Pien Tze Huang Inhibits Tumor Growth And Metastasis Of Colorectal Cancer Through Regulation Of MiRNA And Differential Expressed Genes

Objective:To assess the effect of PZH treatment on colorectal cancer cell growth and metastasis in vitro and in vivo,as well as the expression related miRNA and their target genes and signaling pathways.To assess the clinical significance,biological function and underlying mechanisms of PNO1 in CRC,as well as evaluate the effect of PZH treatment on expression of PNO1 in CRC cells.Methods:1.Using CRC xenograft mouse model,we assessed the effect of PZH treatment on tumor growth by determination of tumor volume and tumor weight.MTT assay,colony formation assay and PI staining were used to detect the proliferation,survival and cell cycle progression of CRC cells with the treatment of PZH.Q-PCR and Western-blot analyses were performed to determine the expression of miR-34c-5p and its target genes in CRC cells after PZH treatment.2.Wound-healing assay,migration and invasion analyses were performed to determine the effect of PZH treatment on metastasis of CRC cells in vitro.By construction the orthotopic metastasis model of CRC cells,IVIS system,tumor weight,metastatic tumor number counting were performed to assess the effect of PZH treatment on metastasis of CRC cells in vivo.Migration and invasion analysis were performed to determine the metastasis of HCT-8/5-FU cells with the treatment of PZH.Q-PCR assay and western-blot were used to determine the regulatory effect of PZH treatment on TGF-?1/miR-200/ZEB1 signaling pathway in both HCT-8/5-FU and HCT-8 cells.3.cDNA Microarray was performed to determine the differential expressed genes between CRC tissues and adjacent normal tissues.TCGA database,Q-PCR analysis,IHC assay,Tissue cDNA array and Tissue microarray were performed to determine the expression of PNO1 in CRC samples and cells;we further analyze the correlation of PNOl expression and survival of CRC patients.Q-PCR and western-blot analyses were performed to examine the expression of PNO1 in CRC cells after transduced with 3 different sh-PNO1 lentivirus.HCS assay,CCK-8 assay,colony formation assay,PI staining and AnnxinV staining were performed to evaluate the effect of PNO1 knockdown on cell proliferation,survival and apoptosis in CRC cells.Using CRC xenograft mouse model,determination of tumor volume,tumor weight and fluorescence intensity of GFP were performed to assess the effect of PNO1 knockdown on tumor growth in vivo.cDNA microarray,Q-PCR assay,Pathway analysis and Western-blot were used to explore the down-stream of signaling pathways and genes after PNO1 knockdown,Q-PCR and Western-blot analyses were performed to determine the expression of PNO1 in CRC cells after PZH treatment.Results:1.PZH treatment significantly reduced the tumor volume and tumor weight of CRC cells in vivo.PZH treatment reduced cell viability,cell survival and induced cell cycle arrest at the checkpoint from G0/G1 to S phase of CRC cells.PZH treatment up-regulated the expression of miR-34c-5p,and down-regulated the expression of c-Myc,CDK4 and CyclinDl on both mRNA and protein levels.2.PZH treatment suppressed the migration and invasion of CRC cells in vitro and Inhibited the tumor growth and metastasis of CRC cells in vivo.PZH treatment obviously suppressed 5-FU resistant enhanced migration and invasion of 5-FU resistant HCT-8(HCT-8/5-FU)cells.PZH treatment significantly down-regulated the expression of TGF-?1,ZEB1,N-cadherin and up-regulated the expression of E-cadherin,as well as increased the expression of miR-200a?miR-200band miR-200c in both 5-FU resistant CRC cells and its parental cells.3.The expression of PNO1 in CRC tissues and cells was significantly increased comparing with nontumor colorectal tissues and cells respectively.Increase of PNO1 in CRC tissues was significantly associated with shorter survival of CRC patients.PNO1 knockdown significantly suppressed tumor growth in vivo and in vitro by reducing cell viability,survival and inducing cell cycle arrest and apoptosis in CRC cells,and increased the expression of p53 and its downstream p21.PZH treatment significantly decreased the expression of PNO1 in CRC cells.Conclusion:PZH treatment significantly suppressed tumor growth in vitro and in vivo by modulation the expression of miR-34c-5p and its target genes.PZH treatment significantly inhibited the metastasis of CRC in vivo and in vitro by suppression TGF-?1/miR-200/ZEB1 signaling pathway.The expression of PNO1 was significantly increased in CRC tissues and cells,and increase of PNO1 in CRC tissues indicates poor prognosis of CRC patients.Knockdown of PNO1 suppresses tumor growth in vitro and in vivo by inhibiting cell proliferation and inducing cell apoptosis,and by activation of p53 signaling pathway.PZH treatment significantly down-regulated the expression of PNO1 in CRC cells.