| Research backgroundGastric cancer,gastrointestinal solid tumors of higher malignancy,is the fourth most common malignant tumor in the world.Globally,930,000 new cases of stomach cancer occur every year,of which 730,000 people die,making it a highly lethal malignancy tumor.Studies found that more than half of the new cases occurred in the developing countries,including east Asia,eastern Europe,central and South America as high-risk areas,especially in east Asian countries such as China and Singapore.At present,gastric cancer patients in Japan have a higher five-year survival rate,which can be as high as 90%,in other countries,the five-year survival rate of patients with gastric cancer is only 10%-30%.Among them,the high survival rate of Japanese gastric cancer patients might be related to the early detection of tumors through endoscopy and early resection.In China,since there is no obvious clinical symptoms in the early stage of gastric cancer patients and early diagnostic markers are not available,most gastric cancer patients are diagnosed as advanced gastric cancer,and patients often have distal metastasis and invasion of tumors,thus losing the possibility of surgery.In addition,with multiple drug resistance to chemotherapy,patients with gastric cancer have extremely low five-year survival rate and poor prognosis.Therefore,to explore the specific molecular mechanism of the occurrence and development of gastric cancer is expected to provide new directions for the earlydiagnosis,treatment and prognosis judgment of gastric cancer patients,which is of great significance for the survival of gastric cancer patients.ObjectivesOur study aims to observe the expression of miR-374b-5p in cancerous tissue and serum of gastric cancer patients and its role in distinguishing pancreatic cancer from healthy control,to assess the prossibility of serum miR-374b-5p in diagnosing gastric cancer and prognosis of survive,to explore the target gene of miR-374b-5p and analyze the effect of miR-374b-5p and its target gene on the invasion of gastric cancer cell.The following problems will be studied in detail:(1)the expression of miR-374b-5p in cancerous tissue and adjacent gastric mucosa of gastric cancer patients was observed,and the relationship between the expression level of miR-374b-5p and the clinicopathological data was analyzed.(2)The expression level of miR-374b-5p in the serum of patients with gastric cancer and healthy control group was analyzed,to explore the role of serum miR-374b-5p in the diagnosis and survival time of patients with gastric cancer.(3)Biological information analysis was used to forecast the target gene of miR-374b-5p,which was verified by endogenous and exogenous experiments.(4)The effects of miR-374b-5p and its target gene on migration and invasion of gastric cancer cells were observed.Materials and Methods(1)60 gastric cancer patients from 2013 to 2015 were collected in department of gastrointestinal surgery,the first affiliated hospital of zhengzhou university,informed consent was obtained from all patients who received no radiotherapy or chemotherapywas before operation,there are 30 males and 30 females,with an average age of 41.6 years.The cancerous tissue and adjacent gastric mucosa of gastric cancer patients were collected and confirmed by pathology.A total of 3-5 ml of peripheral blood was extracted from all the 60 patients,in addition,60 healthy controls(30 males and 30 females,with an average age of 42.5 years)were selected,and there was no statistical difference in gender and age between the gastric cancer patients and the healthy control group.qRT-PCR was used to detected the expression of miR-374b-5p in cancerous tissue and adjacent gastric mucosa of gastric cancerpatients,in the serum of gastric cancer patients and healthy control.?2 test was applied to explore the relationship between the serum expression level of miR-374b-5p and the clinicopathological data in patients with gastric cancer,ROC curve was used to observe the specificity and sensitivity of serum miR-374b-5p as a diagnostic marker of gastric cancer,kaplan-meier survival curve was applied to analyze serum miR-374b-5p in the overall survival time of gastric cancer patients.(2)miRanda and Targetscan miRNA prediction sites were applied to predicte the target gene of miR-374b-5p,the results predicted by both miRanda and TargetScan prediction sites were selected as part of the intersection,system classification was conducted by PANTHER analysis software.Finally,the classification related to the invasion and movement of our subject was selected,the target gene of miR-374b-5p was selected by mapping the target gene network with Cytoscape software and combining the predicted values of Targetscan,the target gene of miR-374b-5p was identified by exogenous luciferase,endogenous qRT-PCR and Western Blot,respectively.(3)The expression of miR-374b-5p in gastic cancer was verified in gastic cancer cell,MTT?Wound Healing ? Transwell assay were used to observe the proliferation and invasion of gastic cancer cell,and the effect of miR-374b-5p/target gene on gastric cancer invasion was further confirmed.Results1,The expression level of miR-374b-5p in the tumor tissues and serum of gastric cancer patients and its clinical significanceqRT-PCR analysis was used to observe the expression of miR-374b-5p in the tumor tissues and adjacent gastric mucosa of gastric cancer patients,the results showed that compared with adjacent gastric mucosa,the expression of miR-374b-5p in tumor tissues was elevated significantly.The relationship between the expression level of miR-374b-5p in the tumor tissue of gastric cancer patients and the clinical pathological was further analyzed,the result showed that the expression level of miR-374b-5p in the cancer tissue of gastric cancer patients is closely related to tumor staging and metastasis of gastric cancer patients.qRT-PCR was used to detecte theserum expression of miR-365 in gastic cancer patients and healthy control,the result showed that compared with the healthy control,the expression level of mir-374b-5p in the serum of patients with gastric cancer was significantly increase.By drawing the ROC curve,it was found that the area under the ROC curve(AUC)was 0.81,and the serum mir-374b-5p as a biomarker for the diagnosis of gastric cancer with 74.6.9%specific and 73.8% sensitive.Results of kaplan-meier survival curve analysis found that the survival time of gastric cancer patients with low serum expression of miR-374b-5p was significantly higher than those with high serum expression of miR-374b-5p.2,Prediction of miR-374b-5p target genes and validation of endogeneity and exogenicityThe target genes of miR-374b-5p were predicted by using the database of Targetscan and miRanda target genes,the predicted results were integrated to obtain a large number of target genes.In order to facilitate screening and analysis,the PANTHER gene function clustering system was further applied to analyze and cluster the common parts predicted by the two databases.The relationship between miR-374b-5p and its motion-related target genes was visualized using Cytoscape software network,and the predicted value of Targetscan was integrated.NCKAP1 was selected as the target gene of miR-374b-5p;The results of luciferase experiment showed that miR-374b-5p could negatively regulate NCKAP1.The results of qRT-PCR and Western Blot experiments showed that miR-374b-5p up-regulated significantly inhibited the mRNA and protein expression level of NCKAP1,while miR-374b-5p down-regulated significantly promoted the mRNA and protein expression level of NCKAP1.3,Effect of miR-374b-5p /NCKAP1 on gastric cancer cell invasion and its mechanismThe expression level of miR-374b-5p in gastric cancer cell line was detected by qRT-PCR,and the results showed that the expression level of miR-374b-5p was up-regulated in the gastric cancer cell line.Up-regulation the expression of miR-374b-5p in gastric cancer cell lines significantly promoted the proliferation,migration and invasion of gastric cancer cells,and down-regulation the expression of miR-374b-5p in gastric cancer cell lines significantly inhibit the proliferation,migration and invasion of gastric cancer cells.Up-regulation the expression of NCKAP1 in gastric cancer cell lines significantly inhitbited the proliferation and invasion of gastric cancer cells,and interference the expression of NCKAP1 in gastric cancer cell lines significantly promoted the proliferation and invasion of gastric cancer cells.Interference of NCKAP1 could reverse the inhibitory effect of miR-374b-5p on the invasion of gastric cancer cells,suggesting that miR-374b-5p/NCKAP1 might involve in the invasion of gastric cancer cells.Conclusions1,The expression level of miR-374b-5p in the cancerous tissues of gastric cancer patients was significantly increased,and the expression level of miR-374b-5p in the cancerous tissues of gastric cancer patients with metastasis was higher than that of patients without metastasis.The expression level of miR-374b-5p in the serum of gastric cancer patients was significantly increased,which might be a potential biomarker for the early diagnosis of gastric cancer with high specificity and sensitivity.The survival time of gastric cancer patients with high serum expression of miR-374b-5p was significantly lower than that of low serum expression of miR-374b-5p,suggesting that the higher serum expression level of miR-374b-5p had the shorter overall survival time.2,Up-regulated miR-374b-5p significantly inhibited the mRNA and protein expression level of NCKAP1,while miR-374b-5p down-regulated significantly promoted the mRNA and protein expression level of NCKAP1.3,Upregulation of miR-374b-5p in gastric cancer cells promoted the proliferation,migration and invasion of gastric cancer cells,and downregulation of miR-374b-5p significantly inhibited the proliferation,migration and invasion of gastric cancer cells.Up-regulation of NCKAP1 inhibited proliferation and invasion of gastric cancer cells,and interference of NCKAP1 expression promoted the proliferation and invasion of gastric cancer cells.Interference with NCKAP1 couldreverse the inhibitory effect of miR-374b-5p on the invasion of gastric cancer cells,which suggested that miR-374b-5p/NCKAP1 was involved in the invasion of gastric cancer cells. |
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