| BackgroundGastric cancer(GC)is one of the most frequently diagnosed cancers worldwide and remains the major contributor to global burden of health.Even though diagnostic approaches and treatment strategies has been improved gradually,the prognosis of GC patients in advanced stages is still dismal.Thus,elucidations of the causative mechanisms behind tumorigenic and metastatic processes of GC are desperately needed.MicroRNAs represent an endogenous group of small non-coding RNAs that post-transcriptionally repress the expression of protein-coding gene.By complementally binding with corresponding 3' untranslated region(3'UTR)of m RNA transcribed from protein-code gene,miRNAs serve as specific repressor of translation or cleavage inducer of RNA transcripts.It is estimated miRNAs regulate up to 60% of human protein-coding genes.Specific subsets of miRNAs or miRNAs clusters have been reported to be dysregulated in various tumors.Recently,the role of miR-140-5p has attracted wide interest.miR-140-5p has been characterized as a tumor suppressor in colorectal cancer,biliary tract cancer and hypopharyngeal cancer.In GC,high miR-140-5p level was detected in the plasma of patients on the one hand,while low miR-140-5p level was defined in 20 tissue samples and one cell line HGC-27 on the other hand.However,the prognostic relevance and functions of miR-140-5p in GC are largely unknown.Noteworthy,given the variety of protein-code genes targeted by one miRNA,the underlying mechanisms behind miR-140-5p mediated phenotypic changes of GC cells remain to be deciphered.Aberrant signaling events frequently occur in human malignancies and contribute immensely to the processes of oncogenesis.The members of Src family tyrosine kinase(SFKs)are prototypical non-receptor protein tyrosine kinases.Up-regulated Src signaling,with proportional increase to the progressive TNM stages,has been reported in a variety of human cancers.YES proto-oncogene 1(YES1),a notable member of SFKs,serves as seminal regulator of cell growth,adhesion and differentiation.The central role of YES1 involved in tumor progression is evidenced by the fact that decreased YES1 expression correlated with impaired growth abilities of several malignancies including malignant mesothelioma,rhabdomyosarcoma and pancreatic cancer.Moreover,YES1 confers chemotherapy resistance in several cancers.For instance,YES1 is able to promote the nuclear translocation of epidermal growth factor receptor which induces the resistance to cetuximab in non-small cell lung cancer line NCI-H226.However,previous studies remain controversial on the expression level of YES1 in GC tissues.Seki et al observed the up-regulation of YES1 in GC tissues compared to adjacent normal tissues,whereas Wang et al drew an opposite conclusion.In addition to these inconsistent reports,the reasons behind the aberrant expression of YES1 in GC are currently unclear.Herein,we aimed to evaluate the prognostic relevance of miR-140-5p in GC and identify whether YES1 is direct target of miR-140-5p in the regulation of tumor progression.Material and methods1 miR-140-5p level was determined in 20 paired frozen specimens using quantitative real-time PCR,and analyzed by in situ hybridization in tissue microarrays from 144 GC patients and 43 normal gastric mucosa tissues from benign disease.2 Database Targetscan was retrieved to search for the potential target of miR-140-5p.and YES1 protein level was determined in AGS and BGC823 cells by western blotting after the transfection of miR-140-5p mimics or control mimics.A dual luciferase reporter assay was applied to verify the target of miR-140-5p.3 The phenotypic changes of GC cells treated with miR-140-5p mimics were determined by MTT,colony formation,wound healing and transwell assays.4 The proliferation,migration and invasion activities of GC cells were evaluated after the transfection of YES1 over-expressing vectors together with miR-140-5p mimics.5 The mouse xenograft model was established in 6 week-old BALB/c-nu mice,and BGC823 cells were injected subcutaneously into the left dorsal flanks of mice.After the treatment,the volumes of tumors were measured every three days and the mice were weighed every four days.On day 19 the mice were sacrificed and the tumors were dissected,weighed and photographed.Tumor tissues were fixed for further immunohistochemical staining of YES1 protein.Results1 miR-140-5p is down-regulated in GC tissues and correlates with overall survival of patients.We investigated miR-140-5p expressions in 20 fresh GC tissues and matched adjacent normal tissue by q RT-PCR and found the expressions of miR-140-5p were significantly down-regulated in tumor tissue(P<0.001).Subsequent in situ hybridization analysis of miR-140-5p levels in 144 GC tissues and 43 normal gastric tissues revealed similar results.Staining intensities of miR-140-5p were reduced in 32.6% of GC tissues(47 of144),while only 11.6%(5 of 43)of normal gastric tissues showed down-regulations (P=0.007).Cox proportional hazards regression model were established to explore the prognostic significance of miR-140-5p and other clinical parameters.The results showed that beside parameter of tumor differentiation,invasion depth and lymph node metastasis,miR-140-5p level was another independent favorable factor for predicting overall survival.Further correlation analysis showed that down-regulated miR-140-5p correlated with reduced overall survival of GC patients(P=0.0084).Specifically,the patients with lower miR-140-5p expressions had shorter median survival time(20months)than those with higher miR-140-5p expressions(59 months).2 miR-140-5p directly targets one evolutionary conserved sequence within 3'UTR of YES1To explore the potential target of miR-140-5p,a combination of prediction database(Target Scan,miRDB,ComiR)was applied and YES1 was shown to be possibly targeted.We thereafter utilized a panel of five GC cell lines and one normal gastric cell line to verify the prediction.We first screened the expression of miR-140-5p in these cell lines.Then a conversely correlated relation between the miR-140-5p level and YES1 protein expression was confirmed in AGS and BGC823 cells(P<0.05).Next,we performed luciferase reporter assay to investigate whether 3?-UTR of YES1 gene was direct target of miR-140-5p in gastric cancer.Transient transfection of AGS cells with miR-140-5p together with YES1 3?-UTR plasmids led to a significant decrease in relative luciferase intensity compared with miRNA control.While vector contained site mutated 3?-UTR sequence was not affected by miR-140-5p.These results indicated 3?-UTR of YES1 is a direct target for miR-140-5p.3 miR-140-5p suppresses cell proliferation,migration and invasion in gastric cancer in vitroTo determine the effects of miR-140-5p on GC cells proliferation,we performed MTT assay and colony formation assay.In AGS and BGC823 cells,over-expression of miR-140-5p by transfection significantly suppressed the proliferation ability of GC cells over time as compared with control groups in vitro.For colony formation assay,AGS and BGC823 transfected with miR-140-5p showed remarkable decrease of colony formation ability,as compared with control groups.Next,we investigated whether miR-140-5p affected GC cell migration and invasion.Wound healing assay was conducted and miR-140-5p over-expressing cells were less proficient than non-transfected cells in closing an artificial wound over a confluent monolayer.In addition,we examined the impact of miR-140-5p on GC cell invasion by Transwell assay.We found that numbers of GC cells invading through the Matrigel were dramatically decreased after miR-140-5p administration compared with the negative control.These results indicated that miR-140-5p attenuated the migration and invasion of GC cells.4 Reconstitution of YES1 rescues miR-140-5p mediated inhibition in GC cellsTo examine whether miR-140-5p exerts inhibitory effects on GC cells through YES1,we transected constructed YES1 expression vector together with miR-140-5p.Subsequent MTT and colony forming assay revealed that reintroduction of YES1 restored miR-140-5p mediated growth repression of GC cells.Likewise,the repressive impact of miR-140-5p on GC cell migration and invasion can also be rescued by reconstitution of YES1.Collectively,these data provided the evidence that miR-140-5p repressed cell proliferation,migration and invasion of GC cells partially through inhibiting YES1.5 miR-140-5p suppresses the growth of BGC-823 xenograft tumorsBased on the results in vitro,we further explored the inhibitory potential of miR-140-5p on GC growth in vivo.BGC-823 cells were subcutaneously implanted into the flank of nude mice and the size of the xenograft tumors was measured after the intratumoral injection of miR-140-5p mimics or control mimics with a blank control.The growth curve showed a tendency towards lower tumor volumes in miR-140-5p group compared with the control groups.Subsequently,xenograft tumors were harvested and weighted as soon as the mice were sacrificed.Consistent with the tumor volume,the mean mass of the tumors was significantly lighter in miR-140-5p group than in control groups.We next examined YES1 level in tumor tissues by immunohistochemistry staining.In line with the in vitro results,YES1 protein staining was obviously weaker in miR-140-5p treated tumors compared to the control tumors.ConclusionsmiR-140-5p serves as potential prognostic factor for GC patients and inhibits tumorigenesis of GC by directly targeting YES1.Our findings will provide further insight to the pathogenesis of GC and may open new avenues for the improvement of therapeutic approaches in this tumor entity. |
PDF Full Text Request