PGC-1? Promotes The Growth,migration And Invasion Of Human Colorectal Carcinoma Cells And The Migration,invasion And Angiogenesis Of Human Umbilical Vein Endothelial Cells

Background and purposeColorectal carcinoma(CRC)mainly includes colon cancer and rectal cancer,which always occurs in the rectum and sigmoid colon.As the most common pathological type of CRC,colorectal adenocarcinoma accounts for more than 95%of all types.CRC has become the third leading cause of death in the world.Recently,with the development of an aging society and changes in diet structure,the incidence and mortality of CRC are increasing year by year in China,which seriously threatens human health.40%-50%of CRC patients always have distant metastases when first diagnosed.Even if they have undergone radical surgery,more than 50%patients still have recurrent metastases.Therefore,finding the key proteins of CRC metastases can provide an effective and promising treatment strategy.Peroxisome proliferator-activated receptor gamma coactivator 1?(PGC-1?)is a transcriptional coactivator of peroxisome proliferation activated receptor gamma(PPAR?).It can bind to multiple nuclear receptors and transcription factors to participate in the regulation of some biological processes,such as energy metabolism and heat production.Altered metabolism has become one of the hallmarks in cancer,which is associated with the development of cancer.In order to meet the high metabolic needs,tumor cells often adaptively increase mitochondrial metabolism.As an important regulator of mitochondrial metabolism,PGC-1? can increase the metabolic capacity of mitochondria to satisfy the high metabolic needs of tumor cells.The role of PGC-1? in tumorigenesis has already been reported.What caught our attention are its different roles in different tumors.At present,current research on the role of PGC-1? in CRC is limited to its effect on tumor growth,and the results are controversial.On the one hand,it is reported that PGC-1? may affect the transition of mitochondrial metabolism towards fatty acid metabolism to promote tumor growth.On the other hand,PGC-1? has been reported to inhibit tumor growth by inducing apoptosis pathways.The tissue chip results of this study show that PGC-1? is highly expressed in colon adenocarcinoma tissues and the expression level is positively related to the degree of lymph node metastasis,indicating that PGC-1? may affect the metastasis of CRC.Moreover,angiogenesis is an indispensable factor for the continuous growth,invasion and metastasis of solid tumor cells.PGC-1? has been reported to regulate angiogenesis in normal muscle and vasculature,but its role in CRC angiogenesis is still unknown.Based on it,this study is designed to explore the role of PGC-1? in the growth,migration and invasion of human colorectal carcinoma cells and the migration,invasion and angiogenesis of human umbilical vein endothelial cells(HUVECs),and then provide a new promising treatment target for CRC metastasis and angiogenesis.Methods1.Tissue microarray was used to detect the expression levels of PGC-1? in human colorectal adenocarcinoma tissues and adjacent tissues by immunohistochemistry.The relationship between PGC-1? expression level and clinical features was analyzed.2.PGC-la expression levels in human CRC cell lines and normal colorectal epithelial cell line were detected by western blotting.PGC-1? expression levels in CRC tissues and adjacent tissues were also detected.3.Three PLKO.1 plasmids containing different PGC-1? shRNA sequences and one PGC-la overexpression PHBLV plasmid were purchased.Two CRC cell lines(DLD-1 and LoVo)expressing medium protein levels of PGC-1? were randomly selected.Using lentivirus transfection technology,CRC cell lines with stable knockdown and overexpression of PGC-1? were established.The knockdown and overexpression effects of PGC-1? were determined to check if the stable cell lines have been successfully established.4.The effect of PGC-1? on the proliferation ability of human CRC cells was detected by cell proliferation assay.5.The effect of PGC-1? on the colony formation ability of human CRC cells was detected by colony formation assay.6.The effect of PGC-1? on the migration and invasion abilities of human CRC cells was detected by transwell assay.The effect of PGC-1? knockdown and overexpression CRC cell conditioned medium on the migration and invasion abilities of HUVECs was examined by transwell assay.7.The effect of PGC-1? on the expression levels of proliferation,invasion and epithelial-mesenchymal transition(EMT)markers was detected by western blotting and immunofluorescence.8.The effect of PGC-1? in human CRC cell conditioned medium on the migration ability of HUVECs was detected by wound healing assay.9.The effect of PGC-1? in human CRC cell conditioned medium on the tube formation ability of HUVECs was detected by tube formation assay.10.DLD-1(PLKO.1)and DLD-1 knockdown PGC-1?(sh1)cell lines were used to perform subcutaneous tumor formation assay in nude mice.The effect of PGC-1?on tumor volume and tumor weight was observed.The expression levels of proliferation,invasion,EMT,and angiogenesis markers were also detected.Results1.Tissue microarray results showed that the expression levels of PGC-1? in human colon adenocarcinoma tissues were significantly higher than those of adjacent tissues(P<0.001).The expression levels of PGC-la in colon adenocarcinoma tissues were positively correlated with lymph node metastasis(P<0.05).2.The expression levels of PGC-1? in 7 human CRC cell lines were higher than that of normal colorectal epithelial cell.Western blotting results in 20 pairs of clinical CRC tissues proved that the expression levels of PGC-1? were significantly higher in 13 CRC samples than those of adjacent tissues.For 10 pairs of CRC samples with lymph node metastasis,the expression levels of PGC-1? in 6 CRC samples were higher than those of adjacent tissues.3.Western blotting results proved that the stable knockdown and overexpression of PGC-1? human CRC cell lines were successfully constructed.4.Knocking down PGC-1? inhibited the proliferation ability of human CRC cells(DLD-1 and LoVo).Overexpression of PGC-1? enhanced the proliferation ability of human CRC cells(DLD-1 and LoVo).5.Knocking down PGC-1? inhibited the colony formation ability of human CRC cells(DLD-1 and LoVo).Overexpression of PGC-1? enhanced the colony formation ability of human CRC cells(DLD-1 and LoVo).6.Knocking down PGC-1? inhibited the migration and invasion abilities of human CRC cells(DLD-1 and LoVo),and its conditioned medium inhibited the migration and invasion abilities of HUVECs.Overexpression of PGC-1?enhanced the migration and invasion abilities of human CRC cells(DLD-1 and LoVo),and its conditioned medium inhibited the migration and invasion abilities of HUVECs.7.Knocking down PGC-1? inhibited the expression levels of Ki67,MMP9,N-cadherin,Vimentin in human CRC cells and promoted the expression level of E-cadherin.Overexpression of PGC-1? promoted the expression levels of Ki67,MMP9,N-cadherin,Vimentin in human CRC cells and inhibited the expression level of E-cadherin.8.Knocking down PGC-1? in human CRC cell conditioned medium inhibited the migration ability of HUVECs.Overexpression of PGC-1? in human CRC cell conditioned medium enhanced the migration ability of HUVECs.9.Knocking down PGC-1? in human CRC cell conditioned medium inhibited the tube formation ability of HUVECs.Overexpression of PGC-1? in human CRC cell conditioned medium enhanced the tube formation ability of HUVECs.10.Tumor xenograft model:The tumor weight of nude mice in the PGC-1?knockdown(DLD-1 sh1)group was significantly reduced,and the tumor volume was smaller compared with the control group.The differences were statistically significant(P<0.001),The tumor microvessel density and CD31 protein level decreased in the PGC-1? knockdown(DLD-1 sh1)group,and the differences were statistically significant(P<0.01).Knocking down PGC-1? inhibited the expression of Ki67,MMP9,N-cadherin,Vimentin and CD31 in CRC tumor xenograft model,and promoted the expression of E-cadherin.Conclusions1.PGC-1? is highly expressed in human CRC tissues and cell lines.Its expression level is positively correlated with lymph node metastasis.2.PGC-1? promotes the growth,migration and invasion of human colorectal carcinoma cells and the migration,invasion and angiogenesis of HUVECs.