Effect And The Related Mechanisms Of PRDX2 On Vasclogenic Mimicry In Colorectal Carcinoma

Part one Expressions of PRDX2 and vasculogenic mimicry in colorectal carcinoma and their clinical significance Objective:To investigate the tissue expressions of PRDX2, vasculogenic mimicry and their related factors in colorectal carcinoma, explore their clinical significance. Methods:1.The protein expressions of PRDX2 and VM related factors VEGFR2, MMP-2 and LN-5γ2 in seventy cases of colorectal carcinoma tissues and their corresponding paracarcinoma tissues were detected by immunohistochemistry, then statistically analyzed the correlation between PRDX2 and VM related factors.2. The Relationship between protein expressions of PRDX2, VEGFR2, MMP-2 and LN-5γ2 with their clinicopathologic features were statistically analyzed.3. Vasculogenic mimicry was detected in colorectal carcinoma tissues by using CD34/periodic acid Schiff double staining, then the correlation between PRDX2 protein expression and vasculogenic mimicry were statistically analyzed. Results:1. The protein expressions of PRDX2, VEGFR2, MMP-2 and LN-5γ2 were relatively higher in colorectal carcinoma tissues as compared to their corresponding paracarcinoma tissues(P<0.05). The protein expression of PRDX2 was positively correlated with the protein expression of VEGFR2, MMP-2 and LN-5γ2(P<0.05).2. The protein expressions of PRDX2, VEGFR2, MMP-2 and LN-5γ2 were correlated with TNM classification and lymph node metastasis(P<0.05). The protein expressions of MMP-2 and LN-5γ2 were also correlated with the degree of tumor differentiation(P<0.05), but the protein expressions of PRDX2 and VEGFR2 were not correlated with the degree of tumor differentiation(P>0.05). All of them were not correlated with age, gender and tumor size(P>0.05).3. The positive cases of vasculogenic mimicry were 16(22.86%) in seventy cases of colorectal carcinoma tissues. The protein expression of PRDX2 was positively correlated with the expression of vasculogenic mimicry in colorectal carcinoma(P<0.05). Conclusion:The protein expressions of PRDX2, VEGFR2, MMP-2 and LN-5γ2 were relatively higher in colorectal carcinoma tissues as compared to their corresponding paracarcinoma tissues. The protein expression of PRDX2 was positively correlated with the protein expressions of VEGFR2, MMP-2 and LN-5γ2. The protein expressions of PRDX2, VEGFR2, MMP-2 and LN-5γ2 were correlated with their unfavourable clinicopathologic features. So the formation of vasculogenic mimicry was correlated with the protein expression of PRDX2.Part two Effect of PRDX2 on vasculogenic mimicry in colorectal carcinoma cells and its significance Objective: To explore effect and significance of PRDX2 on vasculogenic mimicry in colorectal carcinoma cells. Methods:1. The formation abilities of vasculogenic mimicry were observed by using in vitro three-dimensional cell culture in colorectal carcinoma cells.2. Effect of interference PRDX2 lentivirus on expression level of PRDX2 m RNA and protein were determined by Real-Time PCR and Western blot after interference PRDX2 lentivirus and negative control lentivirus transfected HCT116 cells.3. The formation abilities of vasculogenic mimicry were determined by using in vitro three-dimensional cell culture in the exogeneous VEGF simulated tumor microenvironment after interference PRDX2 lentivirus and negative control lentivirus transfected HCT116 cells.4. The abilities of cell migration and invasion were determined by using transwell chambers in the exogeneous VEGF simulated tumor microenvironment after interference PRDX2 lentivirus and negative control lentivirus transfected HCT116 cells.5. Vasculogenic mimicry related factors were determined by Real-Time PCR and Western blot after interference PRDX2 lentivirus and negative control lentivirus transfected HCT116 cells.6. The subcutaneous tumor model of nude mice was constructed to evaluate the PRDX2 interference on the changes of tumor growth. The pulmonary metastasis model of nude mice was constructed to evaluate the PRDX2 interference on the changes of tumor metastasis. Results:1. Only the poorly differentiated HCT116 cells can form vasculogenic mimicry among three colorectal carcinoma cell lines as compared to human umbilical vein Endothelial HUVEC cells.2. The m RNA and protein expression levels of PRDX2 reduced obviously after interference PRDX2 lentivirus transfected HCT116 cells as compared with the blank group and and negative control lentivirus group(P<0.05).3.The formation ability of vasculogenic mimicry in HCT116 cells increased after VEGF induction as compared with negative control(P<0.05). The formation ability of vasculogenic mimicry reduced obviously after interference PRDX2 lentivirus transfected HCT116 cells(P<0.05).4. The abilities of cell migration and invasion were in HCT116 cells increased after VEGF induction as compared with negative control(P<0.05). The abilities of cell migration and invasion were reduced obviously after interference PRDX2 lentivirus transfected HCT116 cells as compared with negative control(P<0.05).5. The protein expression level of VEGFR2 had no obvious change after interference PRDX2 lentivirus transfected HCT116 cells(P>0.05), and the expression level of phosphorylated VEGFR2 protein increased gradually with the increase of time after VEGF induction, but the expression level of phosphorylated VEGFR2 protein reduced obviously after interference PRDX2 lentivirus transfected HCT116 cells as compared with negative control(P<0.05). The m RNA and protein expression levels of MMP-2, MMP-9 and LN-5γ2 increased after VEGF induction, but the m RNA and protein expression levels of MMP-2, MMP-9 and LN-5γ2 reduced obviously after interference PRDX2 lentivirus transfected HCT116 cells as compared with negative control(P<0.05).6. The average subcutaneous tumor volume(0.82 ± 0.11cm3) and the number of pulmonary metastatic tumor(27.13 ± 3.55)decreased after interference PRDX2 lentivirus transfected HCT116 cells as compared with negative control(1.43 ± 0.17 cm3;43.62 ± 5.81)which had statistically significance(P<0.05). PRDX2 interference can inhibit tumor growth and metastasis in colorectal carcinoma. Conclusion:PRDX2 can enhance abilities of vasculogenic mimicry formation, cell migration and invasion which promote colorectal carcinoma growth and metastasis.Part three PRDX2 promotes vasculogenic mimicry in colorectal carcinoma through ERK1/2 signal pathway Objective:To investigate effect of ERK1/2 signal pathway on the formation of vasculogenic mimicry and its relationship with PRDX2 in colorectal carcinoma. Methods:1. The protein expression levels of ERK1/2 and phosphorylated ERK1/2 were detected by Western blot after PRDX2 interference and exogeneous VEGF induction.2. The formation abilities of vasculogenic mimicry were determined by using in vitro three-dimensional cell culture in the exogeneous VEGF simulated tumor microenvironment after using ERK1/2 signal pathway inhibitor PD98059 interference in HCT116 cells.3. The abilities of cell migration and invasion were determined by using transwell chambers in the exogeneous VEGF simulated tumor microenvironment after using ERK1/2 signal pathway inhibitor PD98059 interference in HCT116 cells.4. Vasculogenic mimicry related factors were detected by Real-Time PCR and Western blot after using ERK1/2 signal pathway inhibitor PD98059 interference in HCT116 cells.5. The protein expressions of MMP-2, MMP-9 and LN-5γ2 in subcutaneous tumor tissues were detected by immunohistochemistry. Results:1. The protein expression level of ERK1/2 had no obvious change after interference PRDX2 lentivirus transfected HCT116 cells as compared with negative control(P>0.05), and the expression level of phosphorylated ERK1/2 protein increased gradually with the increase of time after VEGF induction, but the expression level of phosphorylated ERK1/2 protein reduced obviously after interference PRDX2 lentivirus transfected HCT116 cells as compared with negative control(P<0.05). PRDX2 promotes vasculogenic mimicry in colorectal carcinoma is correlated with ERK1/2 signal pathway.2.The formation ability of vasculogenic mimicry reduced obviously after ERK1/2 signal pathway inhibitor PD98059 interference in HCT116 cells as compared with negative control(P<0.05).3. The abilities of cell migration and invasion reduced obviously after ERK1/2 signal pathway inhibitor PD98059 interference in HCT116 cells as compared with negative control(P<0.05).4.The m RNA and protein expression levels of MMP-2, MMP-9 and LN-5γ2 reduced obviously after ERK1/2 signal pathway inhibitor PD98059 interference in HCT116 cells as compared with negative control(P<0.05).5. The protein expression levels of MMP-2, MMP-9 and LN-5γ2 reduced obviously after PRDX2 interference as compared with negative control(P<0.05). Conclusion:PRDX2 can promote vasculogenic mimicry through activating ERK1/2 signal pathway in colorectal carcinoma. PRDX2 interference can suppress the activation of ERK1/2 signal pathway, reduce the expression level of vasculogenic mimicry related factors, and inhibits vasculogenic mimicry,tumor growth and metastasis in colorectal carcinoma.