Low Expression Of Mir-30a-5p Induced The Proliferation And Invasion Of Oral Cancer Via Promoting The Expression Of FAP

Part 1 Study on the expression of miR-30a-5p and FAP in oral cancer tissues and their relationshipObjective:Discussion and analysis of the expression of miR-30a-5p and FAP in oral cancer tissue and their relationshipMethods:66 oral cancer tissues and 25 adjacent normal tissues(at least 2-3 cm from the tumor margin,verified to be free of tumor)were obtained from surgical resection.Inclusion criteria were applied as described in previous study(20).In brief,patients were diagnosed with cancer of the oral cavity but did not receive radiotherapy and chemotherapy before.Written consents were confirmed.Two cohorts of human oral cancer collected at Renmin Hospital of Wuhan University in the year of 2015.All clinical specimens preserved in liquid nitrogen until RNA extraction.This study was approved by Renmin Hospital of Wuhan University and all participants signed an informed consent agreement.All clinical information for human oral cancer tissues was presented,The target relationship between miR-30a-5p and FAP was verified by dual luciferase reporter assay and biotin conjugated miRNA pull-down test(in vitro protein binding assay)..Results:On average,miR-30a-5p expression in cancer tissues was 0.35 times as that of adjacent tissues while FAP mRNA expression in cancer tissues was 3.3 times higher than adjacent tissues(P<0.05).Besides,miR-30a-5p and FAP were negatively correlated in adjacent tissues as well as in OSCC cells(P<0.05).Western blot confirmed that FAP was high expressed in cancer tissues.Besides,FAP was also high expressed in different OSCC cell lines Tca-8113,SCC-4,SCC-15,SCC-25 compared with normal cell line NHOEC.We further detected RNA expression of miR-30a-5p in different cell lines,among which Tca-8113 and SCC-15 cells showed lowest expression.And mRNA level of FAP in different cell lines was most highly upregulated in Tca-8113 and SCC-15 cells.Targetscan(http://www.targetscan.org/)predicted the targeting sites for miR-30a-5p and FAP 3'-UTR,and HEK-293T cells,which served as a vector,were co-transfected with pGL-3FAP-WT,pGL-3FAP-MUT,miR-30a-5p mimics and mimics control.The relative luciferase activity of pGL-3-FAP-WT group was significantly lower than pGL-3-FAP-MUT group,indicting a direct target relationship between miR-30a-5p and FAP(P<0.01).In addition,HEK-293T cells stably expressing FAP after FAP cDNA transfection were transiently transfected with biotinylated miR-30a-5p(Bi-miR-30a-5p)or biotinylated nonspecific miRNA(Bi-NC).RT-qPCR analysis of FAP mRNA level indicated FAP mRNA was 3.5 times higher than that in bi-NC group(normalized as 1)(P<0.05),further verifying the target relationship.Cells were divided into eight groups:control(no treatment),NC(LipofectamineTM2000 treatment),mimics(miR-30a-5p mimics),inhibitor(miR-30a-5p inhibitor),FAP(pcDNA3.1-FAP),siFAP(FAP siRNA),mimics+siFAP(miR-30a-5p mimics and FAP siRNA)and mimics+FAP(miR-30a-5p mimics and pcDNA3.1-FAP),each group was compared with the control(normalized as 1).At 48h after transfection,miR-30a-5p mRNA expressions drastically increased in miR-30a-5p mimics group,miR-30a-5p mimics+siFAP group and miR-30a-5p mimics+FAP group(all P<0.01)while decreased in miR-30a-5p inhibitor group(P<0.05).FAP mRNA expressions significantly increased in miR-30a-5p inhibitor group and FAP group while decreased in mimics group,siFAP group and miR-30a-5p mimics+siFAP group(all P<0.01).Protein expression changes in miR-30a-5p mimics+siFAP and miR-30a-5p mimics+FAP group also confirmed that FAP siRNA or cDNA had no effect on miR-30a-5p expression but conversely miR-30a-5p could decrease FAP expression.Conclusion:MiR-30a-5p was low expressed while FAP was high expressed in oral cancer,MiR-30a-5p directly targeted at FAP and suppressed its expressionPart 2 The effect of miR-30a-5 and FAP on cell proliferation,invasion and migration in oral cancerObjective:To investigate the effect of miR-30a-5 and FAP on cell proliferation,invasion and migration in oral cancer..Methods:66 oral cancer tissues and 25 adjacent normal tissues(at least 2-3 cm from the tumor margin,verified to be free of tumor)were obtained from surgical resection.Inclusion criteria were applied as described in previous study(20).In brief,patients were diagnosed with cancer of the oral cavity but did not receive radiotherapy and chemotherapy before.Written consents were confirmed.Two cohorts of human oral cancer collected at Renmin Hospital of Wuhan University in the year of 2015.All clinical specimens preserved in liquid nitrogen until RNA extraction.This study was approved by Renmin Hospital of Wuhan University and all participants signed an informed consent agreement.All clinical information for human oral cancer tissues was presented.After transfection in Tca-8113 cells and SCC-15 cells,MTT,colony formation,Transwell assay and wound healing assay were performed to study how miR-30a-5p and FAP regulate proliferation,invasion and migration respectively.Results:MTT assay revealed that miR-30a-5p mimics,FAP siRNA,miR-30a-5p mimics+ FAP siRNA could significantly reduce the cell viability of both Tca-8113 and SCC-15 cells,but miR-30a-5p inhibitor and FAP could both increase the cell viability.Therefore,FAP may accelerate cell proliferation which could reversely be reduced by miR-30a-5p.Clone formation assay validated the same conclusion considering smaller colony number in miR-30a-5p mimics,FAP siRNA,miR-30a-5p mimics+ FAP siRNA groups and larger colony number in miR-30a-5p inhibitor and FAP groups.Smaller wound healing area indicated stronger migration ability,therefore,miR-30a-5p mimics,FAP siRNA,miR-30a-5p mimicsH+ FAP siRNA groups shared weaker migration ability while miR-30a-5p inhibitor and FAP group shared stronger invasion ability(all P<0.01,Figure 4A).Invasion ability was valued by invading cells in Tca-8113 and SCC-15 cell lines.As shown in Figure 4B,miR-30a-5p mimics,FAP siRNA,miR-30a-5p mimics+ FAP siRNA groups showed weaker invasion ability while miR-30a-5p inhibitor and FAP groups displayed stronger invasion ability.Conclusions:MiR-30a-5p suppressed the cell proliferation,migration and invasion of oral cancer cells via downregulating FAP...