The Effect Of SiRNA Interfering Caveolin-1Gene Expression In Fibroblasts On The Proliferation Of Breast Cancer Cells And The Investigation Of It’s Mechanism

Background and ObjectAround the world,breast cancer has became the most common malignant tumorand the first health risk toward urban women. The predisposing factors for breastcancer is very complicated. Although the related research has made great progress,the etiology and pathogenesis are still poorly understood.Caveolin-1is a membraneprotein and it is a essnetial structural and functional molecule of caveolae. It plays aregulatory role toward those proteins that gather in caveolae. Caveolin-1is closelyrelated to the transformation of breast fibroblast，and also affect the genesis anddevelopment of breast cancer. Normally, Caveolin-1expresses highly in themammary stromal fibroblasts; but in chronic mastitis, mammary fibroplasia andmalignant transformation of microenviroment, Caveolin-1expression declines or evenis negative. In clinic,the patients with breast cancer whose Caveolin-1expressiondecline or is negative have a poor prognosis and low survival rate. Caveolin-1isalways negative in the advanced breast cancer patients. Inversely,Caveolin-1postiveexpression means good prognosis and high survival rate.89%of breast cancerpatients whose Caveolin-1postive expression were no recurrence in the15years offollow-up time. In breast cancer,often show the epigenetic abnormalities such aschromosome loss or rupture, DNA methylation. Autophagy is a biologicalphenomenon which widely exists in eukaryotic cells. It is a process that degradesabnormal proteins and organelles.The vary of autophagy is associated with variousdiseases such as tumor, neurode generative diseases, infection and so on. Autophgygenes abnormally expressed by the fibroblasts of malignant tumor microenviromentcan induce autophagy disfunction in fibroblasts, for example, degradingmacromolecular proteins to provide nutrition for precancerous cells and modifyingcell secretion to change cell microenviroment, so that the disfunction causes geneticinstability and malignant choice, and promote the development of tumor.From what has been discussed above，in this study, the interaction betweenCaveolin-1and autophagy in fibroblast has been investigated, and the effect of thisinteraction on the growth of breast cancer cells also investigated. In our study, the co-culture system of fibroblast ESF/breast cancer cell BT474was established and Caveolin-1expression was interfered by siRNA in fibroblast ESFto investigate the interaction between Caveolin-1and autophagy in fibroblast and theeffect on the proliferation of adjacent breast cancer cells. We hope that new thinkingfor breast cancer research will be provided.Methods1. Caveolin-1gene interference in fibroblasts was mediated using siRNA, whichwas transfected by liposome. The interferencing effect was detected by qRT-PCR andwestern blot methods.2. ESF/BT474co-culture system was established using transwell method.3. BT474cell proliferation was tested by CCK-8method in the condition ofCaveolin-1gene interference and ESF/BT474co-culture.4. The effect of Caveolin-1gene interference and ESF/BT474co-culture on theautophagy body in ESF cells was examinated using immune fluorescent confocallaser microscopy. The relation between Caveolin-1and autophagy body wasanalyzed.Results1. qRT-PCR and western blot results show that Caveolin-1mRNA expressionlevel was significantly lower in siRNA N.2group than other each group (p<0.05),and Caveolin-1mRNA expression level also had significant difference (p<0.05) insiRNA N.1group and siRNA N.3group, respectively, compared with other eachgroup. Caveolin-1protein expression level was significantly lower in siRNA N.1,siRNA N.2and siRNA N.3gruops than control group (p<0.05). These results indicatethat siRNA interfere effectively Caveolin-1expression in ESF cells. According to theresults of qRT-PCR and western blot, siRNA-2group was selected for theexperiments in this study.2. Cell proliferation experiment results show that after48hours or72hours ofcell culture, BT474cell proliferation significantly raised in BT474/ESF+siRNAN.2group, compared with other each group.3. The results of immune fluorescent confocal laser microscope show as follows.①Autophagy bodies were significantly higher in BT474/ESF+siRNA N.2group than other groups (p<0.05), but Caveolin-1expression was significantly lower inBT474/ESF+siRNA N.2group than other groups (p<0.05).②Autophagy bodieswere significantly lower in ESF group than other groups (p<0.05), but Caveolin-1expression was significantly higher in ESF group than other groups (p<0.05).③Although autophagy bodies and Caveolin-1expression had statistical difference inESF+siRNA N.2(p<0.05), compared with other groups, the range of autophagy bodyup-regulation and Caveolin-1down-regulation was far less in ESF+siRNA N.2groupthan BT474/ESF+siRNA N.2group.④Autophagy bodies were significantly higher inBT474/ESFgroup than ESF group and ESF+siRNA N.2group (p<0.05), butsignificantly lower than BT474/ESF+siRNA N.2group (p<0.05). Caveolin-1expression was significantly higher in BT474/ESF group than BT474/ESF+siRNA N.2group and ESF+siRNA N.2, but is significantly lower than ESF group (p<0.05).Conclusions:1. special siRNA Caveolin-1has emerged as a powerful tool for the interferenceof Caveolin-1gene expression．2. The expression suppression of Caveolin-1in fibroblasts can promotes breastcancer cell proliferation. The co-culture of breast cancer cells and Caveolin-1down-regulated fibroblasts can enhance the proliferation of breast cancer cells.3. Caveolin-1expression down-regulates; conversely, autophagy body increasesin fibroblasts; the level of autophagy body has a negative correlation with that ofCaveolin-1. The co-culture of breast cancer cells with Caveolin-1interferedfibroblasts enhances the antagonism effect of autophagy body with Caveolin-1.