Significance Of Hypoxia Inducible Factor-1 Alpha And FasL Expression In The Invasion And Metastasis Of Colorectal Carcinoma

Background:Colorectal carcinoma is one of the common malignancies with poor prognosis and low survival rate,and the infiltration and metastasis are the key agents leading to death of patients.The metastasis of colorectal carcinoma is a comprehensive biological procedure with multiple factors,multiple stages and multiple steps.The tolerance and accommodation of cells to intratumoral hypoxia is the basement of tumor metastasis. Hypoxia inducible factor-1 alpha(HIF-1α),which is the center regulator of oxygen pressure, can adjust related genetic transcription,participate in tumor invasion and metastasis as well as adjust the intracellular oxygen steady state.The regulation mechanism of HIF-1αin invasion and metastasis of tumor is so complex that it hasn't been understood clearly.Objective:To study the expression of HIF-1αand FasL,to expolre the mechanisms of invasion and metastasis in colorectal carcinoma,and to provid a new target and theory basement for tumor therapy.Methods:1.With the method of molecular cloning,the FasL-pcDNA3.1(+) and pcDNA3.1(-) plasmid were reconstructed to get the new FasL-pcDNA3.1(-) plasmid which had been verificated.With the method of lipid infection,the empty plasmid,FasL-pcDNA3.1(+) and FasL-pcDNA3.1(-) were transfected into human rectal carcinoma cells HR-8348,then HR-8348 cells with different invasive ability,HR-8348_L,HR-8348_F and HR-8348_As were constructed and verificated,and the non-transfected cell HR-8348_B was blank.The cells shape were observed,the invasion ability and proliferation speed were detected in all groups.2.Hypoxia models for HR-8348_B,HR-8348_L,HR-8348_F and HR-8348_As were constructed with chemical modelling.Then the expression of HIF-1αin all groups was quantitated at 0h,6h,12h and 24h after hypoxia with Western blot.At 12h after hypoxia, the HIF-1αlevels were detected with immunocytochemistry and the percents of positive cell were calculated;besides,cell distributions of different cell life cycle were detected with flow cytometry;cell reproductive activities were detected with the method of MTT;and cell apoptosis status was assessed with TUNEL.3.The expression of HIF-1αand FasL were detected by immunohistochemistry in the specimens from 120 colon cancer patients and 30 colon adenoma patients,and the association of HIF-1αexpression level and clinical pathological characteristics,including FasL expression level of colorectal cancer were analyzed.Results1.The new constructed plasmid FasL-pcDNA3.1(-) was consistent with the requirement,which size of enzyme cutting parts was right,and the exactitude ratio of sequencing was 99.2%.The part of FasL and As-FasL could be got from HR-8348_F cell and HR-8348_As cell with RT-PCR,respectively,certificating the transfections of all plasmids were successful.With inverted microscope,the heteromorphism of HR-8348_F cell was more obviously.The number of HR-8348_F cells(12.930±2.434) which penetrated Transwell was significantly more than that of HR-8348_B(8.133±1.959),HR-8348_L(7.670±2.093) and HR-8348_As cells(7.870±1.685),respectively(P＜0.05) in cell monolayer intrafiltration experiment in vitro.The population doubling time of HR-8348_B,HR-8348_L,HR-8348_F and HR-8348_As was 28.3h,41.0h,24h and 35.4h,respectively.2.At 12h after hypoxia,the positive rate of HIF-1αexpression was higher in HR-8348_F cells(76.0%) than in HR-8348_B(48.5%),HR-8348_L(43.0%) and HR-8348_As cells(39.5%),respectively(P＜0.05),and there was no significant differences among those latter 3 groups(P＞0.05).HIF-1αprotein was coloration at 120kD by Western blot,and the expression level of HIF-1αwas not markedly different in all groups at 0h and 6h after hypoxia(P＞0.05),but was significantly higher in HR-8348_F cell than in HR-8348_B, HR-8348_L and HR-8348_As cell(P＜0.01) at 12h and 24h after hypoxia.Under the environment of hypoxia,the proliferation index was significantly higher in HR-8348_F (60.43±3.61) than in HR-8348_B(40.01±3.22),HR-8348_L(41.30±3.96) and HR-8348_As cell (35.87±4.28),respectively(P＜0.05),however,both inhibition rate of proliferation and apoptotic index were significantly lower in HR-8348_F(17.30±2.08 and 13.10±1.06) thanin HR-8348_B(33.70±4.56 and 21.60±1.33),HR-8348_L(34.20±4.12 and 20.10±1.17), HR-8348_As(38.00±4.79 and 23.90±1.25),respectively(P＜0.05).3.The positive rate of HIF-1αexpression was 71.7%(86/120) in colorectal cancer and 89.3%(25/28) in hepatic metastasis,which were obviously higher than that in colon adenoma(20.0%,6/30) and normal tissue(5.8%,7/120)(P＜0.01).Similarly,the positive rate of FasL expression in colorectal cancer(69.2%,83/120) and hepatic metastasis(100.0%, 28/28) were obviously higher than that in colon adenoma(23.3%,7/30) and normal tissue (15.0%,18/120).There was no relation between HIF-1αexpression and the sex,age, pathohistology type and differentiation degree of colon cancer patients(P＞0.05),but close relation with lymph nodes state and Dukes staging(P＜0.05).Furthermore,the positive rate of HIF-1αand FasL expression in primary focus with hepatic metastasis(92.9%,26/28 and 85.7%,24/28) were markedly higher than those without hepatic metastasis(65.2%,60/92 and 64.1%,59/92)(P＜0.01).Expression of HIF-1αand FasL were positive correlation in metastasis of colorectal carcinoma(r=0.924,P＜0.01).Conclusions:1.The expression intensity of intracellular FasL is positive correlation with the invasive ability in colorectal carcinoma.2.The expression enhancement of intracellular FasL in colorectal carcinoma could increase the expression of HIF-1α,which could promote tumor cells to adapt hypoxia,and lead to accelerated proliferation and reduced apoptosis of cells.3.The expression level of HIF-1αin colorectal cancer tissue is positive correlated to the level of FasL,which is highly correlated to the cytobiologic characteristics of colorectal carcinoma,and may be a valuable reference to estimate tumor invasion and metastasis.