| Part I: The expression level of miR-214in gastric cancer cell lines, tissueand the relationship with clinicopathologic featuresObjective To explore the expression level of miR-214in gastric cancer cell lines and tissue,and evaluate the relationship between the expression level of miR-214and clinicopathologicfeatures in gastric cancer tissue, elucidate preliminary the miR-214function in the progessionof gastric cancer.Methods To detect the expression level difference by quantivative Real-Time PCR assaybetween gastric cancer tissue and paracancerous tissue, human gastric cancer cell linesSGC-7901, BGC-823and gastric normal mucosal cell line, respectively. To analyze therelationship between the expression level of miR-214and the clinicopathologic featuresincluding age, sex, histological type, T, N, M, clinical stage and prognosis byindependent-samples T test, Kaplan-Meier survival curve and Log-rank test.Results Compared to human gastric normal mucosal cell line GES-1, the2-△△Ctvalue ofmiR-214expression level in SGC-7901,BGC-823gastric cancer cell lines were(4.20±0.25)(P<0.01)and(3.3±0.24)(P<0.05), respectively, there was significant difference betweentwo groups; compared to gastric normal mucosal tissue, the2-△△Ctvalue of miR-214expression level in gastric cancer tissue was(4.22±1.45), there was significant differencebetween two groups (P<0.01); The expression level of miR-214in gastric cancer tissue wasrelated to tuomor invasion depth, more deeper of tumor invaded, more higher of miR-214expressed in tumor tissue, there was significant difference (P=0.001); it also related tometastasis and TNM stage, more higher of miR-214expressed in metastatic and more laterTNM stage tumor tissue, there was significant difference (P=0.005, P=0.001, respectively);The median survival time was2.7years in low miR-214expression group,1.4years in highmiR-214expression group, the risk ratio was1.929(95%CI:1.497-2.630), there wassignificant difference between two groups (P=0.012).Conlusions The expression level of miR-214is high in gastric cancer tissue and cell linescompare to paracancerous tissue and normal gastric mucosal cell line, and the expressionlevel is positively correlated to tumor invasion, metatasis, is negtively correlated to clinicalprognosis of patients.Part II: The effects of miR-214aberrant expression to gastric cancer cellline biological behaviors and cisplatin chemoresistance Objective To estimated the effects of miR-214aberrant expression to gastric cancer cell lineproliferation, apoptosis, migration, invasion and cisplatin chemoresistance.Methods Transfected miR-214mimics and inhibitor into BGC-823gastric cancer cell line bylipofectin-mediated transfection assay to upregulate and downregulate miR-214expressionlevel, determined BGC-823cell line proliferation change by MTT and clonogenic assay,detected cell cycle and apoptosisi change by flow cytometry, observed cell migration andinvasion change by Transwell assay, estimated cell cisplatin chemoresistance change bycalculating IC50via MTT assay.Results The miR-214mimics group OD values of BGC-823cell line were higher thanmimics control group, there was significant difference between two group each time point(P<0.05), The miR-214inhibitor group OD values of BGC-823cell line were lower thaninhibitor control group, there was significant difference between two group each time point(P<0.05); The miR-214mimics group PI and CFE values of BGC-823cell line were higherthan mimics control group, there was significant difference between two group (P=0.04,P=0.016, respectively), The miR-214inhibitor group PI and CFE values of BGC-823cellline were lower than inhibitor control group, there was significant difference between twogroup (P=0.02, P=0.042, respectively); The filter membrane and matrigel matrix penetrationcell count of miR-214mimics group were more than mimics control, there was significantdifference between two group (P=0.031, P=0.037, respectively), the filter membrane andmatrigel matrix penetration cell count of miR-214inhibitor group were less than inhibitorcontrol group, there was significant difference between two group (P=0.041, P=0.032,respectively); There was no significant between miR-214mimics and mimics control,miR-214inhibitor and inhibitor control group in cell apoptosisi (P>0.05); The IC50value ofmiR-214mimics and mimics control were16.26and8.63mg/L,95%CI were (11.82,20.14)and (5.63,11.49) mg/L, there was significant difference between two group (P<0.05), TheIC50value of miR-214inhibitor and inhibitor control were10.24and4.83mg/L,95%CIwere (6.64,13.64) and (3.28,6.49) mg/L, there was significant difference between two group(P<0.05).Conclusions The upregulation of miR-214expression level could promote gastric caner cellline proliferation, migration, invasion and cisplatin chemoresistance, as while thedownregulation of miR-214expression level could inhibit gastric caner cell line proliferation,migration and invasion, moreover, it could reduce the cisplatin chemoresistance for gastric cancer cell, but there was no effects on gastric cell line apoptosis.Part III: miR-214targeted PTEN gene expression and regulation functionsObjetive To predict and verify that PTEN is a target gene of miR-214, demonstrated theeffects of miR-214on the biological function of BGC-823gastric cancer cell line by targetedregulation to PTEN gene expression.Methods Predicted and screened the target gene according to miRNA target gene database ofbioinformatics, detected the PTEN mRNA and protein expression level change aftertransfection by qRT-PCR and Western-Blot assay, observed gastric cancer cell proliferation,migration, invasion and cisplatin resistance change after cotransfection of pcDNA-PTEN andmiR-214mimics, and verified by luciferase reporter gene assay.Results PTEN gene is potential target gene of miR-214by bioinformatics database. TheqRT-PCR results showed that there was no significant change of PTEN mRNA expressionlevel in gastric cancer cell line between miR-214mimics group and mimics control group,there was no significant differences between two group (P>0.05). The Western Blot assayresults showed that the PTEN protein expression level in gastric cancer cell line transfectedmiR-214mimcis was significantly lower than that transfected mimics control (P<0.01), thePTEN protein expression level in gastric cancer cell line transfected miR-214inhibitor wassignificantly higher than that transfected inhibitor control (P<0.05). The luciferase reportergene assay results showed that the luciferin protein expression level of cell lines transfectedPTEN3'-UTR+miR-214mimics recombinant plamid significantly reduced, the luciferinprotein expression level of gastric cancer cell lines transfected PTEN3'-UTR+miR-214inhibitor recombinant plamid significantly increased (P<0.05). The PI value in gastric cancercell lines transfected miR-214mimics+pcDNA-PTEN (40.97±4.12)%was significantly lowerthan that transfected miR-214mimics+pcDNA (53.89±4.87)%, there was significantdifferences between two group (P=0.025). The CFE value in gastric cancer cell linestransfected miR-214mimics+pcDNA-PTEN (62.33±5.13)%was significantly lower than thattransfected miR-214mimics+pcDNA (47.67±6.43)%, there was significant differencesbetween two group (P=0.037). The cell count penetrated filter membrane of miR-214mimics+pcDNA-PTEN group was significantly less than that in miR-214mimics+pcDNA,cell count were (81.00±9.54)/HPF and (107.33±11.37)/HPF, respectively, there wassignificant differences between two group (P=0.037). The cell count penetrated matrigelmatrix of miR-214mimics+pcDNA-PTEN group was significantly less than that in miR-214 mimics+pcDNA, cell count were (39.00±6.56)/HPF and (24.33±5.69)/HPF,respectively,there was significant differences between two group (P=0.043). The gastric cancer inhibitionrate for cisplatin of miR-214mimics+pcDNA-PTEN group and miR-214mimics+pcDNAgroup were (22.01±4.55)%and (24.04±5.21)%, respectively, and there was no significantdifferences between two groups (P=0.64).Conclusion miR-214influenced the proliferation, migration and invasion of gastric cancercell lines by target regulation of PTEN protein expression on gene translation level, but themolecular mechanism of gastric cancer cisplatin resistance was unclear.
|
PDF Full Text Request