Identification And Functional Analysis Of A Novel Splice Variant Of AC3-33

Objectives RNA alternative splicing not only increases the diversity of mRNA expression,also plays an important role in regulating gene function.In this study,we based on the primer expressed sequence tags,using the MCF-7 breast cancer cell lines by reverse transcription PCR（rt-pcr）succeeded in cloning a new variable spliceosome AC3-33（NCBI reference sequence: NM₀01308229.1）,we named svAC3-33.By studying svAC3-33 basic structure and function,and further explore the mechanism of its function.Providing theoretical basis for the clinical diagnosis and treatment of breast cancer.Hope for gene therapy and genetic engineering drugs to lay the theoretical foundation and may provides new ideas.Methods 1 svAC3-33 bioinformatics analysis: DNA and other bioinformatics software analysis and prediction of gene localization,signal peptide;svAC3-33 cDNA and the corresponding protein sequence alignment;protein sequence alignment;in the third chromosome Comparison of exons with introns.2 Construction of eukaryotic expression vector: The target gene sv-AC3-33 was inserted into eukaryotic expression vector p EGFPC3 vector by molecular cloning technique to construct eukaryotic expression vector;Construction of silencing sub-plasmid si-svAC3-33 and its blank control si-NC plasmid.3 Cell sub-localization analysis: cell sub-localization of sv-AC3-33 protein.4 Cell proliferation assay: CCK-8 technique and Edu were used to detect the cell viability and the growth vigor curve was drawn.To investigate the effect of overexpression of svAC3-33 on the proliferation of MCF-7 cells.5 The effect of svAC3-33 and si-svAC3-33 on cell migration was determined by Transwell assay.6 Luciferase activity to detect the activity of each transcription factor: Screening svAC3-33 can inhibit the activity of transcription factor AP-1,and further use of luciferase activity detection of transcription factor AP-1 activity.7 The effects of C-JUN and C-FOS on svAC3-33 and si-svAC3-33 were analyzed by Western blot.8 The expression levels of P15,P21,P27,P53 and cyclin-D1 were normalized by real-time fluorescence quantitative RTq-PCR and GAPDH-80 was used as internal reference.Transcription level of each factor in MCF-7 cells was detected.9 Effect of silencing svAC3-33 on transcriptional level of P21 in MCF-7 cells.Results 1 The gene sequence is 1825 bp,consisting of 5 exons and 4 introns.Compared with AC3-33,the second exon is found,located at 3q25.31,and the CDS region is 26-920 bp.A total of 884 bp encoding 294 amino acids,more than AC3-33 encoding 43 amino acids.2 This study first demonstrated the presence of AC3-33 variable splice svAC3-33.3 By comparing the information of svAC3-33 and AC3-33,it was found that svAC3-33 lacked exon2.4 The recombinant plasmid p EGFP-C3-svAC3-33 was successfully constructed and confirmed to be expressed in MCF-7 cells.The silencing plasmid sisvAC3-33 and the si-NC plasmid were successfully constructed.Western blot showed that si-svAC3-33 was successfully constructed and expressed.5 Determine the sv-AC3-33 protein subcellular localization in the cytoplasm.6 CCK-8 for 5 days to analyze cell proliferation trends.The inhibitory effect of svAC3-33 on the proliferation of MCF-7 cells was confirmed.The presence of si-svAC-33 after silencing showed a synergistic effect on si-NC group.7 Compared with the overexpression gene p EGFP-C3-svAC3-33 group,the cell division rate was slower than that of the overexpression gene.In contrast,the sisvAC3-33 plasmid group transfected with the silencing target gene was smaller than that of the no-loaded si-NC Plasmid group,cell proliferation faster.8 Through the transwell experiments confirmed that svAC3-33 can significantly inhibit cell migration,while the use of RNA interference technology silencing sv-AC3-33 expression can significantly promote cell migration.9 The activity of each transcription factor was detected by double luciferase activity assay.Compared with other groups,the p AP-1-Luc group had more obvious trend changes,and the experiment was repeated three times.It was concluded that svAC3-33 could inhibit the activity of transcription factor AP-1.While silencing svAC3-33 can promote the activity of AP-1.10 Western blot showed that the protein expression level of C-JUN was significantly inhibited by the transfection of svAC3-33 group,but there was no significant difference compared with C-FOS group.11 The expression of P21,P21,P27,P53 and cyclin-D1 was normalized by real-time fluorescence quantitative qRTPCR,and GAPDH-80 was used as the internal reference.The activity of P21 was the highest and the other transcriptional levels had effect on it.If the expression of sv-AC3-33 is reduced,the expression of P21 will be decressed.Conclusions The sv-AC3-33 gene sequence is 1825 bp in length and consists of 5 exons and 4 introns.The second exon is absent from AC3-33,located at 3q25.31,and the CDS region is 26-920 total 884 bp encoding 294 amino acids.PCR confirmed the presence of AC3-33 variable splice svAC3-33.It was confirmed that sv-AC3-33 protein was subcellularly localized in cytoplasm.It was confirmed that svAC3-33 inhibited the proliferation of MCF-7 cells and could significantly inhibit cell migration The activity of transcription factor AP-1 was found to be inhibited by the activity of each transcription factor by double luciferase activity assay.Further experiments showed that the protein expression level of C-JUN was significantly transfected into svAC3-33,compared with the C-FOS there was no significant difference;then quantitative RT-PCR was performed,and the result showed that P21 expression will be reduced.To sum up,our experimental results showed that it can inhibit AP-1 signaling pathway,and inhibit MCF-7 in cell proliferation,we hope that it will be provided a certain theoretical basis for breast cancer in the future.Figure 23;Table 12;Reference 82...