Inhibitory Effect Of Decorin On Invasion Of Colorectal Adenocarcinoma Cell HCT116 In Vitro

Background Colorectal Cancer?CRC?is the fifth leading cause of cancer-related death in China.CRC‘s recurrence and metastasis are the main reasons of death in patients with colorectal cancer.The edge of the solid tumors always have adequate blood supply,and high oxygen partial pressure.But the lower the oxygen partial pressure is closer to the central region,which causes the central region of hypoxic tumor cells necrosis,resulting in hypoxia tolerance.And different oxygen partial pressures have different effects on the cells‘ growth.So there are a variety of microenvironment such as normoxia and hypoxia for a solid tumor.Therefore,it is of great significance to study the invasion and metastasis mechanism of CRC under different microenvironment.Research shows that: decorin?DCN?is a core glycoprotein,with related receptors and internalize the decomposition of RTK induced resistance,thereby inhibiting the proliferation and migration of tumor cells in vitro,such as lung cancer,renal carcinoma,colorectal cancer.But DCN inhibitory effect and mechanism of invasion of CRC cells in vitro is unclear,therefore,this study will explore DCN effect on invasion of colorectal cancer cells HCT116 in normoxic and hypoxic conditions in vitro and its possible mechanism.Objective In this paper,we intend to explore the function of DCN on the invasion of colorectal cancer cells HCT116 in vitro,in normoxia and hypoxia induced by CoCl₂,and to explore the relationship between DCN and HIF-1? / CD133 / TIMP-2 pathway in normoxia and hypoxia induced by CoCl₂,so as to provide a new idea for the treatment of clinical solid tumors.Method Using the chemical hypoxia mimetic agent CoCl₂ to establish the chemical hypoxia model;then HCT116 cells were treated with decorin,based on the model of hypoxia.?1?Using the MTT assay to detect the effect of DCN on the proliferation of HCT116 cells in vitro in the normal and hypoxic environment;?2?Using the Transwell invasion assay to detect the effect of DCN on the invasive ability of HCT116 cells in the normal and hypoxic environment;?3?Using the Real-time PCR method to detect the expression of CD133 and TIMP-2 mRNA in HCT116 cells under normoxic and hypoxic conditions;?4?Using the Western blot method to detect the expression of HIF-1??CD133 and TIMP-2 protein of HCT116 cells under normoxic and hypoxic conditions.Result 1 Screening the appropriate concentration of CoCl₂ to establish the chemical hypoxia model1.1 The results of MTT showed that the inhibitory effect of CoCl₂ on HCT116 cells was dose-dependent,and the inhibitory effect increased with the increase of CoCl₂ concentration?P <0.05?.1.2 The results of Western blot showed that the expression of HIF-1? / CD133 / TIMP-2 protein increased first and then decreased with the increase of CoCl₂ concentration.In this study,we investigated the invasion mechanism of DCN in the colorectal adenocarcinoma cell line HCT116 induced by CoCl₂-induced hypoxia in vitro.Therefore,combined with the above results,we selected the concentration of CoCl₂?100 ?mol/L?for subsequent experiments which has a small effect on the invasion of HCT116 and the larger changes the HIF-1? / CD133 / TIMP-2 protein.2 The effects of different concentrations of DCN on the proliferation of HCT116 cellsMTT assay was used to detect the effect of different concentrations of DCN on the proliferation of HCT116 cells.The results showed that when the concentration of DCN was 0.5,1 and 3 mg/L,the proliferation of HCT116 cells was not significantly inhibited?P 0.05?;when the DCN concentration was 6 mg/L,the proliferation ability of HCT116 cells was significantly decreased?P <0.01?.Subsequent experiments were performed with the concentration of DCN was 1 and 3 mg/L for the invasion experiments in vitro.3 The effects of DCN on the invasion of HCT116 cells in normoxia and hypoxia3.1 Transwell invasion assay was used to detect the effect of DCN on the invasive ability of HCT116 cells under normoxia.The results showed that the number of HCT116 cells was?241±46?,?168±46?,?51±17?fields in each well when the concentration of DCN was 0,1 and 3 mg/L?10 × 10 times??P <0.01?.3.2 Transwell invasion assay was used to detect the effect of DCN on the invasive ability of HCT116 cells under hypoxic conditions.The results showed that the number of CN,CoCl₂,CoCl₂ + DCN?1 mg / L?,CoCl₂ + DCN?3 mg / L?was?207±61?,?213±64?,?156±54?,?44±17?fields in each well?10 × 10 times??P <0.01?.4 The effects of DCN on the expression of TIMP-2 and CD133 mRNA in HCT116 cells under normoxia and hypoxia4.1 Real-time PCR results showed that DCN?3 mg/L?significantly up-regulated the expression of TIMP-2 mRNA in HCT116 cells under the normoxic condition?P <0.01?,but had no significant effect on the CD133 mRNA expression?P> 0.05?;4.2 Real-time PCR results showed that DCN?3 mg/L?significantly down-regulated the expression of TIMP-2 mRNA in HCT116 cells under the CoCl₂-induced hypoxia environment?P <0.01?,but had no significant effect on the CD133 mRNA expression?P> 0.05?.5 The effects of DCN on the protein expression of HIF-1? / CD133 / TIMP-2 in HCT116 cells under normoxic and hypoxic conditions5.1 The results of Western Blot showed that the expression of TIMP-2?P <0.01?and HIF-1??P <0.05?was significantly up-regulated by DCN?3 mg/L?under normoxic conditions,but had no significant effect on CD133 expression?P 0.05?.5.2 The results of Western Blot showed that DCN?3mg/L?significantly downregulated the expression of HIF-1?,CD133 and TIMP-2 in HCT116 cells in the CoCl₂-induced hypoxic environment?P <0.01?.Conclusion1.DCN can inhibit the proliferation of human colorectal cancer cell HCT116 in vitro,and in a concentration-dependent manner;2.DCN could inhibit the invasive ability of human colorectal adenocarcinoma cell line HCT116 in normoxia and hypoxic environment induced by CoCl₂,and it was concentration-dependent.3.In normoxia and hypoxic environment induced by CoCl₂,DCN inhibits the invasive ability of human colorectal adenocarcinoma cell line HCT116 in vitro by different mechanisms.In hypoxia condition it may play a role by regulating the HIF-1? / CD133 / TIMP-2 pathway;Under normoxic conditions,it may play a role mainly by promoting the expression of TIMP-2,and not with HIF-1a / CD133 pathway.