Bidirectional Regulation Of Erythropoietin-producing Hepatocyte Receptor A3(EPHA3)and MiR-7Involved In Multidrug Resistance In Small Cell Lung Cancer

BackgroundLung cancer is the leading cause of cancer-related deaths in the worldwide. Approximately15%of all histological types consist of small cell lung cancer (SCLC), which is the undifferentiated type bearing poorest outcome. The extreme aggressiveness of SCLC is due to its early and widespread metastases and development of multidrug resistance (MDR) to chemotherapy. The current front-line standard chemotherapy regimen for SCLC, etoposide or irinotecan plus cisplatin, is active in most SCLC cases, but the disease recurs shortly after the first successful treatment with multidrug resistance(MDR) phenotype. Therefore, chemoresistance has become one of the major problems in the treatment of SCLC and is clinically a very important issue for improving poor prognosis of SCLC. The molecular mechanisms involved in MDR processes of SCLC are diverse and complex, such as drug trasport, drug metabolism, DNA synthesis and repair, cell survival and apoptosis. But the specific molecules mechanism is not fully understood.EPHA3belongs to the Eph receptor family with2subclasses(A and B) and16members(A1-A10,Bl-B6), which is the largest subgroup of the receptor tyrosine kinase (RTK) family. EPHA3encodes a transmembrane protein with983amino acids, which is widely expressed during embryonic development, overexpressed in nervous system and heart, lower-expressed in the brain, lung, bladder, prostate and colon.It was reported that many EPHA3mutations identified in lung, colorectal and hepatocellular cancers, melanoma, and glioblastoma impair kinase activity or ephrin ligand binding and/or decrease the level of receptor cell surface localization.Our previous study showed that a number of mRNAs are involved in the drug resistance of SCLC cells using mRNA chip we found that EPHA3is down-regulation in H69AR cells than in the H69cells by mRNA chip, qRT-PCR and Western blot. The results suggested EPHA3may be involved in the regulation of resistant SCLC. At present, the expression of EphA3in SCLC tumors as well as the role of EPHA3in multidrug resistance mechanisms in SCLC cell lines are not yet clear.MicroRNAs (miRNAs), a class of small non-coding RNAs of18-24nucleotides, which are responsible for the posttranscriptional regulation of their target mRNA by either translational inhibition or mRNA degradation. Recent evidence has shown that more than50%of miRNAs are located in cancer-associated genomic break points, and can function as tumor suppressor genes or oncogenes depending on their targets. The differential expression profiles of miRNAs from healthy tissue across cancers, and the surprising strength of these profiles play an important role in tumor therapy response. Chen has reported that down-regulation of miR-100could lead to PIK1over-expression and eventually to docetaxel chemoresistance of human lung adenocarcinoma.By bioinformatics analysis, we found that EPHA3might be a potential target gene for miR-7. MiR-7is an intronic miRNA that resides in the first intron of heterogenous ribonuclear protein K gene on chromosome9and is conserved across all species.Sequence of MiR-7:UGGAAGACUAGUGAUUUUGUUGU. Hsa-miR-7includes:hsa-miR-7-1, hsa-miR-7-2, hsa-miR-7-3. Studies have shown that miR-7is preferentially expressed in endocrine cells of developing and adult human pancreas.Moreover miR-7was reported to play a substantial role in inhibiting the tumorigenesis and reversing the metastasis of HCC as a tumor suppressor through the PI3K/Akt/mTOR signaling pathway in vitro and in vivo. However, miR-7was proven to be an "oncomiR" to modulate EGFR mediated oncogenesis through a Ras/ERK/Myc pathway that targets the Ets2transcriptional repressor ERF. No data are available about miR-7functions in SCLC drug resistance.The objective of this study was to explore the role and the relationship between EPHA3and miR-7in the drug resistance mechanism of SCLC cells and the effect on the PI3K/BMX/STAT3signaling pathway.Materials and Methods1.Clinical samplesA total of44formalin-fixed, paraffin-embedded (FFPE) tissues were obtained from patients who had received chemotherapy or bronchofiberscopy or biopsy for SCLC between the period2004.7and2013.2and receiving care and follow-up at Armed Police Hospital of Guangdong Province and The Third Affiliated Hospital of Guangzhou Medical University. Informed consent was obtained from each patient, and the research protocols were approved by the Ethics Committee of the two Hospitals.2. Cell cultureHuman SCLC cell line NCI-H69and the drug-resistant subline NCI-H69AR, NCI-H446, NCI-H146and NCI-H1688were purchased from the American Type Culture Collection (ATCC, USA) and maintained in RPMI1640medium contain in L-Glutamine with10%and20%fetal calf serum respectively in an incubator at37℃with5%CO2. The drug-resistant cells were maintained in drug-free medium for at least2weeks before any experiment.3. Cell transfectionCells were transiently transfected with100nmol/L of miR-7angomir (Genepharma, Shanghai, China), inhibitor (Qiagen, Germany) and miRNA negative control (miR-NC) by using Lipofectamine2000and OPTI-MEM (Invitrogen). For stable transfection, EPHA3expression plasmid (EPHA3-PEX2-EcoRI/BamHI), EPHA3-PEX2empty plasmid, EPHA3shRNA and EPHA3shRNA negative control were respectively transfected into NCI-H69AR, NCI-H446, NCI-H146and NCI-H1688cells by using Lipofectamine2000and OPTI-MEM. Positive transfectants were selected in100-500g/ml G418(Sigma-Aldrich) respectively.4.RNA isolation and quantitative reverse transcription-PCR Total RNA, including miRNAs, was isolated from cell lines and tissues using RNAiso Plus (Takara) and a RNeasy FFPE kit (Qiagen) according to the manufacturer’s instructions respectively. cDNA synthesis was carried out according to PrimeScript RT reagent Kit with gDNA Eraser (Takara). qRT-PCR for EPHA3and miR-7were performed according to SYBR Preimix Ex TaqTMⅡ (Takara). GAPDH or U6snRNA was used as an endogenous control.5.Western blot analysisTotal protein was isolated with a Total protein extraction kit and quantitated using BCA assay. The protein lysates were separated by8%-10%SDS-PAGE, and electrophoretically transferred to PVDF membrane. Then, the membrane was incubated with antibodies and detected chemiluminescence. The intensity of protein fragments was quantified with Quantity One software.6. Cell counting kit-8(CCK-8) assayCells were plated in96-well plates at5×103cells per well for NCI-H69, NCI-H69AR, NCI-H446and NCI-H1688, while20×103cells per well for NCI-H146. After transient transfection or adherence of stable transfected cells, cells were treated with drugs for24h. A total of three chemotherapy drugs (Cisplatin (DDP; Shandong, China), Etoposide (VP-16; Jiangshu, China), Adriamycin (ADM; Jiangshu, China) were used. The absorbance at450nm was measured after incubation with10μl of CCK-8reagent (Dojindo, Kumamato, Japan) for4h. The cells incubated without drugs were set at100%survival and were used to calculate the concentration of each chemotherapeutic drug IC50(ug/ml). The assay was conducted in five replicate wells for each sample and three parallel experiments were performed.7.Flow cytometric analysisCells were treated with3chemotherapy drugs above-mentioned and2small molecule inhibitors including LY294002(Sigma-Aldrich) and LFM-A13(Sigma-Aldrich) for24h after transfection, and then collected for early apoptosis assay performed by using AnnexinV/propidium iodide detection kit (Becton-Dickinson (BD), San Jose, CA, USA) on FACS-Calibur systems (BD).8. Luciferase reporter assay The cells were seeded in24-well plates and cotransfected with pGL3constructs with or without miR-7inhibitor or precursor for24h. Each sample was also cotransfected with pRL-CMV plasmid expressing Renilla luciferase to monitor the transfection efficiency. At24h post-transfection, the activity of firefly luciferase was measured by using the dual-luciferase reporter assay system as described by the manufacturer (Promega Corporation). Relative luciferase activity was normalized with Renilla luciferase activity and then compared with the pGL3control.9. Immunohistochemistry stainingFormalin-fixed, paraffin-embedded tissues of SCLC clinical patients samples were sectioned at4-mm thickness and analyzed for EPHA3(1:200, Santa cruz biotechnology) expression. Visualization was achieved using the EnVision peroxidase system (Dako).A sample was considered positive if more than10%of the tumor cells retained cytoplasmic staining, and5fields were randomly selected according to semiquantitative scales. The intensity of staining was scored manually (high,3;medium,2; low,1; no staining,0) by-2independent experienced pathologists, and only tumor cells were scored. Negative controls were performed by replacing the primary antibodies stated above with PBS.10. In vivo tumor xenograft modelMale BALB/c nude mices aged30-40days (purchased from the Medical Experimental Animal Center of Guangdong Province, China) were raised under pathogen-free conditions. All procedures were performed according to guidelines of Association for Assessment and Accreditation of Laboratory Animal Care International. Cells in RPMI-1640were subcutaneously inoculated into the shoulder-back of nude mices in the light of1×l07/0.1ml/site to establish the tumor model respectively. The tumor volume was determined every3days over the course of21days by direct measurement with sliding caliper and calculated by the equation (V=0.4×ab2) to construct the growth curves of the tumors.21days later, mices were sacrificed and tumors were excised.11. Statistical analysisAll results were treated by SPSS19.0statistical software. The experiments were repeated independently for at least three times. The selected charts were a result of repeated experiments. Data was showed as mean±standard deviation. Difference between samples in quantitative real-time PCR, western blotting, CCK8, early apoptosis experiment and luciferase reporter gene assay was used one-way ANOVA analysis if data homogenity of variance,and F test/Welch method if data heterogeneity of variance. LSD/Dunnett’s T3methods for multiple comparisons. Chi-square test was used to evaluate the relations between EPHA3expression and the clinical and pathological features. Kaplain-Meier analysis was used to evaluate the relationship between EPHA3expression and survival time. The relationship between EPHA3and miR-7expression was explored by Spearman’s correlation. Cox regression analysis was used to evaluate the prognostic impact factors of SCLC. All experiments were done at least in triplicate and the level of statistical significance was set at p value<0.05for all tests.ResultsWe found that EPHA3expression was localized in the cytoplasm of the cancer cells. No positive staining for EPHA3was presented in the normal lung alveolar epithelium. By Chi-square test, no significant differences were observed in EPHA3expression with respect to gender, age, disease stage and survival status. For overall survival, the Kaplan-Meier method reveals that EPHA3expression level and disease stage correlates with significant overall survival time of the44SCLC patients. Cox regression analysis indicated that EPHA3expression and disease stage (P<0.001) were found to be significantly independent prognostic factors for the SCLC patients. EPHA3expression was an independent predictor of survival with a hazard ratio of0.192and a95%confidence interval ranging from0.072to0.508.The expression of EPHA3in mRNA and protein level were significantly increased in H69cells compared to H69AR cells. Transfecting shRNAs (EPHA3-1690,-1286,-2635,-2934) and NC into H69, EPHA3-1690which showed the most significant inhibition was selected for the followed study. EPHA3-1690reduced EPHA3expression both in mRNA level and protein level. As expected, knockdown of EPHA3resulted in resistant phenotype.The cell survival and the IC50 values of EPHA3-shRNA transfected SCLC cells significantly increased, accompanied by decreased cell early apoptosis by flow cytometry analysis, with treatment of chemotherapeutic drugs including ADM, DDP or VP-16. down-regulation of EPHA3in H69resulted in the decreased expression of PI3K-p110, but increased expression of BMX and p-STAT3in protein levels, in addition to the decreased cell early apoptosis with treatment of LY294002or LFM-A13. By contrast, up-regulation of EPHA3by transfection with EPHA3-PEX2-EcoRI/BamHI in SCLC cells resulted in cell IC50values, survival and the resistance to ADM, DDP or VP-16were significantly decreased, along with cell early apoptosis by flow cytometry analysis was increased. Also, overexpression of EPHA3inH69AR increased PI3K-p110, but decreased BMX and p-STAT3in protein levels, and increased cell early apoptosis by treated with LY294002or LFM-A13.Subsequently, the BALB/C-nude mice xenografts models analysis was found that higher expression of EPHA3significantly enhanced the ability of xenografts formation in SCLC cell except for H1688.By searching PicTar and TarScan database, we found that3’-UTR of human EPHA3contain putative regions that match to the seed sequence of several miRNAs, including miR-7, suggesting a possible modulation of EPHA3by miR-7.To evaluate whether miR-7can affect the endogenous expression of EPHA3, we examined EPHA3expression after up-regulation or knockdown of miR-7by using miR-7angomiror inhibitor respectively, down-regulation of EPHA3in mRNA and protein levels were revealed after the introduction of miR-7angomir in H69cells. Transfection of miR-7inhibitor in H69AR cells restored EPHA3expression in mRNA and protein level. The findings suggested that miR-7can directly target the3`UTR of EPHA3.To directly test the role of miR-7on chemoresistance, SCLC cells were transfected with either miR-7angomir or miR-7inhibitor to against EPHA3by CCK8and flow cytometry analysis. Examinations revealed that SCLC cells transfected with miR-7inhibitor showed a considerably heightened sensitivity to ADM, DDP or VP-16. By contrast, up-regulation of miR-7by transfection with miR-7angomir in SCLC cells resulted in increased resistance to ADM, DDP or VP-16. Moreover, up-regulation of miR-7in H69resulted in the decreased expression of PI3K-p110, but increased expression of BMX and p-STAT3in protein levels, and the decreased cell early apoptosis with treatment of LY294002or LFM-A13. The opposite results were gotten by inhibition of miR-7in H69AR that the protein expression levels of PI3K-p110was increased, but BMX and p-STAT3were decreased, and the cell early apoptosis with treatment of LY294002or LFM-A13was increased.In31cases of SCLC tissues samples, miR-7and EPHA3expressions showed a significant inverse correlation(Spearman correlation, r=-0.444, P=0.012). To further elucidate whether the regulation of miR-7were mediated by EPHA3in SCLC cells, we detected the miR-7expressions of SCLC cells transfected with EPHA3shRNA or EPHA3-PEX2-EcoRI/BamHI. EPHA3knockdown led to miR-7expressions significantly increased, otherwise, EPHA3up-regulation led to miR-7expressions significantly decreased.To verify whether EPHA3is a direct target of miR-7, we constructed luciferase reporters with wide-type (psiCHECK2-EPHA3-3’UTR and psiCHECK-2-EPHA3-3’UTR-R) and mutated3’-UTR (psiCHECK2-EPHA3-mut-3’ UTR) of EPHA3. Both the wild-type and the mutant-type reporters were introduced into H69AR cells and cotransfected with miR-7angomir or inhibitor. The Luciferase activity of EPHA3was measured by the dual luciferase assays. Luciferase activity was demonstrated a significant decrease or increasing compared to either mutant or empty vector controls when cotransfected with miR-7angomiror inhibitor.Conclusions1. We firstly analyzed the expression of EPHA3in human SCLC tissues. EPHA3expression level correlates with significantly overall survival time of the SCLC but is not associated with gender, age, disease stage and survival status. Our results may provide independent prognostic factor for SCLC patients.2. Our study revealed a coordinated down-regulation of EPHA3gene expression in SCLC multidrug-resistance cells, up-regulation EPHA3can reduce the resistance of chemotherapy drugs such as ADM, DDP and VP-16, and its mechanism may be involed in changing the cell apoptosis and PI3K/BMX/STAT3cell signaling pathways. We revealed EPHA3was a direct target of miR-7and furthermore reversly regulated miR-7. Besides, higher expression of EPHA3significantly enhanced the ability of xenografts formation in SCLC cells except for H1688.3. This study revealed that bidirectional regulation of EPHA3and miR-7involving in the chemoresistance effects in SCLC cells.