Hypoxic Response Of MMP-9 In Esophageal Squamous Cell Carcinoma And Its Role In Invasion And Metastasis

Background Esophageal carcinoma(esophageal carcinoma,EC),one of the most common digestive system malignancies in China whose mortality rate of patients accounts for the sixth rate of cancer mortality in the whole world.With the early diagnosis and early treatment of esophageal cancer,although the improvement of its prognosis,but the five-year survival rate of patients is still unoptimistic,meanwhile tumor invasion and metastasis is an important reason that leading to postoperative recurrence and death of patients.The studys in recent years shows that HIF-1?(hypoxia-inducible factor-1?)and MMP-9(matrix metalloproteinase-9)have played an important role in the invasion and metastasis of various human malignant tumors.HIF-1?and MMP-9 are highly expressed in many malignancies and are associated with tumor invasion and metastasis.It has been reported that HIF-1?can promote the invasion of tumor cells by up-regulating the expression of MMP-9 in breast cancer and lung cancer cells.Up to now,in esophageal squamous cell carcinoma HIF-1?can regulate the expression of MMP-9 and thus affect the invasion of tumor cells has not been reported.Methods 1.Immunohistochemical SP method was used to detect the expression of HIF-1? and MMP-9 protein in 91 cases of esophageal squamous cell carcinoma and adjacent normal tissues,and to analyze the relationship between HIF-1? and MMP-9.2.To investigate the correlation between HIF-1?and MMP-9.The correlation between each of them with clinic pathologic parameters including gender,age,histological grade,invasion depth,lymph node metastasis and TNM staging were analyzed in ESCC tissues.Furthermore,the correlation of HIF-1?protein expression with MMP-9 were analyzed in ESCC tissues.3.After we estabilsh a chemical hypoxia model with using 150 ? mol/L Co Cl2,Real-time PCR and Western blot were used to detect the expression on m RNA level and protein level of HIF-1a and MMP-9 respectively.Changes of MMP-9 protein expression in supernatant was also detected by ELISA.4.After the transfection with HIF-1?si RNA,the expression of HIF-1a and MMP-9 m RNA and protein in EC-1 cell were detected by Real-time PCR and Western blot.The expression of MMP-9 protein in the supernatant were detected by ELISA.Transwell in vitro invasion assay was used to detect the ability of cell invasion.Result 1.In 91 cases of esophageal squamous cell carcinoma,the expression of HIF-l? in esophageal squamous cell carcinoma(74.725%)was significantly higher than that in normal esophageal epithelium(39.560%),and the expression of MMP-9 in esophageal squamous cell carcinoma(76.923%)was significantly higher than that in normal esophageal epithelium(24.175%)too(P <0.05).2.In 91 cases of ESCC tissues,The expression of HIF-l? was positively correlated with TNM staging(I vs II vs III: 44.4% vs 78.4% vs 90.9%)(P<0.05),but not correlated with gender,age and tumor histological grade or tumor invasion depth and lymph node metastasis(P>0.05).The expression of MMP-9 was positively correlated with histological grade(?vs?vs?:64.7% vs 76.0% vs 87.5%,P<0.05),tumor invasion depth(T1 vs T2 vs T3:58.3% vs 62.5% vs 87.3%,P<0.05),lymph node metastasis(no vs yes:71.4% vs 95.2%,P<0.05)and TNM staging(?vs?vs?:55.6% vs 76.5% vs 95.5%,P<0.05),but not correlated with gender,age or tumor histological grade(P>0.05).HIF-l? protein expression was positively correlated with MMP-9 protein(?s=0.406,P<0.05).3.The m RNA expression of HIF-1? and MMP-9 at 0h,12 h and 24 h after Co Cl2 stimulation was detected by Real-time PCR.The results showed that the relative expression of HIF-1? m RNA at 0h,12 h and 24 h were 1±0.023,1.873±0.231,2.714±0.301(P <0.05).The expression of HIF-1? at 24 h was significantly higher than that of 0h(P <0.05).The relative expression levels of MMP-9 m RNA at 0h,12 h and 24 h were 1 ± 0.035,1.773±0.255,2.932±0.407,and the expression of MMP-9 m RNA at 24 h was significantly higher than that of 0h(P <0.05).Western blot was used to detect the protein expression of HIF-1a and MMP-9 at 0h,12 h and 24 h after Co Cl2 stimulation.The results showed that the relative expression of HIF-1? protein at 0h,12 h and 24 h were 0.615 ± 0.092,1.679 ± 0.104,2.105±0.111(P <0.05).Compared to 0h,the expression at 24 h was significantly increased(P <0.05).The relative expression levels of MMP-9 protein were 0.754 ± 0.085,1.735 ± 0.255,2.132 ± 0.346 at the time of 0h,12 h and 24 h after Co Cl2 stimulation,and the expression of MMP-9 protein at 24 h was significantly higher than that of 0h.After stimulated by Co Cl2 the secretion of MMP-9 in supernatant at 0h,12 h and 24 h was detected by enzyme-linked immunosorbent assay(ELISA).The secretion of MMP-9 in EC-1 supernatant were 386.974±20.260 pg/ml,632.817±35.335 pg / ml,702.141±43.771 pg/ ml respectively.The expression of 12 h and 24 h was significantly higher than that of 0h(P <0.05).4.The expression of HIF-1? m RNA(0.262 ± 0.023 vs 1 ± 0.048)and protein(0.464 ± 0.086 vs 0.694± 0.085)were significantly decreased in HIF-1? si RNA transfected group compared with normoxia group after transfection of HIF-1a si RNA into esophageal cancer cell EC-1 cells(P <0.05).The expression of HIF-1? m RNA(0.353 ± 0.025 vs 2.351 ± 0.331)and protein(0.519±0.154 vs 2.165±0.137)in dual group was significantly lower than that of hypoxia group(P <0.05).The expression of MMP-9 m RNA(0.315 ± 0.038 vs 1 ± 0.052)and protein(0.549 ± 0.075 vs 0.774 ± 0.107)in HIF-1? si RNA transfection group was significantly lower than that in normoxia group,and compared with hypoxia group,the expression of MMP-9 m RNA in dual group(0.418 ± 0.044 vs 2.601 ± 0.417)and protein(0.623 ± 0.159 vs 2.251 ± 0.133)was significantly decreased(P <0.05).The results of ELISA showed that the secretion of MMP-9 protein was significantly decreased in HIF-1? si RNA transfection group compared with normoxia group(128.455 ± 14.156 pg / ml vs375.974 ± 22.346 pg / ml)while compared with hypoxia group,tthe secretion of MMP-9 protein in dual group(235.354 ± 28.742 pg / ml vs 661.654 ± 32.551 pg / ml)was significantly decreased(P <0.05).Transwell invasion assay showed that the number of transmembrane cells in HIF-1? si RNA transfected group was significantly lower than that in normoxia group(32 ± 6.52 vs 72 ± 7.15),and compared with hypoxia group the number of transmembrane cells in dual group(45 ± 6.91 vs 156 ± 15.24)was significantly decreased(P <0.05).Conclusion 1.HIF-1? and MMP-9 were highly expressed in esophageal squamous cell carcinoma and correlated with tumor invasion and metastasis.2.HIF-1? may enhance the invasion of esophageal squamous cell carcinoma EC-1 cells by regulating the expression of MMP-9.