| Gastric cancer is one of the most common malignant tumors in China.The incidence of gastric cancer in malignant tumors is the second place. It isthe third leading cause of cancer-related death in2009. Gastric cancer has thehigh incidence, the low rate of early diagnosis and the5-year survival in China.The early diagnostic rate of gastric cancer is still hovering around10%-20%,which is also the main reason of high mortality rate in China. At present,surgery is the only method to be proven to cure gastric cancer. But even if theyhad accepted the radical operation, most of patients with gastric cancer wouldinduce recurrence or metastasis. Postoperative adjuvant radiation andchemotherapy may be available to control the recurrence or metastasis ofgastric cancer. Chemotherapy plays an important role in comprehensivetreatment of advanced gastric cancer, but the drug resistance of chemotherapyhas become a big bottleneck. Therefore, we concluded that the early diagnosisand timely effective treatment is the key to improve the5-year survival rate ingastric cancer, so it is the most crucial task to look for a sensitive and specificmolecular markers, clarifing molecular targets of treatment and correctingprognosis assessment. MicroRNAs （miRNAs） are a new class of endogenousand non-coding small RNA, the length of which is approximately18-25nucleotides. These miRNAs account for only1%of human genes, but theymay regulate more than a third of gene expression, modification, transcriptionand translation in human. These miRNAs may have the potential applicationvalue of molecular markers, for the early diagnosis and prognosis assessmentof malignant tumor to open up a new road. Currently, the miRNA-200familyhas become the focus of miRNAs study. The family includes five members,which are miRNA-200a, miRNA-200b, miRNA-200c, miRNA-141andmiRNA-429. They have several biological characteristics, including the highlyconservative, gene cluster phenomenon, timing, and tissue specificity. Some studies have shown that the passive expression alone of each member in themiRNA-200family can prevent epithelial mesenchymal transformation （EMT）change, while they can adjust ZEB1and SIP1（ZEB2） expression if they worktogether, but specific role of the family in human malignant tumor has notbeen clarified. In this study, we detected the expression level of themiRNA-200family in gastric cancer tissue and adjacent normal tissue by thereal-time quantitative PCR technique, and analyzed the correlation betweenthe expression level and clinical pathological parameters of gastric cancer. Weselected the miRNA-200c, which is the representative members of the family,as study subject. We detected the expression level of miRNA-200c in4casesof gastric cancer cells using real-time quantitative PCR, and adopted themethod of transient transfection to transfer the miRNA-200c mimics ornegative control into the gastric cancer cell lines in order to obtain highexpression cell model of the miRNA-200c. In order to understand themiRNA-200c effects on gastric cancer cell biological behavior, and to furtherclarify the role of miRNA-200c in gastric cancer occurrence and development,we explored the proliferation of tumor cells by MTT method, detected the cellcycle change using flow cytometry instrument, and studied the tumor cellmigration ability by wound healing assay. Then we screened the potentialtarget genes by bioinformatics software, and at the same time used dualluciferase report gene method to validate the candidate target gene, forintensive study the molecular mechanism of the miRNA-200c.Part One The expression level of the miRNA-200family and thecorrelation with clinical pathological parameters in gastric cancerObjective: To explore the expression level of the miRNA-200family ingastric cancer tissue and adjacent normal tissue, and to analyze the correlationbetween the expression level and clinical pathological parameters of gastriccancer.Methods: The expression level of the miRNA-200family was detectedby real-time quantitative PCR in46cases of gastric cancer tissues andadjacent normal tissues. The correlation between the expression level of cancer tissues and clinical pathological parameters of gastric cancer wereanalyzed by independent-samples T tests and one-way ANOVA.Results:1We detected the expression level of the miRNA-200family in46casesof gastric cancer tissues and adjacent normal tissues by real-time quantitativePCR. Through the analysis of data, the results demonstrated that five membersof miRNA-200family, including miRNA-200a, miRNA-200b, miRNA-200c,miRNA-141and miRNA-429, were all significantly lower in cancerous tissuesthan those in the corresponding normal tissues （P<0.05）. Among46patientswith gastric cancer,86.96%（40/46）,91.30%（42/46）,93.48%（43/46）,93.48%（43/46） and80.43%（37/46） cases were showed low expression of themiRNA-200family, respectively.2We analyzed the correlation between the expression level of cancertissues and clinical pathological parameters of gastric cancer byindependent-samples T tests and one-way ANOVA. Medians of the relativeexpression values and clinicopathological factors were presented in Table. Thestatistical analysis showed that the expression of five members wereassociated with histologic grade and intravascular cancer embolus （P<0.05）.However, no association was observed about gender, age, tumor size, lymphnode metastasis, invasive depth, TNM stage, Lauren type and Borrmanntype（P>0.05）.Conclusion:1The expression level of the miRNA-200family was down-regulated ingastric cancer tissues compared with adjacent normal tissues, suggesting thatthe down-regulated of the miRNA-200family might become a therapeutic andpreventive tool in patients with gastric cancer, but might not be a promisingbiomarker in the early diagnosis of gastric cancer.2Lower levels of five members were associated with histologic gradeand intravascular cancer embolus, suggesting that the five members of themiRNA-200family might play an important role in the occurrence anddevelopment of gastric cancer. Part Two The effects of the miRNA-200c on biological behaviors inhuman gastric cancer cell lineObjective: The miRNA-200c, which is the representative members of thefamily, was as the study subject. To estimate the effects of the miRNA-200family on biological behaviors in human gastric cancer cell line, including thecell proliferation, the cell cycle change and the tumor cell migration ability.Methods: The expression level in4cases of gastric cancer cell lines weredetected by real-time quantitative PCR, and the transient transfection wasadopt to transfer the miRNA-200c mimics or negative control into the gastriccancer cell lines in order to obtain high expression cell model of themiRNA-200c. The proliferation of tumor cells was explored by MTT, the cellcycle was detected using flow cytometry, and the tumor cell migration abilitywas studied by wound healing assay.Results:1We detected the expression level of miRNA-200c in4cases of gastriccancer cell lines, including MGC803, HGC27, SGC7901and AGS. Comparedwith adjacent normal tissue, the results showed that the miRNA-200c wassignificantly lower in MGC803, HGC27, SGC7901and AGS. The mediandecreased about9.93,9.69,7.18and2.93folds （P<0.0001）.2The SGC7901cell was found to be a suitable cell model in vitro. Wedetected the expression level of miRNA-200c in SGC7901cell transferred themiRNA-200c mimics or negative control after48hours. The results displayedthat the expression level of miRNA-200c transferred the miRNA-200c mimicswas significantly higher than those in the negative control and blank controlgroup, with the change of median about10.2folds （P<0.05）. These suggestedthat the transient transfection of miRNA-200c was successful and could beused in the next studies.3The MTT assays showed that24h,48h and72h after transfection ofthe miRNA-200c mimics or negative control in SGC7901cell, the cellproliferation in miRNA-200c mimics group was significantly inhibitedcompared to the negative control group（P<0.05）. The OD values of miRNA-200c mimics group were0.308±0.010,0.360±0.016and0.457±0.029,while the OD values of negative control group were0.339±0.008,0.505±0.006and0.775±0.010. In three time points after transfection, the rateof inhibition in miRNA-200c mimics group were10.76±2.50%,30.83±2.58%and33.86±15.9%, while the rate of inhibition in negative control group were1.78±2.70%,3.01±2.15%and0.64±0.69%. They had significant difference ineach time point （P<0.05）.4The flow cytometry showed that48h after transfection of themiRNA-200c mimics or negative control in SGC7901cell, the cell cycle ofmiRNA-200c mimics group was significantly changed compared to thenegative control group（P<0.05）. The ratio in G0/G1phase, S phase and G2/Mphase of miRNA-200c mimics group were72.77±0.85%,8.87±0.45%and18.36±1.29%, while the ratio of negative control group were42.10±0.80%,43.57±1.27%and14.33±2.06%, respectively. There was significant differencein G0/G1phase and S phase （P<0.05）. The G0/G1phase arrest suggested thatover-expression of miRNA-200c in SGC7901cell could inhibit the cell cyclefrom G0/G1phase to S phase.5The wound healing assays showed that24h after transfection of themiRNA-200c mimics or negative control in SGC7901cell, the tumor cellmigration ability of miRNA-200c mimics group was significantly decreasedcompared to the negative control group（P<0.05）. The width in miRNA-200cmimics group was344.40±17.24μm, while the width in negative control groupwas270.51±18.21μm. There was significant difference between miRNA-200cmimics group and negative control group （P<0.05）.Conclusion:1The expression level of miRNA-200c in4cases of gastric cancer celllines, was significant down-regulated. The results were consistent with theexpression in gastric cancer tissue.2The transient transfection of miRNA-200c was successful andup-regulated the expression level of miRNA-200c in SGC7901cell.3The over-expression of miRNA-200c in SGC7901cell inhibited the cell proliferation, suggesting that the down-regulation of miRNA-200c might playa role in molecule regulation of cell proliferation.4The over-expression of miRNA-200c in SGC7901cell inhibited the cellcycle from G0/G1phase to S phase, suggesting that the down-regulation ofmiRNA-200c might inhibit the cell proliferation through cell cycle change.5The over-expression of miRNA-200c in SGC7901cell decreased thetumor cell migration ability, suggesting that the down-regulation ofmiRNA-200c might affect the cell migration ability and could provide theproof for searching the molecular targets of treatment in gastric cancer.Part Three The prediction and verification of miRNA-200c target genesObjective: To predict and verify that RhoE is a target gene ofmiRNA-200c. To make a foundation for further research on moleculemechanism of miRNA-200c.Methods: The potential target gene was predicted and screenedaccording to the bioinformatics software, and at the same time the candidatetarget gene was verified by dual luciferase report gene assay.Results:1We used the bioinformatics software （TargetScan, PicTar, mirBase andmiRanda） to predict and screen the potential target gene of miRNA-200c. Theresults showed that miRNA-200c belongs to the chromosome12and islocated to12:7072862-7072929[+]. There were1057and639target genes ofmiRNA-200c by TargetScan and PicTar bioinformatics software.Comprehensive analysis we chose RhoE （NM005168） as the research subject.We found that RhoE3’UTR has two binding sites of miRNA-200c, so we willbuild the report carrier of RhoE3’UTR and verify the target gene by dualluciferase report gene assay.2We verified that the RhoE is a target gene of miRNA-200c by theclassical method. Firstly, the RhoE3’UTR vector and RhoE mut3’UTRvector were successfully constructed. We divided into four groups throughtransferring the RhoE3’UTR/RhoE mut3’UTR and miRNA-200c mimics/miRNA-200c NC in SGC7901cell. They were RhoE3’UTR+miRNA-200c mimics group, RhoE3’UTR+miRNA-200c NC group, RhoE mut3’UTR+miRNA-200c mimics group and RhoE mut3’UTR+miRNA-200c NC group.The dual luciferase report gene assays showed that48h after transfection, theluciferin protein expression level of RhoE3’UTR+miRNA-200c mimicsgroup was reduced compared to other groups （P<0.05）. The results showedthat miRNA-200c could inhibit the activity of RhoE3’UTR, suggesting thatRhoE was the target gene of miRNA-200c.Conclusion:The miRNA-200c could inhibit the activity of RhoE3’UTR, verifyingthat RhoE was the target gene of miRNA-200c. This suggested thatmiRNA-200c has the inhibitory function to RhoE, and may exert its biologicalbehaviors by RhoE. Thus they may affect the occurrence, development,invasion and metastasis of gastric cancer. This provide a foundation for furtherclarify the molecule mechanism of miRNA-200c in gastric cancer. |
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