Subcelluar Localization Of P-Snail Mediated By C14orf166 Was Crucial For IL-8induced Breast Cancer Metastasis

Objective To investigate the role of IL-8 in promoting breast cancer EMT through C14orf166 mediated Snail activation,and clarify the unearthed mechanisms of C14orf166 in breast cancer tumorigenesis and progression.Methods 1.Human breast cancer cell lines MCF-7 and SK-BR-3 were treated with human recombinant cytokine IL-8,one group of which was treated with signaling pathway inhibitor LY294002(PI3K/AKT)at the same time.The expression of C14orf166 was detected by Western blotting and Semi-quantitative Real-time PCR.Besides,cells were transfected with small interfering RNA(si RNA)of C14orf166 to detect the protein level of Snail under the stimulation of IL-8.2.MCF-7 and SK-BR-3 cells were transfected with C14orf166 si RNA or overexpression plasmids(p CMV-tag2B-C14orf166).Western blotting and semi-quantitative RT-PCR were applied to detect Snail,EMT associated markers E-cadherin,Vimentin and Fibronectin.Meanwhile,Scratchassay and Transwell assay were applied to test cell functions.3.C14orf166 was overexpressed in breast cancer cells,which were thentreated with translation inhibitor cycloheximid(CHX).The halflives of Snail in different groups were determined by western blotting and gray scale scanning.Then the protein level of phosphrylated Snail(p-Snail(S246))were assessed after IL-8 stimulation.Subsequently,western blotting was used to detect p-Snail when C14orf166 was knocked down by sh RNA lentivirus or overexpressed in breast cancer cells.Furthermore,the destribution of p-Snail in cytoplasm and nucleus was detected.4.MCF-7 cells were infected with C14orf166 sh RNA lentivirus,then were selected with puromycin to aquire C14orf166 stable knockdown cell lines,which were injected into nude mice fat pad.Until were transplanted successfully,the tumors were injected with PBS/IL-8 in situ.Tumor volumes were assessed each week.Mice were killed after 4 weeks,liver,lung tissues were stained by immunohistochemistry(IHC)to observe the expression of C14orf166,Snail,p-Snail and EMT markers in tumors.H&E staining was adopted to observe the metastasis of tumors.5.Different stages of clinic mammary tissues were collected,andthen the expression levels and correlations of CXCRB,C14orf166,Snail and EMT markers were detected by IHC.C14orf166 associated breast cancer survival percentage was analysedby Kaplan-Meier Plotter.Results 1.It was showed that PI3K/Akt signaling pathway inhibitor could block IL-8 induced C14orf166 upregulation.But C14orf166 specificlyinhibited this alteration.2.Western blotting showed that protein level of Snail was reduced in MCF-7 and SK-BR-3 cells when C14orf166 was knocked down,epithelial marker E-cadherin was increased,while mesenchymal markers Vimentin and Fibronectin were reduced accordingly.The abilities of cell invasion and motility were also repressed;protein level of Snail was increased in MCF-7 and SK-BR-3 cells when C14orf166 was overexpressed.EMT makers were transferred accordingly.The abilities of cell invasion and motility were also prompted.However,realtime quantitative PCR showed the m RNA level of Snail was scarcely influenced under the same conditons.3.Western blotting demonstrated that when cells were treated with CHX,halflife of Snail in C14orf166 overexpression group was obversely prolonged.P-Snail was increased in a time dependent manner with IL-8 stimulation.P-Snail as well as the distribution in cell nucleus was decreased when C14orf166 was depleted.Nonetheless,this change could be reversedby C14orf166 overexpressing.4.It was showed that IL-8 could promote the growth of breast tumors,whereas this change could be blocked by the deficiency of C14orf166.IHC determined that IL-8 could promote the expression of C14orf166,Snail,p-Snail,Vimentin,Fibronectin,but inhibite the expression of E-cadherin.Otherwise,the deficiency of C14orf166 repressed the series changes initiated by IL-8.Besides,IL-8 could promote the metastasis of breast cancer,but the deficiency of C14orf166 blocked this proccess.5.IHC showed that,with the malignant degree of mammary tissues increasing,the expression of CXCRB,C14orf166,Snail,Vimentin,Fibronectin were higher and higher,but the expression of E-cadherin became lower and lower.Survival curve showed that C14orf166 level was positively related with of breast cancer patients death percentage.Conclusions 1.IL-8 upregulated C14orf166 through PI3K/Akt signaling pathway to mediate the expression of Snail.2.C14orf166 mediated Snail to have effects on breast cancer cells EMT progression,invasion and motility abilities.3.C14orf166 mediated p-Snail translocation into nucleus,which inhibitedbreast cancer cell invasion and motility abilities.4.Deficiency of C14orf166 could inhibite the growth and metastasis of breast cancer in vivo.5.In clinic specimes,C14orf166 was closely correlated with breast cancer malignancyassociated genes,and higher level of C14orf166 could lead to higher death percentage.