| Background:Gastric cancer is one of the common malignant tumors of the digestive system.Due to the lack of specific clinical symptoms and effective screening method in the early stage,most patients are already in progress at the time of treatment,which seriously affected their prognosis.Therefore,early diagnosis of gastric cancer is particularly important.At present,gastroscopy combined with pathology is an important method in diagnosis of gastric cancer.Although gastroscopy is becoming more common than ever before.There are still many limitations in its application,such as the large population,the sensitivity of the patient to the stimulation and the dependence on the level of the examiner.CEA,CA19-9 and CA72-4 are often used in the auxiliary diagnosis of gastric cancer,but their sensitivity and specificity are not good.Therefore,a new effective biological marker is urgently needed.In recent years,exosomes have become one of the research hotspots.Exosomes are tiny vesicles with a diameter of about 50-150 nm,which have a lipid bilayer structure and can be secreted by most cells of the body.Exosomes are widely existed in the cell microenvironment,and affect the physiological state of cells by transmitting important signal molecules,thus participating in the occurrence and progression of a variety of diseases.Studies have found that exosomes play an important role in the development of gastric cancer,and it is believed that the content of exosomes may be an ideal biomarker.With the popularization of biological detection methods,mRNA has been widely studied as a marker of various diseases,so the exosome mRNA has gradually attracted more attention.Matrix metalloproteinases(MMPs)is a zinc-dependent endopeptidases family,which is one of the main enzymes that cause extracellular matrix degradation.It can be further divided into two types,membrane type and secrete type.Membrane type 1-matrix metalloproteinase(MT1-MMP),also known as MMP14,as the first discoveried proteolytic enzyme of the MT-MMPs,plays an important role in the development of tumor,so it has attracted more and more attention.In recent years,many studies have found that MT1-MMP plays an important role in gastric cancer and the increased expression of MT1-MMP is associated with poor prognosis of gastric cancer.Currently the studies on MT1-MMP mainly focus on tissue samples,although the literature also reports some findings about MT1-MMP at the cyclic level.It was found that exosomes are stable in serum,while the stability of proteins and mRNA in serum is easily affected by the storage time and temperature changes.So we tried to find a reliable biomarker based on exosomes.The importance of MT1-MMP in gastric cancer has been confirmed by many studies and its presence in exosomes has been previously reported.So we think that serum exosomal MT1-MMP mRNA may be a new biological marker of gastric cancer.But the data about serum exosomal MT1-MMP mRNA in gastric cancer is rare,the potential significance in gastric cancer remains unclear.This research is divided into two parts.In the first part,we discussed the determination method of serum exosomal MT1-MMP mRNA,and studied its expression in gastric cancer.In the second part,the clinical significance of serum exosomal MT1-MMP mRNA in gastric cancer was studied to evaluate its value in early evaluation and progress prediction of gastric cancer.Part 1:Detection of Serum Exosomal MT1-MMP mRNA and Expressionin Gastric CancerObjective:Investigate the determination of serum exosomal MT1-MMP mRNA.Study the expression of serum exosomal MT1-MMP mRNA in gastric cancer patients.Methods:1.A total of 33 participants in Qilu Hospital of Shandong University were enrolled in this study from December 2016 to January 2017,including 17 gastric cancer patients and 16 healthy controls.2.Fasting serum of all participants was collected and serum exosomes were extracted.The following observation and analysis were performed on the healthy control group and the gastric cancer group respectively.The morphology of serum exosome was observed by TEM.The size of exosome was determined by Flow Nano Analyzer.Exosome marker proteins CD9,CD63 and TSG101 were detected by Western blot analysis.3.Exosome RNA was extracted and the expression of exosomal MT1-MMP mRNA was analyzed by qRT-PCR.4.At 37?,the exosomes extracted from gastric cancer group and healthy control group were treated with RNase A or Triton X-100+RNase A,respectively.The expression changes of exosomal MT1-MMP mRNA were analyzed with no treatment as the control to verify the existence of MT1-MMP mRNA in the exosome.5.Serum of healthy control group and gastric cancer group were stored at different temperature and time respectively,then analyzed the expression changes of the serum exosomal MT1-MMP mRNA.6.Analyzed the difference in expression of exosomal MT1-MMP mRNA between gastric cancer group and healthy control group.Results:1.The characterization of exosomes in serumExosomes were extracted from the serum in healthy control group and gastric cancer group.Under TEM,the exosomes in both groups appeared the specific morphology with dish-like vesicles and double-lipid layer.Flow Nano Analyzer(FNA)showed that the diameter of serum exosomes in gastric cancer group was 90.07±34.85nm,and healthy control group was 91.19±32.84nm,which were similar in size.Exosome markers CD9,TSG101 and CD63 were detected in both groups by Western blot analysis.2.MT1-MMP mRNA exist in serum exosomeThe expression of MT1-MMP mRNA from exosomes in both gastric cancer group and health control group was little changed after treating with RNase A(all P>0.05),but was significantly decreased when Triton X-100 firstly penetrated exosomesand then treating with RNase A(all P<0.001).The significant decrease of MT1-MMP mRNA expression after exosome destruction indicated that MT1-MMP mRNA does exist in exosomes.3.The expression of serum exosomal MT1-MMP mRNA was not affected by storage time and temperatureSerum in healthy control group and gastric cancer group stored at room temperature(RT)or 4? for 12h to 24h or stored at-80? were freeze-thawed repeatedly for 1 to 3 cycles.Compared with the fresh serum collected after 2h,the expression of exosomal MT1-MMP mRNA in both group were not significantly changed after storage time and temperature changed(all P>0.05),confirming that exosomal MT1-MMP mRNA was stable in serum4.The expression of serum exosomal MT1-MMP mRNA was increased in gastric cancer.Compared the expression of serum exosomal MT1-MMP mRNA in the gastric cancer group with the healthy control group,the result showed that the gastric cancer group was significantly higher than the healthy control group(P<0.001).Conclusions:1.Extraction of exosome and exosome RNA in serum and determination of MT1-MMP mRNA in exosome were stable and reliable.The expression of serum exosomal MT1-MMP mRNA was not affected by storage time and temperature in both healthy control group and gastric cancer group.2.The expression of serum exosomal MT1-MMP mRNA in gastric cancer group was significantly higher than that in healthy control group,suggesting that exosomal MT1-MMP mRNA in the peripheral blood is of certain potential value in the diagnosis of gastric cancer.Part 2:The Clinical Significance of Serum Exosomal MT1-MMP mRNA inGastric CancerObjective:Study the difference of serum exosomal MT1-MMP mRNA expression in gastric cancer and gastric precancerous lesions,to discuss its clinical significance in the early evaluation of gastric cancer.Study the correlation between serum exosomal MT1-MMP mRNA and clinical stage,pathological characteristics in gastric cancer patients,to explore its clinical value as a biological marker of gastric cancer.Methods:1.214 cases in Qilu Hospital of Shandong University were enrolled in this study from January 2017 to December 2017,including 31 healthy controls,31 chronic atrophic gastritis,33 atypical hyperplasia and 119 gastric cancer.Healthy control group was from the health examination center.Chronic atrophic gastritis group,atypical hyperplasia group and gastric cancer group were all from hospitalized patients.2.Fasting serum of all participants was collected,and expression of serum exosomal MT1-MMP mRNA was analyzed by qRT-PCR according to the experimental method in the first part.Meanwhile,serum CEA,CA19-9 and CA72-4 were determined by electrochemical luminescence.3.Expression of serum exosomal MT1-MMP mRNA in gastric cancer group,atypical hyperplasia group,chronic atrophic gastritis group and healthy control group were compared and analyzed,to study the difference in gastric cancer and gastric precancerous lesions.4.Expression of serum CEA,CA19-9,CA72-4 in gastric cancer group,atypical hyperplasia group,chronic atrophic gastritis group and healthy control group were compared and analyzed.5.Study the correlation between serum exosomal MT1-MMP mRNA and CEA,and compare their value in the diagnosis of gastric cancer.6.Demographics and clinicopathological parameters of gastric cancer patients were collected and recorded,such as gender,age,tumor site,tumor diameter,differentiation,Borrmann type,invasion depth,lymphatic metastasis,distant metastasis,TNM stage and so on.TNM stage was performed according to the UICC/AJCC TNM staging system(2010).Summarized and analyzed the correlation between serum exosomal MT1-MMP mRNA and common clinicopathological variables in gastric cancer.Carried out logistic analysis on possible factors affecting lymph node metastasis in gastric cancer.7.Analyzed the correlation between serum exosomal MT1-MMP mRNA and clinical stage of gastric cancer.Serum exosomal MT1-MMP mRNA in different TNM stages of gastric cancer were compared to evaluate its significance in the clinical progress of gastric cancer.Results:1.The expression of serum exosomal MT1-MMP mRNA was significantly increased in gastric cancerThe expression of serum exosomal MT1-MMP mRNA in gastric cancer group was significantly higher than atypical hyperplasia group,chronic gastritis group and healthy control group(all P<0.05).Atypical hyperplasia group was significantly higher than chronic atrophic gastritis group and healthy control group(all P<0.05).Chronic atrophic gastritis group was significantly higher than healthy control group(P<0.05).2.The expression of serum CEA increased significantly in gastric cancerThe expression of serum CEA in gastric cancer group was significantly higher than healthy control,chronic atrophic gastritis and atypical hyperplasia group respectively(all P<0.05).But there was no significant difference in chronic atrophic gastritis,atypical hyperplasia and healthy control group respectively(all P>0.05).Moreover,the expression of serum CA19-9 and CA72-4 were not significantly different in all the four groups(all P>0.05).3.The value of serum exosomal MT1-MMP mRNA in the diagnosis of gastric cancer was superior to CEAROC analysis between exosomal MT1-MMP mRNA and CEA in gastric cancer showed that the AUC,sensitivity and specificity of serum exosomal MT1-MMP mRNA were higher than CEA.Furthermore,ROC analysis showed that the combination of MT1-MMP mRNA and CEA gave an AUC better than them alone,which can significantly improve the diagnosis of gastric cancer.In addition,there was no correlation between exosomal MT1-MMP mRNA and CEA(P>0.05).4.Correlation between serum exosomal MT1-MMP mRNA and clinicopathological features in gastric cancerThe expression of serum exosomal MT1-MMP mRNA was statistically correlated with tumor diameter,differentiation,Borrmann type,invasion depth,lymphatic metastasis,distal metastasis and TNM stage(all P<0.05).In the multiple logistic regression model,the higher expression of exosomal MT1-MMP mRNA was an independent risk factors of lymphatic metastasis(P=0.004).5.Correlation between serum exosomal MT1-MMP mRNA and clinical stage in gastric cancerThere was a significant correlation between the expression of serum exosomal MT1-MMP mRNA and TNM stage in gastric cancer(P<0.001),indicating that the up-regulation of MT1-MMP mRNA might be correlated with the clinical progression of gastric cancer.The expression of MT1-MMP mRNA in stage ? was significantly higher than stage ?,stage ? and stage ?(all P<0.05).Stage ? was higher than stage? and stage ?(all P<0.05).While stage ? was higher than stage ?(P<0.05).Conclusions:1.The expression of serum exosomal MT1-MMP mRNA in gastric cancer was significantly increased,and gastric cancer group>atypical hyperplasia group>chronic atrophic gastritis group>healthy control group,which has potential clinical significance for the early evaluation of gastric cancer.2.The value of serum exosomal MT1-MMP mRNA in the diagnosis of gastric cancer was better than CEA,CA19-9 and CA72-4.3.Serum exosomal MT1-MMP mRNA was associated with lymphatic metastasis in gastric cancer,and was an independent risk factor.4.Significant correlation was observed between exosomal MT1-MMP mRNA and TNM stage in gastric cancer.The expression of exosomal MT1-MMP mRNA was as follows:stage ?>stage ?>stage ?>stage ?,revealing that serum exosomal MT1-MMP mRNA might be a reliable biomarker for gastric cancer stage.Therefore,serum exosomal MT1-MMP mRNA plays an important role in the early evaluation and progression prediction of gastric cancer,and may become a potential and effective biological marker of gastric cancer. |
PDF Full Text Request