The Study Of The Activation Of P-STAT3 In VM Formation And Its Mechanism In Colorectal Cancer

Purpose This paper mainly studies that the STAT3 activation makes effect on the formation of vasculogenic mimicry(VM)in colorectal cancer and the correlative mechanism.Through immunohistochemistry methods,the existance of VM can be found and the p-STAT3 in colorectal cancer can be expressed.And the relationship between p-STAT3 expression and clinical pathological data and clinical outcomes is analysesd.Then through statistical correlation between p-STAT3 and EMT related proteins and VM related proteins.Besides,the formation of STAT3 activation in VM positive data in colorectal cancer is focused.At the same time,several experiments are done in vitro,and the possible molecular mechanism of p-STAT3 activation in the two colorectal cancer cell lines are studied to explore the vasculogenic mimicry by using IL6 and AG490.Methods(1)The expression of p-STAT3,VEGF,VE-cadherin,MMP-2,Vimentin,E-cadherin,Snail,and STAT3 in the colon cancer specimens are detected by immunohistochemical staining and the results of CD31/PAS double staining.(2)In colorectal cancer specimens,analysize the correlation between the activation of p-STAT3 and CD31/PAS double staining,the correlation between the p-STAT3 and VEGF,VE-cadherin,MMP-2,E-cadherin,Vimentin,Cyclin D1 expression.And explore the relationship between p-STAT3 activation and clinical prognosis.(3)Firstly,through the MTT experiment,find the suitable concentration of IL6 and AG490 which does not affect the proliferation of the cells.And then through the Western Blot analysis,find the concentration of IL6 and AG490 treating on the cells that can make the obvious changes on p-STAT3 activation,and choose the most suitable concentration to the experiment in vitro.(4)The morphological changes of cells and the changes of invasion,migration and tube formation are investigated,when using IL6 induced the activation of p-STAT3,in HCT116 cell line and HT29 cell line.(5)The morphological changes of cells and the changes of invasion,migration and tube formation are investigated,when using AG490 down regulated the activation of p-STAT3,in HCT116 cell line and HT29 cell line.(6)Western Blot and RT-PCR are used to detect the expression of EMT markers(E-cadherin,Vimentin)in the protein and m RNA levels of the IL6 and AG490 compared with the control group.(7)The expression of VE-cadherin,MMP-2,Snail and Twist are detected by Western Blot assay under the treatment of IL6 and AG490.(8)Detection of p-STAT3 and Vimentin co-expression under the treatment of IL6 or AG490 in the two colorectal cancer cell lines by immunofluorescence double staining.Result(1)There is no significantly difference between the activation of P-STAT3 in colorectal cancer and the age,gender,tumor size and the grade of tumor.There is significant difference in TNM stage,and the difference is statistically significant(P =0.015).The positive rate of p-STAT3 in the patients with distant metastasis is higher than patients without distant metastasis.And the difference is statistically significant(P =0.040).And p-STAT3 is higher in patients with lymph node metastasis than without that,which is statistically significant(P=0.046).Kaplan-Meier survival analysis showed that the survival time of p-STAT3 positive group is significantly lower than that of negative group(P =0.0045).(2)In colon cancer,P-STAT3 expression in VM positive group is higher than that in the negative group.And the difference is statistically significant(?2=4.045,P=0.044).(3)P-STAT3 activation is and positively correlated with VEGF expression,VE-cadherin expression and MMP2 expression in colorectal cancer.(4)P-STAT3 is negatively correlated with the expression of E-cadherin and positively associated with Vimentin expression and Snail expression in colorectal cancer.(5)To observe the effect of IL6 induced activation of p-STAT3 on colorectal cancer,The results showed that compared with the control group,the experimentalgroup had increased migration,invasion and tube formation in vitro.(6)To explore the effect of AG490 on the inhibition of p-STAT3 activation,the results showed that compared with the control group,cells in the experimental group had decreased migration,invasion and tube formation in the two cell lines.(7)WB results showed that comparing with the control group,IL6 induced the expression of E-cadherin protein is significantly reduced,p-STAT3,Vimentin,Snail,Twist,VE-cadherin,MMP-2 protein expression are significantly increased.(8)Te results of WB showed that in two colorectal cancer cell lines,compared with the control group,the expression of E-cadherin protein with AG490 treated is significantly increased,the expression of p-STAT3,Vimentin,Snail,Twist,VE-cadherin and MMP-2 protein are significantly reduced.(9)RT-PCR results shows that,in the two colorectal cancer cell lines,compared with the control group,IL6 induced STAT3 m RNA expression in the experimental group had no obvious changement,but E-cadherin expression of m RNA decreased significantly,Vimentin increased in the expression level of m RNA.(10)The results of RT-PCR shows that in two colorectal cancer cell lines,compared with the control group,the STAT3 m RNA expression had no obvious change,but E-cadherin m RNA expression is significantly increased and Vimentin m RNA expression level decreased in the AG490 treated group.(11)The immunofluorescence double staining results shows that in two colorectal cancer cell lines,compared with the control group,the experimental group with IL6 induced,p-STAT3 and Vimentin co-expression is increased,but with AG490 p-STAT3 and Vimentin co-expression is decreased.Conclusion(1)p-STAT3 is highly expressed in colorectal cancer,which is significantly correlated with VM,and is associated with poor prognosis.(2)when use IL6 treated on the two cell lines to induce the activation of p-STAT3,the migration,invasion and the ability of tube formation are enhanced.(3)when use AG490 treated on the two cell lines to inhibit the activation of p-STAT3,the migration,invasion and the ability of tube formation are decreased.(4)The activation of p-STAT3 can affect the expression of Vimentin then inducing EMT,and regulate VM related protein VE-cadherin and MMP2,promote the formation of VM.