| Objective: To investigate the regulation of lnc RNA GAS5 on mi R-135b-5p and its role on EMT and migration of cervical cancer Hela cells.Methods:(1)Lnc RNA GAS5 affects the occurrence and migration of EMT in cervical cancer Hela cells: real-time quantitative q RT-PCR analysis of Lnc RNA GAS5 expression in human immortalized epithelial cells Hacat and cervical cancer Hela cells;constructing pc DNA3.1 GAS5 eukaryotic expression plasmid and then transfected into cervical cancer Hela cells,using Western Blot and q RT-PCR to detect changes about EMT-related protein E-cad,N-cad,vimentin expression;Applying Transwell technique to detect Lnc RNA GAS5 high expression on Hela cell migration The impact of capacity.(2)Lnc RNA GAS5 affects EMT changes in cervical cancer Hela cells by regulating the expression of mi R-135b-5p: Star Base v2.0 database for mi RNAs related to Lnc RNA GAS5,bioinformatics cluster analysis for mi RNAs closely related to EMT and migration mi R-135b-5p was screened;q R-PCR was used to detect the expression level of mi R-135b-5p after transfection of pc DNA3.1 GAS5,and pc DNA3.1 GAS5 was detected by luciferase reporter system which analyze whether Lnc RNA GAS5 can regulate the expression of mi R-135b-5p.Transfected mi R-135b-5p mimic or mi R-135b-5p NC into Hela cells,Western Blot and q RT-PCR were used to analyze the expression levels of EMT-related proteins E-cad,N-cad and vimentin.Transwell technique was used to detect the effect of-135b-5p on the migration ability of Hela cells;Lnc RNA GAS5 and mi R-135b-5p were co-transfected into Hela cells,and Western Blot and q RT-PCR were used to detect the reversel effect of Lnc RNA GAS5 on mi R-135b-5p by promoting EMT in Hela cells.(3)Screening the related mi R-135b-5p downstream target genes: searching about mi R-135b-5p mature sequence database(Target Scan,mi RDB,Mirtarbase),downloading all the target genes and select the genes which is most relevant to EMT as a follow-up study objects.The luciferase reporter gene determines the regulation of mi R-135b-5p on the target genes,Lnc RNA GAS5/mi R-135b-5p/downstream target genes regulatory pathway is verified by Western Blot and q RT-PCR.Results:(1)The expression level of Lnc RNA GAS5 in Hela was significantly lower than that of Hacat cells.When Lnc RNA GAS5 was up-regulated,E-cad m RNA level and protein level were significantly increased,while N-cad and Vimentin were significantly decreased.Transwell results showed that the migration ability of Hela cells wassignificantly reduced after overexpression of Lnc RNA GAS5 compared to the control group.(2)The mi RNA-mi R-135b-5p regulated by Lnc RNA GAS5 was successfully screened.The high expression of Lnc RNA GAS5 could down-regulate the expression of mi R-135b-5p.The activity of the wild-type mi R-135b-5p luciferase reporter gene luciferase was significantly lower than that of the control group,and the luciferase activity of the mutant fluorescein reporter gene did not change significantly compared with the control group.(3)Compared with the control group,the m RNA and protein levels of EMT-related protein E-cad was significantly decreased when mi R-135b-5p was highly expressed,,while N-cad and Vimentin were significantly increased.Lnc RNA GAS5 partially reversed the EMT-promoting effect of mi R-135b-5p;(4)Successfully screened mi R-135b-5p downstream target genes-LATS2 and KLF4,and highly expressed mi R-135b-5p,KLF4 was significantly down-regulated,while LATS2 was partially Up.When Lnc RNA GAS5 is highly expressed,the expression level of KLF4 is down-regulated.Conclusions: Lnc RNA GAS5 can inhibit the occurrence and development of EMT in cervical cancer Hela cells and reduce its ability of migration.Inhibition of mi R-135b-5p by Lnc RNA GAS5 and further inhibition of the expression of the downstream target gene KLF4 is a possible regulatory mechanism. |
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