In Vitro Study On Proliferation And Invasion Of Breast Cancer Mediated By PPARG/FABP4 Signal Pathway

Background and objective:Breast cancer has become the highest incidence of malignant tumor among women in the world,Although the incidence of female breast cancer in China is lower than the global incidence,the number of new cases in 2018 is as high as 367,900,ranking first,and the incidence is increasing year by year and showing a younger trend.It is urgent to improve the diagnosis and treatment of breast cancer.The birth and development of gene chip technology makes it possible to study thousands of gene expression and mutual regulation at the same time,which provides a powerful "engine" for the development of gene functional scienceThe role of PPARG/FABP4 pathway in the occurrence and development of malignant tumors has been controversial,but these two genes have been clearly associated with a variety of malignant characteristics of human tumors,such as proliferation,invasion,distant metastasis and so on,and involved in a variety of mechanisms.In order to further explore the relationship between this signal pathway and breast cancer,this study verified tissue samples and breast cancer cell lines on the basis of gene chip data mining and analysis.In summary,this study intends to complete the following objectives from bioinformatics analysis and in vitro experiments:(1)to mine differential genes and pathways related to breast cancer tumorigenesis by bioinformatics analysis;(2)to determine the differential expression of related genes in tissue samples;and(3)to explore the effect of peroxisome proliferator-activated receptor G/fatty-binding protein 4 pathway on the proliferation and invasion of breast cancer.Materials and methods:Clinical samples collection.From October 2018 to May 2019,10 pairs of postoperative breast cancer foci and normal glands were collected in Nanfang Hospital of Southern Medical University were collected.2.Acquisition and analysis of GEO database gene chip expression data.Download 3 samples of gene chip data including cancer tissue and normal gland tissue,use G02R based on R language to calculate the expression of each gene,screen out differentially expressed genes according to P<0.05and lgFC>3 or<-3,make GO analysis,KEGG analysis and PPI network drawing,use Cytoscape software to calculate Hub gene,and finally carry out survival analysis.3.Cell culture.MCF7 cell line and MDA-MB-231 cell line were cultured in DMEM medium of 10%FBS.The cells were digested and passaged when the fusion rate was about 85%in the culture flask,and the subsequent transfection was carried out when the fusion rate was about 90%in the six-well plate.RNA/protein was extracted and functional test.4.Cell transfection.According to the instructions,lipo2000 was used to transfect the two kinds of cells to construct PPARG/FABP4 overexpression and interference cell lines.5.RNA extraction and q-PCR reaction.Total RNA was extracted in tissue samples and cells.SYBR method was used to detect the expression of PPARG and FABP4 in each sample6.Protein extraction and Western blotting.Protein lysate was used to extract proteins from breast cancer,normal gland specimens and various cell lines.Western blotting was used to detect the expression and correlation of PPARG and FABP4 proteins in each sample.7.Cell function test.CCK8,scratch test and cell chamber experiment were used to verify the effect of PPARG/FABP4 pathway on the proliferation,invasion and metastasis of breast cancer cells.8.Statistics.All the data were analyzed by SPSS 21.0 statistical software,and the counting data were analyzed by two independent sample t-test.P<0.05 was considered as statistical differenceResults:Gene chip data analysis.A total of 146 differentially expressed genes were obtained from the three microarrays.After survival analysis,5 genes related to the prognosis of breast cancer patients were obtained.The low expression of PPARG,LPL,PLIN1,CIDEC and FABP4,genes were related to shorter overall survival time.2.Expression of PPARG and FABP4 in tissue specimens.10 pairs of postoperative breast cancer specimens and normal gland specimens were verified by q-PCR.It was found that the RNA levels of PPARG and FABP4 in cancer lesions were lower than those in normal glands.3.PPARG positively regulates the expression of FABP4.After overexpression and interference of PPARG and FABP4 in MCF7 and MDA-MB-231 cells,q-PCR and WB were used to confirm that PPARG positively regulated the expression of FABP4.4.PPARG/FABP4 pathway affects the proliferation,invasion and metastasis of breast cancer.CCK8,scratch test and cell chamber test were carried out on MCF7 and MDA-MB-231 cells which were overexpressed or interfered by PPARG and FABP4.The results showed that up-regulation of PPARG/FABP4 pathway could inhibit breast cancer proliferation and invade the ability of metastasis.Conclusion:1.The results of bioinformatics analysis showed that the down-regulation of PPARG and FABP4 and PPARs pathway were associated with the tumorigenesis of breast cancer,and the down-regulation of these two genes predicted a shorter overall survival time of breast cancer patients2.PPARG positively regulates the expression of FABP4,and the upregulation of this pathway can inhibit the proliferation,invasion and metastasis of breast cancer.