| Background Gastric cancer is one of the most frequently reported cancers in the world,and incidence of gastric cancer is much higher in China than in any other country,constituting an important public health problem.Despite promising developments in multimodal treatment strategies,the survival rates of gastric cancer are still low,because of a lack of validated screening programs and high rates of tumor invasion and metastasis.Therefore,most GC cases are diagnosed at an advanced stage,required palliative chemotherapy.Cisplatin is one of the most commonly used drugs in gastric cancer,but the effective rate of platinum-based chemotherapy regimens are no more than 50%,existence of platinum primary or secondary drug resistance limited its clinical application.Numerous studies showed that drug resistance of gastric cancer cells could be modulated by the abnormal expression of micro RNAs(miRNAs)which target multiple cell signaling pathways.Previous study have been confirmed that miR-138 could regulate drug resistance in lung cancer cells,by targeted ERCC1,reduced nucleotide excision repair lead to cisplatin sensitive.However,whether miR-138-5p can lead to cisplatin resistance in gastric cancer remains unknown,we hypothesized that the acquisition of chemoresistance by gastric cancer cells may also be modulated via the changes in miR-138-5p levels,by targeted at the nucleotide excision repair,and could be applied to treat chemotherapy resistance in gastric cancer patients.In the early stage of clinical data collection of this study,we found that the prognosis of advanced gastric cancer patients with hyponatremia was significantly worse,hyponatremia seems to be associated with the effective rate of gastric cancer chemotherapy.Although it is not yet clear whether serum sodium correction reflects exclusively the activity of anticancer drug or it has a real role in determining an increase of survival,need to study.So we further investigate the effect of hyponatremia on first-line chemotherapy in patients with advanced gastric cancer,and the relationship between hyponatremia and miR-138-5p expression,By combining the laboratory tests in patients with the expression of micro RNAs in order to better guiding the clinical individualized treatment of gastric cancer patients.This study contains three parts.Part 1: Expression of miR-138-5p and relationship with cisplatin resistance in gastric cancer cellObjective Investigate the expression of miR-138-5p and relationship with cisplatin resistance in gastric cancer cell.Methods Microarray system was used to analyze the miRNA expression in SGC7901/DDP cell compared with SGC7901 cell.Bioinformatics analysis software was used to predicte the target of miR-138-5p.Quantificational real-time PCR was used to detect expression of miR-138-5p in gastric cancer cells(SGC7901 and SGC7901/DDP).Western-blot was used to detect the expresstion of ERCC1,ERCC4 protein.The SGC7901 and SGC7901/DDP cells were transfected with the lentiviral vector which had the target gene Interfere expression of miR-138-5p or negative control RNA(NC).Microscope was used to detected cell transfection efficiency,MTT was used to analyze drug sensitivity.Results 1.The expression of miR-138-5p was an average of 5.006 fold higher in SGC7901 cells than in SGC7901/DDP cells and there is a significant difference between the two groups(P<0.01),consistent with microarray system analysis results.2.The analysis of bioinformatics software showed the ERCC1 and ERCC4 were the direct target genes of miR-138-5p.The expression of ERCC1 and ERCC4 protein was upreagulated in SGC7901/DDP cell than in SGC7901 cell and there is a significant difference between the two groups(P<0.01).3.The expression of miR-138-5p in SGC7901/DDP-LV-miR-138-5p-OE cells upregulated,fluorescent microscopy showed green,was an average of 4.17 fold and 4.36 fold higher than in SGC7901/DDP cells and SGC7901/DDP-LV-NC-OE cells.The expression of miR-138-5p in SGC7901-LV-miR-138-5p-KD cells downregulated,fluorescent microscopy showed red,was an average of 0.209 fold and 0.211 fold higher than in SGC7901 cells and SGC7901-LV-NC-KD cells,there are a significant difference between them(P<0.05).4.Western-blot detected the expression of ERCC1,ERCC4 protein was upreagulated in SGC7901-LV-miR138-5p-KD cells than in SGC7901 cells and SGC7901-LV-NC-KD cells,the expression of ERCC1,ERCC4 protein was downregulated in SGC7901/DDP-LV-miR138-5p-OE cells than in SGC7901/DDP cells and SGC7901/DDP-LV-NC-OE cells,and there was a significant difference between them(P<0.01).5.SGC7901/DDP-LV-miR138-5p-OE cells showed that the IC50(3.14±0.32μg/m L)was lower than the SGC7901 cells(6.81±0.12μg/m L)and SGC7901/DDP-LV-NC-OE cells(6.89±0.14μg/m L),SGC7901-LV-miR138-5p-KD cells showed that the IC50(0.39±0.04μg/m L)was more than the SGC7901 cells(0.20±0.01μg/m L)and SGC7901-LV-NC-KD cells(0.25±0.05μg/m L),and there was a significant difference between them(P<0.01).Conclusion The expression of miR-138-5p was downregulated in SGC7901/DDP cells.Transfected lentivirus lead to miR-138-5p upregulated can reverse cisplatin resistance in SGC7901/DDP cells,by decreasing the ERCC1,ERCC4 protein levels,reduce the nucleotide excision repair.Downregulated miR-138-5p can lead to cisplatin resistance in SGC7901 cells.Part2: Mi R-138-5p expression predicts efficacy of platinum-based chemotherapy in patients with advanced gastric cancerObjective: There is increasing evidence that micro RNA expression is closely related to the sensitivity of cancer drug therapy.The aim of this study is to investigate the relationship between miR-138-5p expression in cancerous tissue and plasma and the efficacy of first-line platinum-based chemotherapy and survival in patients with advanced gastric cancer.Methods: Mi R-138-5p expression was measured by q RT-PCR in cancerous tissue and plasma from 51 patients with advanced gastric cancer.The paracancerous tissues from 20 patients and plasma from 20 healthy volunteers were used as control.All the enrolled patients were received platinum-based chemotherapy,the chemotherapeutic efficacy,first-line progression-free survival(PFS),and the overall survival(OS)were evaluated.Results: Mi R-138-5p expression was lower both in cancerous tissue and plasma than those in control group(P<0.05).The differences of tissue miR-138-5p relative expression among partial response(PR),stable disease(SD)and progressive disease(PD)patients were statistically significant.The area under the curve(AUC)in receiver operating characteristics(ROC)curves for predicting objective response rate(ORR)was 0.907,with the cut-off tissue miR-138-5p relative level 0.104.The plasma miR-138-5p relative expression in PR patients was higher than the expression in SD and PD patients.The AUC in ROC curves for predicting ORR was 0.973,with the cut-off plasma miR-138-5p relative level 0.091.The PFS and OS both in tissue and plasma miR-138-5p high expression group(≥0.104 in tissus,and ≥0.091 in plasma)were longer than that in low expression group(P<0.05).There was a positive correlation between miR-138-5p expression in tissue and plasma of gastric cancer patients(r =0.899,P<0.05).Conclusion: Mi R-138-5p expression both in cancerous tissue and plasma can be a potential biomarker for predicting the efficacy of platinum-based chemotherapy and survival in patients with advanced gastric cancer.Part3: Hyponatremia predicts efficacy of chemotherapy in patients with advanced gastric cancer and the relationship with expression of miR-138-5pObjective: To investigate the effect of hyponatremia on chemotherapy efficacy in patients with advanced gastric cancer,and the relationship with expression of miR-138-5p.Methods: A total of 44 patients with advanced gastric cancer complicated with hyponatremia(low-sodium group),and 45 cases of advanced gastric cancer with normal blood sodium(control group)were selected from the hospitalized patients in the First Affiliated Hospital of Anhui Medical University,from Jun 2009 to Dec 2015.The clinical data include the first-line chemotherapeutic efficacy,first-line progression-free survival(PFS),and the incidence rate of side effects of chemotherapy were retrospectively analyzed and compared between two groups.q RT-PCR was used to detect expression of miR-138-5p in 5 gastric patients with hyponatremia,and 10 patients without hyponatremia.Results: In patients with advanced gastric cancer,female patients or who have liver or peritoneal metastasis and low ECOG score were more easily complicated with hyponatremia.The objective response rate(ORR)of first-line chemotherapy in low-sodium group and control group were 25.0%(11/44)and 40.0%(18/45),respectively(P=0.131).Disease control rate(DCR)in low-sodium group(40.9%,18/44)was statistically lower than DCR in control group(73.3%,33/45)(P=0.002).The median PFS of first-line chemotherapy in low-sodium group lasted for 3 months(95% CI: 2.8-3.7),while it lasted for 4.5 months in control group(95% CI: 4.3-6.2).The difference between two groups was statistically significant(χ2=14.618,P<0.001).The median PFS was lower in persistent hyponatremia group than the group which sodium levels return to normal.After the first-line chemotherapy,the occurrence rate of complication(anemia,nausea,emesis,fatigue and anorexia)in low-sodium group was statistically higher than that in control group.The expression of miR-138-5p is lower in low-sodium group than in control group,but the difference between two groups was without statistically significant.Conclusion: Hyponatremia is one of the poor predictive factors for chemotherapy efficacy,Through combine the miR-138-5p expression both in cancerous tissue and plasma and the serum sodium levels can be used as potential biomarkers for predicting the efficacy of platinum-based chemotherapy and survival in patients with advanced gastric cancer. |
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