there is no research available to explain the modulating role and mechanism of miR-137 in SCLC MDR. We wished the present study could explain how miR-137 modulated drug sensitivity of SCLC cells. We found that miR-137 expression was closely related to the MDR of SCLC for its deregulated expression in multi-drug-resistant cells and the positive results of gain-of-function and loss-offunction experiments. Besides, our results also showed that the sensitivity of H446/CDDP cells to cisplatin markedly increased after being transfected with miR-137 mimics, while the sensitivities of the H446 cells significantly reduced when transfected with
no KIT expressed in H446 cells whether transfected with miR-137 inhibitor or not, while miR-137 mimics transfection significantly suppressed KIT expression in H446/CDDP cells (*P < 0.05). 3.4. Drug sensitivity comparison before and after transfection In order to confirm the relation between miR-137 expression and drug sensitivity of the two cell lines, IC50 of the cells before and after transfection was examined (Fig. 4). Results showed that H446 cells were sensitive to cisplatin and inhibition of miR-137 expression could cause the sensitivity to decrease (**P < 0.05). But the upregulated miR-137 expression by miR-137 mimics sharply increased the sensitivity of H446/CDDP cells to cisplatin (##P < 0.05). 3.5. Association between miR-137 and cisplatin-induced apoptosis H446 and H446/CDDP cells apoptosis were assessed before and after transfection. The apoptosis rate of H446/CDDP cells was 5.36% 48 h after 0.2 m/ml cisplatin exposure, and the up-regulation of miR-137 expression by miR-137 mimics significantly increased H446/CDDP cell apoptosis to 16.41%. On the other hand, miR-137 inhibitor transfection led to a marked decrease of H446 cell death from 27.68% to 8.49% (Fig. 5). 4. Discussion Chemotherapy becomes the chief treatment for SCLC patients, and multi-drug resistance (MDR) is one of the most important
Fig. 4. Drug sensitivity comparison before and after transfection. In order further confirm the effects of transfection, drug sensitivity of H446 and H446/CDDP cells was evaluated and IC50 of cells in each group was calculated. Results showed that drug sensitivity of H446 cells significantly decreased after transfected with miR137 inhibitor (**P < 0.05) and miR-137 transfection would markedly increase drug sensitivity of H446/CDDP cells (##P < 0.05).
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Fig. 5. Cells apoptosis analysis before and after transfection. In order to evaluate impact of miR-137 expression on cell apoptosis, apoptosis rate was evaluated 48 h after treatment. Apoptosis rate of H446 cells was 27.68%, and dropped to 8.49% after transfected with miR-137 inhibitor (*P < 0.05). On the other hand, apoptosis rate of H446/ CDDP cells was 5.36%, and increased to 16.41% after miR-137 mimics transfection (*P < 0.05).
miR-137 inhibitor. In order to further confirm that miR-137 took part in SCLC MDR, cell apoptosis was evaluated and the results turned out that apoptosis of H446/CDDP cells transfected with miR-137 mimics significantly increased when stimulated by cisplatin, while apoptosis of H446 cells transfected with miR137 inhibitor decreased. All these results confirmed that miR-137 regulated SCLC MDR. In order to explore the mechanisms of how miR-137 modulated SCLC MDR, the target genes of miR-137 have been predicted by using the software on line (Target scan 6.2 http://www.targetscan.org/; http://www.microrna.org/microrna/home.do; and http:// mirdb.org/miRDB/). The sequence alignment of miR-137 indicates that KIT is one of the potential targets of miR-137. Since KIT is a multi-drug resistance gene in GIST, we hypothesized that miR-137 expression might sensitize H446/CDDP cells to cisplatin by repressing KIT expression. We detected KIT expressions in H446 and H446/CDDP cells on both gene and protein levels. Both gene and protein examination indicated that there was no KIT expressed in H446 cells, while the expression of KIT in H446/CDDP cells was very high. KIT, encoded by proto-oncogene c-kit, is a type I transmembrane glycoprotein belonging to the type III transmembrane tyrosine kinase receptor family, and its abnormal expression would be closely related to multiple kinds of tumorgeneous [4– 7,9,8]. It has been believed that KIT expression was a characteristic marker of gastrointestinal stromal tumors, and the total positive rate could reach 85% to 98% [29,30]. Besides, KIT was involved in the process of gastrointestinal stromal tumor MDR. It has been confirmed, in the present research, that KIT gene and protein would express in H446/CDDP cells but not in H446 cells, which indicated that KIT was an acquired MDR gene for SCLC cells. Then, the KIT protein expression was examined in parental and drug resistant cells after being transfected with miR-137 inhibitor and miR-137
mimics. The results showed that no KIT expression was stimulated in H446 cells when transfected with miR-137 inhibitor, while the expression of KIT in H446/CDDP cells was suppressed after miR137 mimics transfection. Notably, apoptosis rate of H446 cells transfected with miR-137 inhibitor decreased, indicating that target genes of miR-137 were far more than KIT, and there perhaps were other MDR related genes in H446/CDDP which called for further research to reveal SCLC MDR mechanisms. 5. Conclusion The present study demonstrated that miR-137 was closely related to MDR of SCLC, and interference of miR-137 expression may attenuate drug resistant of H446/CDDP cells to cisplatin, partially through KIT expression regulation. Besides, it has also been proved that KIT might be only one of the downstream molecules of miR-137 that related to SCLC MDR. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements The study was supported by the National Natural Science Foundation of China (No. 81071933). References [1] Lee H, Jin GY, Han YM, Chung GH, Lee YC, Kwon KS, et al. Comparison of survival rate in primary non-small-cell lung cancer among elderly patients treated with radiofrequency ablation, surgery, or chemotherapy. Cardiovasc Intervent Radiol 2012;35:343–50.
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