| Objective: To explore the effect of Pien Tze Huang(PZH)on lymphangiogenesis of colorectal cancer and Lnc RNA ANRIL/VEGF-C/AKT pathway in vivo and in vitro,and to elucidate its mechanism of prevention and treatment of colorectal cancer metastasis,so as to provide experimental evidence for its clinical application.Method: In vivo: 1.Establish a model of HCT-116 cell subcutaneously transplanted tumor in nude mice.According to the tumor volume,the nude mice were randomly divided into the control group and the PZH group.The PZH group was given 0.25 g/kg/ day,while the control group was given equal volume of normal saline.The tumor volume and weight of nude mice were measured every other day.After 2 weeks of continuous administration,the nude mice were sacrificed,the tumor tissues were stripped and weighed.(1)The expression of LYVE-1,VEGF-C and VEGFR-3 in tumor tissue was detected by immunohistochemistry(IHC)to observe lymphangiogenesis.(2)The expression of VEGF-C and VEGFR-3 protein in tumor tissue was detected by western blot.(3)ELISA assay was used to detect the content of VEGFC secreted in serum.(4)RT-q PCR assay was used to detect the gene expression of Lnc RNA ANRIL in tumor tissues.2.Construct an orthotopic xenograft tumor model of nude mice with HCT-116/luc cells.Grouping and administration were the same as subcutaneous xenograft tumor model.(1)IVIS Spectrum Imaging System was used to observe tumor growth and metastasis.(2)IHC,ELISA and RT-q PCR assays indicators were the same as the subcutaneous xenograft tumor model.(3)HE assay was used to detect the liver metastasis of orthotopic transplantation tumor.In vitro study: 1 RT-q PCR was used to detect the expression of Lnc RNA ANRIL in different colorectal cancer cell lines.HCT-116 cells were silenced with Lipofectamine RNAi MAX and HCT-8 cells were transfected with lentivirus overexpressing Lnc RNA ANRIL.Microscope and CCK-8 assays were used to observe cell morphology and viability.Transwell assay was used to detect cell migration and invasion ability.Western Blot was used to detect VEGF-C protein expression.2.Collect cell culture supernatants of HCT-116/si ANRIL and HCT-8/ANRIL to treat HLEC.Microscope,CCK-8,Transwell and Tube formation assays were used to detect the effect of Lnc RNA ANRIL on HLEC.3.Different concentrations of PZH to treat HCT-8/ANRIL cells.Microscope was used to observe cell morphology.MTT and ELISA assays were used to detect cell proliferation and VEGF-C protein secretion.4.Collect HCT-8/ANRIL cells and PZH(0.25mg/m L)supernatant to culture HLEC.Microscope and CCK-8 assays were used to observe cell morphology and cell viability.Transwell and Tube formation assays were used to detect cell migration,invasion and tube formation capabilities.The expressions of MMP-1,MMP-2,MMP-7,MMP-9,VEGFR-3 and PI3K/AKT pathway were detected by Western Blot.Results: In vivo study: 1.Subcutaneous tumor transplantation model in nude mice:(1)PZH has inhibitory effect on the volume and weight of subcutaneous transplanted tumor of colorectal cancer(P<0.05 or P<0.05),and has no effect on the body weight of nude mice.(2)PZH could significantly inhibit the expression of LYVE-1,VEGF-C and VEGFR-3 in tumor tissues(P<0.05),VEGF-C in serum of nude mice(P<0.05)and Lnc RNA ANRIL gene expression(P<0.05).2.Orthotopic xenograft tumor model in nude mice:(1)The results of IVIS Spectrum Imaging System showed that the fluorescence intensity of tumor in PZH group was obviously weaker than that in control group.(2)PZH could significantly inhibit the expression of LYVE-1,VEGF-C and VEGFR-3 in tumor tissues(P<0.05),VEGF-C in serum of nude mice(P<0.05)and Lnc RNA ANRIL gene expression(P<0.05).(3)The liver cells in the control group had obvious necrosis and inflammatory cell infiltration,with disordered structure of hepatic lobules and irregular arrangement of hepatocytes,while in the PZH group,there was no edema and necrosis,normal structure of hepatic lobules,obvious demarcation,neat arrangement of hepatocyte elements and no infiltration of inflammatory cells.In vitro study: 1.Lnc RNA ANRIL has the highest expression in HT-29 cells and HCT-116 cells(P<0.05),and the lowest expression in HCT-8 cells.The expression of Lnc RNA ANRIL in HCT-116/si ANRIL group was significantly lower than that in HCT-116/si NC group(p<0.05).The expression of Lnc RNA ANRIL in HCT-8/ANRIL group was significantly higher than that in HCT-8/NC group(p<0.05).Lnc RNA ANRIL silencing and overexpression inhibited and promoted cell proliferation,migration and invasion,respectively(P<0.05).Lnc RNA ANRIL silencing and overexpression down-regulated and up-regulated the protein expression of VEGF-C(P<0.05).2.The HLEC cell density,viability,migration,invasion and tube forming ability of HCT-116/si ANRIL group were significantly lower than that of HCT-116/si NC group.HLEC cell density,viability,migration,invasion and tube forming ability of HCT-8/ANRIL group were significantly higher than that of HCT-8/NC group(P<0.05).3.PZH can reduce HCT-8/ANRIL cells density,viability and VEGF-C secreted protein expression(P<0.05),but the concentration of PZH 0.25 mg/m L has no significant effect on HCT-8/ANRIL cell morphology and cytotoxicity.4.In HCT-8/ANRIL group,the viability,migration and invasion ability of HLEC cells were significantly higher than those of HCT-8/NC(P<0.05).In HCT-8/ANRIL+PZH and HCT-8/NC+PZH groups,the migration and invasion ability of HLEC cells decreased significantly(P<0.05).The viability,migration and invasion ability of HLEC cells in HCT-8/ANRIL+PZH group were higher than those in HCT-8/NC+PZH group(P<0.05).The results of tube formation are consistent with the trend of Transwell.Compared with HCT-8/NC,the expressions of MMP-1/-2/-7/-9,VEGFR-3,p-PI3 K and p-AKT in HCT-8/ANRIL group were up-regulated(P<0.05).The expression of these proteins decreased in HCT-8/NC+PZH group and HCT-8/ANRIL+PZH group(P<0.05).Conclusion: Lnc RNA ANRIL is an important target for PZH to inhibit lymphangiogene-sis.PZH inhibits Lnc RNA ANRIL/VEGF-C/PI3K/AKT pathway by down regulating the expression of Lnc RNA ANRIL,inhibits lymphangiogenesis,and the development and metastasis of colorectal cancer. |
