The Role Of CIAP2 In The Growth And Metastasis Of Gallbladder Carcinoma And Its Regulation Mechanism

?Background? The mechanism of gallbladder cancer(GBC)is far from clear.Inhibition of apoptosis is a key factor in tumor cell survival.This process provides tumor cells the ability to develop and metastasize in harsh microenvironments.Inhibitors of Apoptosis Proteins(IAPs)are a class of highly conserved proteins predominantly known for inhibition of apoptosis and closely related with tumor occurrence and development.Currently,the most studied in the IAPs family is the Survivin.and the relationship between c IAP2 and the tumor is relatively rare,and the relationship with GBC has not been reported.Cellular inhibitor of apoptosis 2(c IAP2)is another important member of IAPs family.There is little research on the relationship between c IAP2 and carcinoma,let alone to GBC.After binding to its receptor,TNF-a can recruit RIP1,c IAP2 to form the signal "complex I",which initiates TNF-a-induced cell survival or apoptosis pathways.RIP1 ubiquitin is the important factor that determines cell survival.As a component of "complex I",c IAP2 has the ubiquitin enzyme activity.It is speculated that c IAP2 promotes the survival of tumor cells through ubiquitination of RIP1 and promotes the development of TNFa-induced gallbladder cancer.Therefore,this issue is to investigate the role of c IAP2 in the growth and metastasis of gallbladder carcinoma and its regulatory mechanism.The results are helpful to reveal the mechanism of the development of gallbladder cancer.?Methods? 1.Fluorescence PCR and immunohistochemistry were used to detect the expression of c IAP2 in clinical GBC,and to analyze its relationship with clinicopathological parameters and prognosis.2.Knockdown and overexpression of c IAP2 in GBC cells NOZ,CCK8,scratch test,trsanwell and co-culture with HDLEC were used to test the effect of c IAP2 on the malignant biological behavior in NOZ.3.The orhtotopic xenograft models of GBC in nude mice were established to observe the effect of c IAP2 on the growth and metastasis of GBC in nude mice.4.The dual-luciferase reporter assay,western blot,immunofluorescence and ELISA were used to investigate the correlation between c IAP2 and TNFa-NF-?B(P65),TNFa-VEGF-C.The co-immunoprecipitation and immunofluorescence localization were used to analyze the interaction between c IAP2 and RIP1.The ubiquitin mutants was used to investigate the way of the c IAP2 targeted modified RIP1.?Results?: 1.c IAP2 expression in GBC and its correlation with lymphatic metastasis and prognosis in patients.The m RNA and protein levels of c IAP2 in the GBC tissues were significantly higher than those in the matched non-tumor gallbladder tissues(m RNA: 6.54±0.88 Vs 3.25±0.39,P<0.05;protein: 0.2854 ±0.0061 Vs 0.1722±0.0084,P<0.05).According to the analysis of clinicopathological parameters,the expression of c IAP2 was positively correlated with lymph node metastasis in patients(X 2= 13.227,P<0.001).The median survival time of patients with elevated c IAP2 expression(18 months)was significantly lower than that of patients with lower c IAP2 expression(34 months).The overall survival rate of patients with elevated c IAP2 expression(16.7%)was significantly lower than that of patients with lower c IAP2 expression(64.3%),P<0.05.It is suggested that the high expression of c IAP2 can lead to lymphatic metastasis of GBC and poor prognosis.2.The effect of c IAP2 on malignant biological behavior and lymphangiogenesis of NOZ cells We successfully constructed two groups of NOZ with knockdown expression of c IAP2(sic IAP2#1 and sic IAP2#2)and the group of NOZ with overexpression of c IAP2.CCK-8 assay: Cell proliferation in the sic IAP2 #1(0.840±0.028)and sic IAP2 #2(0.773±0.015)groups was slower than that of the si RNA/NC cells(1.118±0.058);Cell proliferation in the group of c IAP2 overexpression(1.314±0.005)was higher than that in the control group(1.001±0.002).Scratch assays: Cell migration in the sic IAP2 #1(38.67±1.20%)and sic IAP2 #2(40.00±2.08%)groups was lower than that of the si RNA/NC cells(56.67±2.40%).Cell migration in the group of c IAP2 overexpression(84.67±2.60%)was higher than that in the control group(55.0±2.08%).Transwell chamber assays: The number of invaded cells was significantly lower in the sic IAP2 #1(148±8.32)and sic IAP2 #2(154.7±8.37)groups than that in the si RNA/NC group(290.3±11.83);The number was higher in the c IAP2 overexpression group(376.3±7.84)than that in the control group(257.3±9.26).3-D co-culture system of NOZ and HDLEC: the number of tubes was significantly lower in the sic IAP2 #1(11.0±0.58)and sic IAP2 #2(11.33±1.86)groups than that in the si RNA/NC group(18.67±1.76);The number was higher in the c IAP2 overexpression group(27.33±0.88)than that in the control group(17.6±1.45).3.The effect of c IAP2 on the formation of GBC,lymph node metastasis(LNM)and lymphatic vessel number(LVN)in nude mice.Compared with the si RNA/NC group,the average weight of GBC(1.074±0.160 g Vs 1.701±0.228 g,P=0.0405),the number of LNM(2.250±1.11 Vs 7.125±1.977,P=0.0497)and LVN(5.625±1.668 Vs 11.88±2.781,P=0.0436)were reduced in the sic IAP2 #1 groups.Compared with the control group,the average weight of GBC(2.544±0.298 g Vs 1.689±0.239 g,P=0.0421),the number of LNM(18.750±4.843 Vs 7.000+1.982,P= 0.0414)and LVN(17.75±1.509 Vs 11.50±2.22,P=0.0354)were increased in the c IAP2 overexpression group.4.The effect of c IAP2 on TNFa-induced NF-?B and VEGF-C activity,as well as the ubiquitination of RIP1.Dual luciferase report system was used to measure the impact of c IAP2 on NF-?B activity of different groups NOZ cells in response to TNF-a stimulation.The activity of NF-?B in the sic IAP2#1 and sic IAP2#2 groups exhibited lower than that in the si RNA/NC group.The activity of NF-?B in the c IAP2 overexpression group exhibited higher than that in the control group.Westren blot and cellular immunofluorescence were used to detect NF-?B translocation.When knockdown c IAP2 expression,NF-?B translocation from the cytosol into the nucleus was markedly reduced in response to TNF-a stimulation.when overexpression of c IAP2,NF-?B translocation from the cytosol into the nucleus was markedly increased.ELISA was used to detect the activity of VEGF-C.The activity of VEGF-C in the sic IAP2#1 and sic IAP2#2 groups exhibited lower than that in the si RNA/NC group.The activity of VEGF-C in the c IAP2 overexpression group exhibited higher than that in the control group.Co-immunoprecipitation and fluorescence colocation were used to observe that c IAP2 and RIP1 were interacted in NOZ cells,and The ubiquitin mutants indicate that c IAP2 promotes the stability of RIP1 through Lys63-mediatied polyubiquitination.?Conclusions?: 1.The level of c IAP2 was upregulated in GBC,and was associated with lymph node metastasis and prognosis of patients.2.c IAP2 could promote the proliferation,invasion and lymphangiogenesis of GBC cells,thereby promoting the growth of gallbladder cancer,the formation of microlymphangiogenesis and lymph node metastasis.3.c IAP2 could enhance TNFa-induced activity of NF-?B and VEGF-C.4.c IAP2 interacted with RIP1 in NOZ,c IAP2 modified RIP1 through Lys63-mediatied polyubiquitination.