| Background: Breast cancer(BC)has become the largest cancer in the world with the continuous increase of new cases every year.Although the early detection and treatment of BC can reduce the mortality,the late-stage palindromia and lymphatic metastasis of BC keep the mortality rate high.It is indispensable to find novel targets for the treatment of BC as an indicator of BC prognosis.Discs large homolog associated protein 5(DLGAP5)gene encodes a mitotic spindle protein that can regulate cell cycle progression.We found DLGAP5 is highly expressed in BC tissues by GEO and TCGA database,which indicates that DLGAP5 may be used as a marker of BC and play a role in promoting cancer in BC progression.However,its specific mechanism in BC remains unclear.Therefore,this study further determines the mechanism of action of DLGAP5 on BC by deeply exploring the effects of DLGAP5 on the behavior and pathways of BC cell lines,and provides a new direction for BC diagnosis and screening.Research method: 1.Downloading the gene expression data of tissue from GEO and TCGA databases,and using R software to obtain the expression difference of DLGAP5.The differential expression of DLGAP5 was verified in clinical tissues by immunohistochemical.2.Silencing the DLGAP5 gene by small interfering RNA sequence(si-RNA)and overexpressing the DLGAP5 gene by adenovirus.3.The RNA interference sequence and adenovirus were respectively transfected into breast cancer cell lines.Proliferation was detected by CCK-8 and clonal formation.4.Flow cytology was used to detect the effect of DLGAP5 expression level regulated by RNA interference sequence and adenovirus on cell apoptosis rate and cycle progress.5.The RNA interference sequence and adenovirus were respectively transfected into breast cancer cell lines.Cell migration ability was evaluated by scratch method.Transwell compartment was used to reflect changes in cell migration and invasion phenotype.6.DLGAP5 expression difference of two cell lines was detected by q RT-PCR,the degree of regulation of DLGAP5 expression by si-RNA and adenovirus and the changes of markers such as Ki67,Cyclin D1,BAX,BCL2,P53 and IL-6after the change of DLGAP5 expression.7.Using Western Blot to detect the difference in DLGAP5 protein expression in the two cell lines,and the regulation degree of DLGAP5 protein by si-RNA and adenovirus,as well as the changes in markers of BAX,BCL2,P53,MMP2,E-cadherin,N-cadherin,IL-6,p-JAK2 and p-STAT3 after DLGAP5 protein change.Experimental Result: 1.It was found in GEO,TCGA and GEPIA databases that DLGAP5 is highly expressed in BC tissues.Kaplan database predicted that DLGAP5 overexpression was associated with poor prognosis.Clinical tissue samples confirmed that DLGAP5 protein was highly expressed in BC tissues.2.DLGAP5 is under-expressed in the low-invasive cell MCF-7 and over-expressed in the high-invasive cell MDA-MB-231,and we selected MCF-7 cells for the study of overexpression function.3.Silencing DLGAP5 gene can inhibit the reproduction,migration and invasion of BC cells,and promote apoptosis.4.Overexpression of DLGAP5 by adenovirus can promote the proliferation,migration and invasion of BC cells,and inhibit cell apoptosis.5.Silencing or overexpression of DLGAP5 gene can correspondingly down-regulate or up-regulate IL-6,p-JAK2 and p-STAT3.Conclusion: It was found that DLGAP5 can regulate the proliferation,apoptosis,migration,invasion and other behaviors of breast cancer cells,and also affect the activity of IL-6/JAK2/STAT3 signaling pathway,indicating that DLGAP5 is very important for breast cancer progression. |
PDF Full Text Request