| BackgroundThe incidence of lung cancer is the top1all over the world. According the cell type, lung cancer is grouped into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The incidence of SCLC covers15%of primary lung cancer. The treatment of SCLC is mainly in medicine, but because of multidrug resistance (MDR), the lost chemotherapy, or a higher recurrence, a weak prognosis is often occurred in clinic. A two-year survival rate is less than20%and90%of patients is dead in5years.Several kinds of ion channels play important roles in cell development and differentiation, i.e. Kir2.1encoded by KCNJ2gene. In1984, DeCoursey et al discovered the co-relationship between the K channel and tumorigenesis. Pancrazio et al reported that the K channel plays important role in SCLC resistance. Some publications reported that the inner-current K channel had a close relevance with the MDR. All the publications did not answer the mechanism. We discover that the Ras-MAPK signal pathway participates in the regulation of Kir2.1. Additionally some binding sites for anchoring proteins such as the filamin A, psd935, or SAP97, promote the Kir2.1channel expression in cell membrane. Recently, it has been suggested that kinases and phosphatases can be intimately associated to channels in a single regulatory protein complex that modulates channel activity and PKA and PKC regulate the Kir2.1expression. So the Ras-MAPK-PKC signal pathway regulates the KCNJ2expression in SCLC cells.MicroRNAs (miRNAs) are a class of small non-coding RNAs of18-24nucleotides which regulate protein expressions of specific mRNAs 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 classification and prediction of therapy response. More and more evidences confirm that miRNA has close relevance in tumorigenesis and tumor prognosis, the evidence of the roles for miRNAs in determining drug sensitivity/resistance has been emerging. miRNAs regulate a variety of cellular activities through negatively regulating special target genes. Furthermore, it may also be involved in chemoresistance to cancer therapy. Some miRNAs are involved the MDR in lung tumor. The mechanism may be mediated by inhibiting FZD7/β-catenin signal, or ABCB1, ZNRD1protein. In2012, Goldoni D et al reported that Mi-212down-regulated the KCNJ2expression. We ever screened the gene spectra in the H69AR and H69cells.21,522genes were analyzed, showing that miR-7had higher expression in the H69cells as well as the up-regulations of1252genes in the H69AR cells and the down-regulations of1131genes. Of them, KCNJ2gene showed higher expression in the H69AR cells, suggesting that the KCNJ2gene and miR-7were relevant to SCLC MDR. Biological information show that KCNJ2gene is the target of miR-7and miR-7can inhibit the expression of KCNJ2, then, miR-7may regulate small-cell lung cancer multidrug resistance by inhibiting the expression of KCNJ2.This study aims to investigate the expression of the potassium ion channel protein Kir2.1/KCNJ2in small cell lung cancer cell lines and tissues, and to explore the role of Kir2.1/KCNJ2in small cell lung cancer multidrug resistance and its relationship with clinical prognosis relevance, and to further explore the regulatory mechanisms of Kir2.1/KCNJ2regulating of the multidrug resistance of small cell lung cancer, this study may provide new ideas for investigation of multidrug resistance of small cell lung cancer and provide a theoretical basis for clinical treatment and prognosis.ObjectivesTo analyze the expression of Mir-7, KCNJ2and ABCC1in small cell lung cancer specimens and cell lines, and to explore the role of KCNJ2in multidrug resistance in small cell lung cancer and the mechanisms of KCNJ2involved in multidrug resistance and it’s regulation mechanisms and in-depth study of the interaction between KCNJ2and ABCC1; furthere more, to analyze the correlationship between Mir-7, KCNJ2and ABCC1with clinical outcome. To further enriched theoretical molecular mechanisms of multi-drug resistance in SCLC and to provide a theoretical basis for clinical treatment.Materials and methods1. An analysis of KCNJ2gene expression differences in multi-drug resistant SCLC cell lines H69AR, H446AR and there sensitive cell lines H69and H446Multidrug resistance cell lines H69AR, H446AR and sensitive cell lines H69, H446as the research object, and KCNJ2mRNA and protein expression real-time PCR and Western Blot Detection of four cell lines, verify KCNJ2mRNA and protein in four differential expression of cell lines;2. KCNJ2expression of multidrug resistance in small cell lung cancerUsing KCNJ2overexpression plasmids and siRNA increase or decrease multidrug resistant SCLC cell lines H69AR, H446AR and sensitive cell lines H69, H446in KCNJ2expression levels, CCK8analysis on small cell lung cancer cells commonly used chemotherapy drug cisplatin (Cisplatin, DDP), sensitivity to changes etoposide (Etoposide, VP-16) and adriamycin (Doxorubicin, ADM), flow cytometry analysis of changes in cell cycle distribution and apoptosis.3. KCNJ2regulation mechanismIn H69, H69AR, H446and H446AR cell lines, to detect the expression of KCNJ2mRNA and protein using real-time PCR and Western Bloting by inhibiting the key protein kinase PKC and MEK of PKC-MEK-KCNJ2signaling pathway respectively or both. 4. KCNJ2correlation with ABCC1Using immunohistochemical analysis of tissue specimens to express the correlation between KCNJ2and ABCC1,and using co-immunoprecipitation experiments to explore the interaction between KCNJ2and ABCC1in cells, to explore the mechanisms that KCNJ2regulate the small cell lung cancer multidrug resistance.5. In vivo tumor xenograft model:The mice were raised under pathogen-free conditions. All procedures were performed according to guidelines of Association for Assessment and Accreditation of Laboratory Animal Care International. H69AR, H69AR-NC, H69AR-KCNJ2-homo-628, H69AR-KCNJ2-homo-1073, H69, H69-NC, H69-KCNJ2-PEX-1and H446AR, H446AR-NC, H446AR-KCNJ2-homo-628, H446AR-KCNJ2-homo-1073,:H446, H446-NC, H446-KCNJ2-PEX-1cells were subcutaneously inoculated into the legs of nude mice to establish the tumor model, respectively. Three weeks later,5mice of each group were sacrificed and tumors were excised, tumor size were measured and PCNA immune cells were to be detected and analyzed.6. Expression differences of miR-7in H69AR, H446AR and H69, H446cells.Using miRNA microarray to analyze the microRNA expression differences in H69AR, H446AR and H69, H446cells, using miRNA target gene analysis software (miRanda, TagetScan and PicTar)to analyye KCNJ2related miRNA, then select differentially expressed miRNA (miR-7) to study and the miR-7expression difference were validated by real-time quantitative PCR.7. KCNJ2is the target genes of miR-7(1). using miR-7inhibitor (antangomir) and mimics (angomir)to inhibit or increase the expression level of miR-7in H69AR, H446AR and H69, H446cells, using real-time quantitative PCR and Western Bloting to analyze KCNJ2mRNA and protein expression changes.(2)..To construct the psiCHECK-2luciferase vector (psiCHECK-2-KCNJ2-3’UTE) of KCNJ23’-untranslated region (3’-untranslated region,3’-UTR), and to transfect the miR-7mimic or inhibitor into H69, H69AR, H446and H446AR cells, KCNJ2luciferase activity were determined by the method dual luciferase reporter gene assay.8. miR-7involve in multidrug resistance of SCLCmiR-7mimic or inhibitor was transfected into cells to increase or inhibit miR-7expression. Chemosensitivity to drugs were determined by using CCK-8assay.9.expression and clinicopathological significance miR-7, KCNJ2and ABCC1in SCLC tissues52cases of paraffin-embedded tissues of SCLC patients were collected, follow-up clinical data, KCNJ2and ABCC1expression are to be detected by immunohistochemical staining, the expression of miR-7is to be detected by QRT-PCR, to analyze the statistical difference betwteen the expression of KCNJ2, ABCC1and miR-7in resistance group (relapse after three months) and sensitive group (six months without recurrence), limited disease group(LD) and extensive disease group(ED), and further to analyze expression of KCNJ2, miR-7and ABCC1with the clinical characteristics of SCLC patients and survival relationship.10. statistical analysisAll results were confirmed by statistical software SPSS13.0statistics. Data are expressed as mean±standard deviation (x±s), diferences of KCNJ2or miR-7expression between two cells were used independent samples t test to compare; miR-7, KCNJ2or ABCC1expression in multiple groups were compared using one-way ANOVA (homogeneity of variance simultaneously) or Welch method correction (when heterogeneity of variance), multiple comparisons using the SNK method (homogeneity of variance simultaneously) or Dunnett’s T3method (when heterogeneity of variance); miR-7, the relationship between KCNJ2and ABCC1expression with clinicopathological characteristics using paired sample chi-square analysis (McNemar test); using multivariate Logistic regression analysis the effects of age, sex, drug sensitivity, clinical stage and other factors on Mir-7, ABCC1and KCNJ2expression; using Cox proportional hazards model analysis above variables and Mir-7, ABCC1and KCNJ2protein expression levels of survival time for patients with SCLC; influence of various factors on the survival time of patients with SCLC using Kaplain-Meier analysis. P<0.05was considered as statistically significant.Result1. KCNJ2expression increased significantly in multi-drug resistant cell line H69AR and H446AR.Real-time quantitative PCR showed KCNJ2mRNA expression in multidrug resistant SCLC cell lines H69AR, H446AR was significantly higher than the sensitive strain H69and H446(t=16.88, P=0.003; t=27.798, P<0.001), Western Blot detection showed KCNJ2protein was also significantly increased;.2. Forced KCNJ2expression enhanced chemosenresistance of H69and H446cells significantly, while the expression of ABCC1was also increased.(1) We succesfully constructed KCNJ2expression plasmid KCNJ2-PEX-1, by sequencing and comparison showed consistent with human KCNJ2coding (coding sequence, CDS).(2) Estabishement of stably transfected cell line H69-KCNJ2-PEX-1and H446-KCNJ2-PEX-1.KCNJ2-PEX-1or negtive control GM scrambled negative control (NC) were transfected into H69and H446cell, after G418selection to establish stable transfected cell line H69-KCNJ2-PEX-1and H446-KCNJ2-PEX-1, Elevation of KCNJ2mRNA and protein level in H69-KCNJ2-PEX-1and H446-KCNJ2-PEX-1were verified by using using quantitive real-time PCR (4.89times, t=10.184, P=0.006;7.23times, t=13.562, P=0.003);(3) After treatment of ADM, DDP and VP-16, survival and half maximal inhibitory concentration of a substance (IC50) value of H69-KCNJ2-PEX-1and H446-KCNJ2-PEX-1cells increased significantly compared with H69-NC and H446-NC cells(P<0.001).(4) flow cytometry analysis showed that apoptosis of H69-KCNJ2-PEX-1and H446-KCNJ2-PEX-1cell H69AR-KCNJ2cell decreased compared with H69-NC and H446-NC cells(P<0.001) after treatment of ADM, DDP and VP-16, while G0-G1and G2-M phase cells increased. 3. Downregulation of KCNJ2expression significantly increased drug sensitivity of H69AR and H446AR cells(1).KCNJ2-homo-628, KCNJ2-homo-1073significantly reduced the expression of KCNJ2in H69AR and H446AR cells, while ABCC1expression was also significantly reduced;According KCNJ2genome sequence of a human cDNA library, the combination of methods Ambion’S siRNA Target Finder provided and references to design targeting human KCNJ2gene four pairs shRNAg interference plasmid: KCNJ2-homo-514, KCNJ2-homo-628, KCNJ2-homo-1073, KCNJ2-homo-1357, were used to detect expression levels of KCNJ2mRNA and protein changes by quantitative PCR and Western Blot method, the two pairs of shRNA (KCNJ2-homo-628, KCNJ2-homo-1073) which had the most significantly inhibitory effection were chosen to be used in following experiments.(2). CCK-8analysis showed that survival of H69AR and H446AR cell transfeced with KCNJ2-homo-628and KCNJ2-homo-1073were significantly lower compared with negative control (NC) group and the control group (P<0.001), IC50values were also significantly reduced.(3). flow cytometry analysis showed that the apoptosis rate of H69AR and H446AR cells transfeced with KCNJ2-homo-628and KCNJ2-homo-1073after treatment of ADM, DDP and VP-16treatment increased significantly compared with the negative control group and blank control group(P<0.001), while the number of G0-G1phase and G2-M phase cells decreased significantly.4. In vivo tumor xenograft model:following cells:H69AR, H69AR-NC, H69AR-kcnj2-homo-628, H69AR-kcnj2-homo-1073, H69, H69-NC and H69-KCNJ2-PEX-1injected into nude mice, three weeks later,5mice of each group were sacrificed and tumors were excised, tumor size were measured, PCNA was detected and analyzed. The results suggest KCNJ2can enhance tumor cell proliferation,5. PKC-MEK signaling pathway may regulate the expression of KCNJ2In H69AR and H446AR cells, both inhibited the PKC-MEK signaling pathway critical protein kinase PKC and MEK, KCNJ2expression was detected by RT-PCR and Western blot, KCNJ2mRNA levels were reduced by45.8%(P<0.001),51.6%(P <0.001), protein levels decreased by62.3%(P<0.001),45.2%(P<0.001), inhibited PKC alonely, KCNJ2mRNA levels were reduced by26.2%(P=0.023) and38.6%(P=0.003), protein levels decreased by23.5%,(P=0.026),29.6%(P<0.022), respectively, inhibited MEK alonely, KCNJ2mRNA levels were reduced31.3%(P=0.017),25.8%(P=0.029), protein levels decreased29.4%,(P=0.034),21.6%(P=0.036). respectively. Surgesting PKC-MEK signaling pathway may regulate the expression of KCNJ2.6. correlation between KCNJ2and ABCC1(1). An increase KCNJ2expression significantly increased the expression of ABCC1;(2).immunohistochemical analysis of tissue samples showed significantly positive correlation of KCNJ2and ABCCl(χ2=19.396, P<0.001);(3). Co-immunoprecipitation assay verified the interaction of KCNJ2with ABCC1in H69AR cells7. There are no statistically significance between miR-7expression in multidrug resistant SCLC cell lines H69AR, H446AR and sensitive cell lines H69,H446miRNA microarray results showed miR-7expression in multidrug resistant SCLC cell lines H69AR was4.64times lower than that in sensitive cell lines H69, while real-time PCR results suggested that. miR-7expression in H69and H446cells are higher than that in H69AR and H446AR cell. But the defferrenes have no statistically significance (1.09times,2=2.148, P=0.098;1.03times,t=0.478, P=0.657).8. KCNJ2is the target genes of miR-7(1) According to miRNA target gene prediction software (PicTar, TarScan and miRBase), there are complementary binding sites between miR-7and KCNJ23’-UTR.(2) regulation of KCNJ2mRNA and protein expression by miR-7 ①After transfection of mir-7agomir H69, H69AR, H446and H446AR cells to increase the expression of miR-7, showed KCNJ2mRNA expression levels were significantly lower (F=57.780, P=<0.001), KCNJ2expression was significantly reduced (F=254.520, P=<0.001). ABCC1mRNA levels and protein levels was also significantly decreased (F=381.121,P<0.001; F=365.439, P<0.001).②After transfection of mir-7antagomir to reduce the expression of miR-7in H69, H69AR, H446and H446AR cells, the results showed that the expression KCNJ2mRNA levels and protein expression were significantly elevated(F=152.419, P<0.001; F=87.124, P<0.001.); ABCC1mRNA levels and protein levels were also significantly increased (F=3.385, P<0.001; F=786.360, P<0.001).(3) KCNJ2is the target gene of miR-7①successfully constructed psiCHECK-2-KCNJ2-3’UTR and psiCHECK-2-KCNJ2-3’UTR-R carrier.KCNJ2-3’-UTR was amplified by PCR, the product length is1250bp, After it was converted, resistance screened and selected for monoclone, we extracted plasmid. The plasmid was cleaved with Not I and Xho I, and the product of6365bp/1235bp were obtained. Clone1-4of psiCHECK-2-KCNJ2-3’UTE and clone3of psiCHECK-2-KCNJ2-3’UTR-R were verified to be correct by using restriction enzyme digestion, so we selected them to be sequenced. The sequences were consistent with the theoretical sequence. Plasmid was extracted for following experiments.②miR-7mimic and inhibitor regulated luciferase activity of KCNJ2PsiCHECK-2-KCNJ2-3’UTR or psiCHECK-2-KCNJ2-3’UTR-mut was transfected into H69AR cell. Co-transfection of miR-7mimic or inhibitor was performed. Compared with H69AR cell transfeected with miR-NC, luciferase activity of KCNJ2in H69AR cell co-transfected with miR-7mimic and KCNJ23’-UTR reduced significantly (F=437.572, P<0.001), miR-7inhibitor can be significantly increased transfection KCNJ23’-H69AR cells KCNJ2UTR of luciferase activity (multiple comparison P<0.05), miR-7mimic and miR-7inhibitor had no significant effect on transfection KCNJ23’UTR-mut in H69AR cells KCNJ2 luciferase activity (multiple comparison of P>0.05).9. miR-7have no significant correlation with drug sensitivity in H69/H69AR and H446/H446AR cellsAfter treatment of ADM, DDP and VP-16, the differences between survival of H69, H446,H69AR and H446AR cells transfected with miR-7mimic or miR-7inhibitor were no statistically significance (multiple comparisons, P>0.05)..10. Relationships between miR-7, KCNJ2and ABCC1expression and clinicopathological features in SCLC tissues.Mir-7expression was the protective factors for clinical stages of SCLC patients (OR=0.258, P=0.043), while KCNJ2and ABCC1expression were the risk factors for the clinical stages of SCLC patients (OR=11.124, P=0.001; OR=10.710, P=0.001), other indicators were not statistically significance (P>0.05).11. survival analysis of SCLC patients(1) COX proportional hazards regression modelUsing COX stepwise regression analysis, the variables were selected into the " sex "," age"," staging "," sensitive ","Mir-7","KCNJ2" and "ABCC1", the relative risk of the variable "Mir-7" was0.75(P=0.026), i.e.,suggest that death risk of patients with higher Mir-7expression was0.75times compared with patient with lower Mir-7expression, there were no significant correlation between gender, stage, drug sensitivity, KCNJ2and ABCC1expression with the relative risk of SCLC patients.(2). The estimated survival timeUsing the Kaplan-Meier method compares the impact of different factors on the survival time of patients and found that male patients and female patients with no significant difference in survival time (x2=0.473, P=0.492). survival time of patients younger than55years was significantly longer than that of patients over55years of age (χ2=6.867, P=0.009). survival time of patients with limited-stage was significantly longer than patients with extensive stage (x,2=4.065, P=0.0231). survival time of patients with higher Mir-7expression was significantly longer than patients with lower Mir-7expression (χ2=5.825, P=0.016); there were no significant difference between the survival time of patients with KCNJ2and ABCC1positive expression and that of the patients with KCNJ2and ABCC1negative expression (x2=1.016, P=0.313; x2=1.696, P=0.193).Conclusion1. KCNJ2expression of multidrug resistance in SCLC cell lines H69AR, H446AR significantly increased cell KCNJ2increase or decrease the expression of resistance can cause cell changes, indicating KCNJ2involved in multi-drug resistant of SCLC.2. KCNJ2and ABCC1expression in SCLC cell lines and clinical tissue samples showed a significant positive correlation, co-immunoprecipitation display verified the interaction of KCNJ2and ABCC1in H69AR cells, suggesting KCNJ2involved in multi-drug resistant of SCLC possibly through interaction with ABCC1;3.Separately or simultaneously inhibiting PKC or MEK can reduce the expression of KCNJ2in SCLC cell lines, suggesting that PKC-MEK signaling pathway involved in the regulation of KCNJ2;4. KCNJ2is a target gene of miR-7, miR-7expression can regulate the expression of KCNJ2, decrease or increase the expression of miR-7in cells may change the expression of KCNJ2, suggesting that miR-7involved in regulation of KCNJ2;5. The expression of miR-7, KCNJ2and ABCC1in SCLC tissues related to the clinical stages of SCLC patients, whereas miR-7expression was significantly associated with the patient’s survival time as well, suggesting that miR-7is expected to be a clinical prognostic indicator in patients with SCLC and therapeutic target. |
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