The Effect Of Dominant Negtive Mutant And MicroRNA On The Breast Cancer Cell By Down-regulating The STAT3Signaling Pathway

Objective：1.To observe the expression of Stat3β gene in mouse breast cancer cellline4T1cells by construction Stat3β recombinant adenovirus expressionvector，and to study the effect on mouse breast cancer cells by competitiveinhibition of STAT3signal pathway.2. To investigate the regulating role of miR17-5p on the STAT3gene.Meanwhile, the effect of miR17-5p on the malignant biological behaviors ofMCF-7, such as proliferation，apoptosis, migration and invasion, werefurther studied in this research.Methods:1.Stat3β gene from the plasmid pcDNA-Zeo-Stat3β-C4flag, wassubcloned to the shuttle vector pAdTrack–CMV. After recombinant,shuttle vector was linearized by PmeⅠ, and then it was transformed to theBJ5183strain containing pAdEasy-1. The recombinant adenovirusexpression vector was identified and linearized by Pac Ⅰ, and then packaged by HEK293A cells; After4T1cells were infected with theadenovirus expression vector, the expression of the Stat3β gene, MMP2,MMP9and p-STAT3was observed;4T1cells cell cycle and apoptosiswere detected by FCM, and the migration and invasion ability of4T1cellswas investigated by wound heal assay and transwell assay respectively.2. Firstly，the bioinformatical tools were used to detect the conservationof STAT3mRNA3’UTR region among vertebrates. It was predicted thatmicroRNA can regulate STAT3post-transcriptionally. Then, miR17-5p wasselected as the candidate. In order to investigate whether miR17-5p couldtarget STAT3mRNA3’UTR directly or not, the luciferase reporter systemwere be constructed. After the verification, miR17-5p mimics weretransfected into human breast cancer cell lines MCF-7. The mRNA andprotein of STAT3were detected in order to identify the mechanism ofmiR17-5p regulation, and the impact on the STAT3signaling pathwaycompared with mimics control groups and STAT3rescue groups. Transwellassay was used to determine the migration and invasion ability aftertransfected with miR17-5p. MCF-7cell cycle and apoptosis were also testedby means of FCM.Results：1.The recombinant adenovirus expression vector was successfullyconstructed, and GFP was expressed after the recombinant adenovirusbeing transfected into the HEK293A cells. The titer of purified adenovirus was1.1×10~9pfu/ml. The mouse cancer cells4T1were infected with therecombinant adenovirus pAd-Stat3β, and the RT-PCR showed that theStat3β was expressed significantly in treatment group compared withcontrol groups. Except for p-STAT3, Western Blot implied that c-myc、MMP2，MMP9in4T1cells were down-regulated significantly followingSTAT3β expressed. As a result, the invasion ability was inhibited followingSTAT3β block. FCM indicated that there was no noticeable change in thecell cycles and apoptosis after infection of recombinant adenoviruspAd-Stat3β in4T1cells compared with control groups. Similarly, thewound heal assay revealed that there was no obvious difference inmigration ability in4T1cells between the treatment group and controlgroups.2. The bioinformatical analysis confirmed that STAT3mRNA3’UTRregion was highly conserved among homo sapiens, horses, rabbits and othervertebrates. After the successful construction of luciferase vectors, theluciferase reporter system verified that miR17-5p could directly targetSTAT3mRNA3’UTR. RT-PCR and Western Blot displayed that there wasno significant difference in STAT3mRNA expression, but the protein ofSTAT3was different obviously between treatment group transfected withmiR17-5p mimics and control groups, which means miR17-5p couldregulate STAT3in a way of post-transcription. Western Blot indicated thatp-STAT3, MMP2, c-myc, Bcl2, vimentine, which are some downstream genes of STAT3signaling pathway, could be down-regulated significantlyby miR17-5p. What’s more, CD31and AnnexinⅡ could be significantlyup-regulated by miR17-5p. Unfortunately, there is no obvious change inE-cadherin and MMP9.The transwell assay implied that miR17-5p couldreduce the ability of invasion but have no impact on the migration in MCF-7cells. The FCM showed that miR17-5P could promote apoptosis of MCF-7cells, but had no significant effect on cell cycle.Conclution：1.The recombinant adenovirus expression vector was successfullyconstructed and the target gene could express in the4T1cells.Overexpression of Stat3β can competitively inhibit STAT3signalingpathway, suppressing the expression of downstream genes, such as c-myc、MMP2and MMP9. Consequently,4T1migration was also inhibited.2. The STAT3mRNA can be down-regulated by miR17-5p; miR17-5pcould regulate a series of downstream targets of STAT3signaling pathway,which can inhibit the invasion ability and induce apoptosis in MCF-7cells.Furthermore, it also affected some of biomarkers related to epithelial tomesenchymal transition and angiogenesis.