Effects Of MiR-203 To Non-small Cell Lung Cancer On Cisplatin Sensitivity, Cell Proliferation And Apoptosis

Background and objectivesLung cancer is one of the most common malignant diseases with high morbidity and mortality.It is the leading cause of death for neoplastic diseases both in men and women.Non-small cell lung cancer（NSCLC）is the most common type of lung cancer.Despite the high incidence of lung cancer,the early diagnosis is still very difficult,most cases cannot be clearly diagnosed until the advanced stage.As a result,patients often miss the best surgical treatment indications.Recently with the clinical application of molecular targeted therapy,the prognosis of advanced NSCLC has imporved to a certain extent,but so far,cisplatin（DDP）combined with the other chemotherapy medicine is still the main treatment.However,the response of cisplatin chemotherapy is not optimistic,and the rate of inherent or acquired resistance to chemotherapy is high.In order to improve the prognosis of lung cancer,it is particularly important to understand the mechanism of resistance to DDP and then seek for the targeted treatment.micro RNA（miRNA）is a non-coding small RNA with a length of 19-25 nucleotides that can regulate the translation or degradation of target mRNA in the human body.miRNA plays an important role in the proliferation,invasion and apoptosis of malignant tumor cells.A number of studies have shown that miR-203 may be involved in the regulation of the biological behavior of tumor cells in various tumor tissues such as NSCLC;miR-203 shows a lower expression level in lung cancer tissues than normal tissues.What’s more,miR-203 has the potential to affect DDP sensitivity in human tumor cells.The human Dickkopf-1（DKK1）gene is located on the 10q11.2 chromosome and encodes a protein containing 266 amino acids.DKK1 is a secretory protein and an inhibitor of beta-catenin-dependent Wnt signaling pathways.Wnt signaling is a multifunctional signaling pathway that regulates stem cell function,cell differentiation,cell proliferation,survival,migration,developmental polarity and adult tissue homeostasis.Wnt signaling mediates a variety of cellular biological behaviors,and Wnt signaling pathway abnormalities are closely related to tumorigenesis.A number of studies have shown that DKK1 has a higher expression level in lung cancer than that in normal bodies.In some tumors,DKK1 is related to the poor prognosis of tumor and has the effect of promoting cell proliferation.In addition,some studies indicate that DKK1 can affect DDP sensitivity.The aim of this study was to investigate the differential expression of miR-203 and DKK1 in NSCLC with different chemosensitivity,and to explore the effects of miR-203 on the proliferation,apoptosis and cisplatin sensitivity of A549/H460 cell lines.This study includes three parts.In the first part,we detected the expression level of miR-203 and DKK1 in 30 cases of NSCLC with sensitive or insensitive response to cisplatin included chemothrapy.In the second part,we investigated the effects of overexpressing miR-203 to A549/H460 cell lines on cisplatin sensitivity,cell proliferation and apoptosis.In the third part,we performed the preliminary study on the mechanism of miR-203 on DKK1.Part One Expression level of miR-203 and DKK1 in NSCLC tissues with different response to chemotherapyMethods 1.30 cases of advanced NSCLC tissues without EGFR or ALK mutation were collected and divided into two groups according to the response to cisplatin included chemotherapy.2.The relative expression level of miR-203 and DKK1 mRNA in lung cancer tissues was detected by q RT-PCR.The relative expression level of DKK1 protein was detected by Western blot.3.SPSS 21.0 software was used for statistical analysis,the normal distribution data were described with（（?）±s）;t test was used for comparsion of two independent data;Pearson correlation analysis was used for analyzing the correlation between two sets of data,the test level α=0.05.Results 1.The relative expression level of miR-203 in NSCLC tissues insensitive to cisplatin included chemotherapy was lower than that in sensitive tissues（P= 0.035）.The relative expression level of DKK1 mRNA in NSCLC tissues insensitive to cisplatin included chemotherapy was higher that in sensitive tissues（P= 0.003）.2.The relative expression level of DKK1 protein in NSCLC tissues insensitive to cisplatin included chemotherapy was higher than that in sensitive tissues（P<0.001）.3.Expression level of miR-203 was negatively correlated with DKK1 mRNA（R2 = 0.532,P<0.001）,and also negatively correlated with DKK1 protein（R2 = 0.510,P <0.001）.Part Two Effects of overexpressing miR-203 to A549/H460 cell lines on cisplatin sensitivity,cell proliferation and apoptosisMethods 1.The constructed pre-miR-203 or NC sequence lentiviral vector was used to infect A549/H460 cell lines which could stably express the firefly luciferase.Cells were divided into three groups: miR-203 group,NC group,Blank group,then the expression level of miR-203 was identified by q RT-PCR.2.CCK-8 assay was used to detect the absorbance values of three groups of cells with the treatment of different concentrations of cisplatin（0μM,2μM,4μM,6μM,10μM and 20μM）after 24 h.Then the proliferation inhibition rate and IC₅₀ of cisplatin was calculated based on absorbance values.3.The amount of apoptotic cells of three groups with the treatment of different concentrations of cisplatin（0μM,4μM）after 24 h was detected by Anncxinv-FITC/PI double-labeled flow cytometry;and the activity of caspase-3/7 was measured by Caspase-3/7 kit.4.Cells of miR-203 or NC group were inoculated into the axillary tissue of nude mice to construct lung cancer model of nude mice.After successfully inoculation,cisplatin or saline was injected intraperitoneally（3mg/kg once every other day,for a total of three doses）.Small animal live imaging system was used to detect the fluorescence signal of the nude mice;finally the mice were sacrificed 4 weeks after inoculation.5.SPSS 21.0 software was used for statistical analysis.The comparison of multiple sets of data with normality and variance homogeneity was carried out by one-way ANOVA.LSD-t test was used to compare the two pairs among data sets.The IC₅₀ of cisplatin was calculated by normal probability conversion method using the Probit program in Regression analysis of SPSS 21.0 software;α = 0.05.Results 1.Recombinant lentiviral vectors were successfully constructed.miR-203 recombinant lentiviral titer was 1.65×108TU/m L;and NC recombinant lentivirus titer was 2.03×108TU/m L.The A549/H460 cell lines with stable expression level of miR-203 were successfully constructed.2.The proliferation inhibition rate of miR-203 group was significantly higher than that of NC group and Blank group with or without cisplatin treatment（P<0.05）,IC₅₀ value of cisplatin in miR-203 group was significantly lower than that of NC group and Blank group（P<0.05）.3.The number of apoptotic cells in miR-203 group was significantly higher than that in NC group and Blank group（P<0.05）.The number of apoptotic cells in groups with cisplatin was higher than that in the cisplatin untreated groups,and apoptotic cells in miR-203 group was significantly higher than that in NC group and Blank group（P<0.05）.The activity of caspase3/7 in miR-203 group was significantly higher than that in NC group and Blank group with or without cisplatin treatment（P<0.05）.4.The fluorescence signal and tumor weight of nude mice transplanted tumor were significantly lower in cisplantin with overexpressed miR-203 group than other groups（P<0.05）.Part Three Preliminary study on the mechanism of miR-203 on DKK1Methods 1.The target gene of miR-203 was predicted by bioinformatics method,point mutation was designed at the "seed region".2.Wild-type and mutant-type DKK1 3’UTR double-reporter vector（pmir GLO-DKK1-Wt/Mt）was constructed and co-transfected into A549/H460 cells with miR-203 mimics or Scramble.We detected the luciferase activity of each groups 24 h after transfection.Then the effect of overexpression of miR-203 on DKK1 protein expression was detected by western blot.3.The cells were transfected with DKK1-si RNA and/or miR-203 into A549/H460 cells,cisplatin of 4μM or 0μM was added 24 h after transfection.CCK-8 assay was used to detect the absorbance values of cells 24 h after cisplatin treatment.Then the IC₅₀ of cisplatin was calculated based on absorbance values.4.pc DNA3.1-DKK1 recombinant expression vector（lacking 3’UTR）was constructed and transfected into A549/H460 cells alone or co-transfected with miR-203 mimics,cisplatin of 4μM or 0μM was added 24 h after transfection.CCK-8 assay was used to detect the absorbance values of cells 24 h after cisplatin treatment.Then the IC₅₀ of cisplatin was calculated based on absorbance values.5.SPSS 21.0 statistical software was used for data analysis.Results 1.We have predicted that DKK1 may be the target gene of miR-203.2.Double-reporter recombinant vectors were successfully constructed.Luciferase signal of the miR-203+pmir GLO-DKK1-Wt group was significantly lower than those in the other three groups（miR-203+pmir GLO-DKK1-Mt,NC + pmir GLO-DKK1-Wt,and NC+pmir GLO-DKK1-Mt）（P<0.05）,while there was no significant luciferase signal difference amog the three groups.Meanwhile,upregulation of miR-203 reduced DKK1 protein expression.3.The IC₅₀ of miR-203 group,DKK1-si RNA group,DKK1-si RNA + miR-203 group were all significantly lower than the Scramble group（P <0.05）,while there was no significant difference in IC₅₀ value among the three groups.4.pc DNA3.1-DKK1 recombinant vectors were successfully constructed.When comparing with the Scramble group,IC₅₀ value of miR-203 group was significantly lower（P <0.05）,while pc DNA3.1-DKK1 group and pc DNA3.1-DKK1+miR-203 group was higher（P <0.05）.Besides,there was no significant difference in IC₅₀ value between the last two groups.Conclusions 1.The relative expression level of miR-203 in NSCLC tissues insensitive to cisplatin included chemotherapy was lower than that in sensitive tissues.The relative expression level of DKK1 mRNA and DKK1 protein in NSCLC tissues insensitive to cisplatin included chemotherapy was higher than that in sensitive tissues.Expression level of miR-203 was negatively correlated with DKK1.2.Overexpression of miR-203 can increase the sensitivity of A549/H460 cells to cisplatin,inhibit the proliferation of A549/H460 cells,and promote apoptosis of A549/H460 cells.3.miR-203 negatively regulates DKK1 by targeting the 3’UTR of DKK1,thereby increasing the sensitivity of A549/H460 cell lines to cisplatin.