| ?Background and objective? Cell migration and metastasis contribute to the high mortality rate and poor prognosis of colorectal cancer(CRC).The mechanisms of migration and metastasis are complex,involving multiple genes,several factors,and different pathways.The epithelial-tomesenchymal transition(EMT),one of the crux pathways,mediates neoplastic cell dissociation from primary lesions to distant organs,promoting cancer cell migration.Therefore,the EMT process is an important therapeutic target in CRC for inhibiting cancer cell migration.Mi RNAs,a class of small non-protein coding RNAs(19-25 nucleotides long),play an important role in post-transcriptional regulation of gene expression by binding to the 3'-untranslated region(3?UTR)of m RNAs.Many mi RNAs involved in the EMT process have been identified.Mi R-1254 is a novel mi RNA and its expression or function is controversial in different cancers.These observations imply that mi R-1254 may serve the function in a cell-type specific manner,and the role of this mi RNA in colorectal cancer remains unknown.In addition,the detailed molecular pathway between mi R-1254 and cell migration remains elusive.In the current study,we investigate the function and relative mechanism of mi R-1254 in CRC cell migration.The expression of mi R-1254 in CRC tissues helped to evaluate the viability as the diagnostic marker and targeted therapeutics for metastasis.?Methods? 1.Detection of differential mi R-1254 expression in CRC and adjacent tissues.After total RNA was isolated from 21 cases of colorectal cancer and adjacent tissues,the expression of mi R-1254 was detected by real-time PCR.2.Investigation for the function of mi R-1254 in CRC.To investigate the function of mi R-1254 in CRC,by using transwell assay for cell migration,CCK8 assay for cell growth and flow cytometry for cell cycle.3.Verification of the direct regulation of mi R-1254 to PSMD10.Based on the results of RNA-seq and bioinformatics prediction,the possible target gene of mi R-1254 was PSMD10.Their relationship was verified by real-time PCR and western blot.The reporter plasmids containing the binding site and mutant site of PSMD10 3' UTR to mi R-1254 were constructed for the dual luciferase reporter assay.After mi R-1254 mimics and either report plasmid were co-transfected into HEK-293 T cell,luciferase activity was detected to confirm that PSMD10 was the target gene of mi R-1254.4.Mi R-1254 inhibits the migration of CRC cells by regulating PSMD10.The expression of PSMD10 in colorectal cancer and adjacent tissues was detected by real-time PCR and also was correlated with the expression of mi R-1254.PSMD10 was silenced in colon carcinoma cells to determine its contribution to CRC migration by using transwell assay.Moreover,PSMD10 was also overexpressed in colon carcinoma cells overexpressing mi R-1254 to determine whether their interaction contributed to CRC migration via transwell assay and the changes of EMT markers via real-time PCR and western blot.5.Exploration of the differentially expressed genes and pathways regulated by mi R-1254 in CRC After transfection of mi R-1254 mimics or NC in SW1116,total RNA was extracted for RNA-seq analysis,the results of which were analyzed by bioinformatics for differentially expressed genes(DEGs)and pathways involed and also PPI network as well.6.Statistical analysis The statistical analysis was performed in SPSS 21.0.The comparison of normally distributed data between paired subjects was conducted using Student's t-test.Data not normally distributed were compared using the Mann-Whitney tests.The relevant analysis for mi R-1254 and PSMD10 expression was performed using Pearson's correlation analysis.A P <0.05 was considered statistically significant.?Results? 1.Mi R-1254 levels were much lower in CRC lesions than in its adjacent tissues.Mi R-1254 was down-regulated in 80.95%(17/21)of CRC tissue specimens compared with its expression in adjacent tissues,whereas mi R-1254 levels remained unchanged in 19.05%(4/21)of CRC tissue specimens.2.Mi R-1254 inhibited cell migration but not growth of CRC cells.Mi R-1254 mimics and its control were transfected into the colon adenocarcinoma cell lines SW1116 and HCT116.Transwell assay showed that mi R-1254 overexpression inhibited the migration of colon adenocarcinoma cells(P <0.001 and <0.05,respectively).However,exogenous mi R-1254 delivery had no effect on the proliferation and cell cylce of either cancer cell line.3.Mi R-1254 down-regulated the expression of PSMD10.Real-time RT-PCR and western blot results revealed that PSMD10 was downregulated in response to exogenous mi R-1254 delivery(P<0.01).4.Up-regualted mi R-1254 resulted in the reduction of luciferase activity in cells with PSMD10 3?UTR reporter plasmids.The plasmids were co-transfected with mi R-1254 mimicss or NC,and the results showed a 58.5% reduction of luciferase activity in cells simultaneously transfected with PSMD10 3?UTR reporter plasmids and mi R-1254 mimics(P<0.05).In contrast,in cells transfected with plasmids containing a mutated version of the mi R-1254 binding site of PSMD10 3?UTR,mi R-1254 mimics failed to suppress luciferase activity.These results indicated that mi R-1254 bound PSMD10 3' UTR to regulate its expression.5.There was a negative correlation between mi R-1254 levels and PSMD10 transcription in CRC.PSMD10 was up-regulated in 76.19%(16/21)of CRC tissue specimens compared with its expression in adjacent tissues,and we observed a negative correlation between mi R-1254 levels and PSMD10 transcription(P<0.001).6.Down-regulated PSMD10 inhibited migration of CRC cells,whereas overexpression of PSMD10 attenuated the anti-migration activity of mi R-1254.Lentivirus-mediated PSMD10 knockdown significantly impaired the migratory behavior of the colon adenocarcinoma cell lines SW1116 and HCT116(P<0.001).In addition,overexpression of PSMD10 in colon adenocarcinoma SW1116 cells promoted cell migration and attenuated the anti-migration activity of mi R-1254 7.The regulatory effect of mi R-1254 and PSMD10 on cancer cell migration is mediated by EMT.Mi R-1254 overexpression not only promoted the expression of E-cadherin,a marker of the epithelial phenotype,but also down-regulated N-cadherin,vimentin,and Snail,which are indicators of the mesenchymal phenotype,at the RNA and protein levels.Additionally,we transfected the CRC cell line with mi R-1254 mimics followed by overexpressed PSMD10,and we found that PSMD10 overexpression significantly decreased mi R-1254-induced changes in the m RNA transcript and protein levels of all EMT indicators analyzed.8.The differentially expressed genes and pathways involved regulated by mi R-1254 in CRC.The RNA-seq results showed 217 DEGs,including 121 down-regulated genes and 96 up-regulated ones.According to the bioinformatics,DEGs were enriched in p53 signaling pathway,aminoacyl-t RNA biosynthesis,endocytosis,N-glycan biosynthesis and glycine,serine and threonine metabolism.And also PPI was analyzed to find the top 5 hub nodes including UMPS(Uridine 5'-monophosphate synthase),EGR1(Early growth response protein 1),CDKN1A(Cyclin-dependent kinase inhibitor 1),YARS(Tyrosine--t RNA ligase)and ASNS(asparagine synthetase).These results could be helpful to the further study on CRC carcinogenesis and clinical diagnosis and treatment.?Conclusions? Mi R-1254 bound PSMD10 to inhibit CRC cells migration mediated by EMT. |
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