| Objectives:To investigate the relationship between the expression of cysteine-rich motoneuron protein 1(CRIM1)and angiogenesis in cervical squamous cell carcinoma and its molecular mechanism.Methods:1.The expression of CRIM1 protein in cervical squamous cell carcinoma and chronic cervicitis tissue were detected by immunohistochemistry.Microvessel density(MVD)was assessed by CD34 endothelial labeling in combination with Weinner counting to analyze the relationship between CRIM1 expression and MVD.2.SiHa cells were transiently transfected with pCMV3-CRIM1 plasmid.Western blot was used to identify the transfection efficiency to obtain CRIM1 gene overexpression.Then western blot was used to detect the expression of vascular endothelial growth factor protein and ELISA was used to detect the change of VEGF content in the supernatant.The supernantan of those groups of cancer cells mentioned above was extracted to treat HVECs.Then the endothelial proliferative ability,migration ability and tabulation capcity was evaluated to observe the influence of CRIM1 on the agniogenesis-inducing ability of cervical squamous carcinoma cells by performing CCK8 assay,transwell migration assay and Magrigel in vitro tube formation asay,respectively.3.The CRIM1 gene was transient transfection in SiHa cells.After 36 housr of CRIM1 gene transfection,the SiHa cells were treated with the inhibitor of p-ERK,U0126,and the change of the expression level of CRIM1,ERK,p-ERK and VEGF was measured to explore the signaling mechanism of CRIM1-mediated VEGF up-reguation.The cellular supernatant of the cancers with ERK signaling inhibition was also extracted to treat HUVECs and the angiogenic functions of endothelial cells(including proliferation,migration and tabulation)was also evaluated with the methods mentioned above to further reveal the role of ERK/VEGF signaling in the promoting effects of CRIM1 upon the angiogenesis-inducing capacity of cervicalsquamous carcinoma.4.Y15,an inhibitor of p-FAK,was used to treat SiHa cells that transfected with CRIM1,and the expression level of CRIM1,FAK,p-FAK,ERK,p-ERK and VEGF were detected by Western blot to observe whether CRIM1 regulated VEGF expression by activating the FAK/ERK signal axis,thus clarifying the molecular mechanism through which CRIM1 promoted the angiogenesis of cervical squamous cell carcinoma.5.Human recombinant CRIM1 protein was used to directly stimulate SiHa cells.Western blot and ELISA were used to detect the level of VEGF in the cells and supernatant,and to observe whether CRIM1 could function as an exocrine protein to influence the vascularity of cervical cancer cells.Results:1.The expression level of CRIM1 in cervical squamous cell carcinoma was significantly higher than that in chronic cervicitis tissue,and the difference was statistically significant(t = 5.349,P < 0.001).Correlation analysis showed that the expression of CRIM1 was positively correlated with the MVD of cervical squamous cell carcinoma.(P < 0.01,r = 0.546).2.Western blot results showed that the expression level of CRIM1(0.819 ± 0.086)and VEGF(0.404 ± 0.039)were significantly higher in the cancer cells of positive transfection group than in the cancer cells of control group(0.092 ± 0.021,0.059 ±0.008)(t = 11.609,P <0.001;t = 12.077,P <0.001)and vector transfection group(0.122 ± 0.019,0.056 ± 0.008)(t = 11.167,P < 0.001;t = 10.636,P < 0.001).Correspondingly,ELISA results showed that the VEGF content in the supernatant of the positive transfection group(763.751 ± 25.816 pg/ml)was also significantly higher than that of the control group(653.501 ± 25.487 pg/ml)(t = 5.264,P < 0.01).)and vector group(649.006 ± 36.539pg/ml)(t = 4.113,P < 0.01).The HUVECs stimulated by supernatants of the cancer cells with CRIM1 gene transfection had a higher proliferative capacity,migration ability and tube formation ability than the control group(t = 4.107,P < 0.01;t = 4.566,P < 0.05;t = 2.915,P < 0.05)and the vector transfection group(t = 4.28,P < 0.01;t = 5.013,P < 0.05;t = 2.4,P < 0.05),respetively.3.Between the cancers cells with pCMV3-CRIM1 transfection plus U0126 treatment and the cancer cells with only pCMV3-CRIM1 transfection,there was no siginifcant difference of CRIM1 expression(t = 1.631,P > 0.05).However,the expression level of p-ERK and VEGF in the former cells(0.218 ± 0.008,0.061 ±0.008)was significantly lower than in the latter cells(0.416 ± 0.016,0.244 ± 0.014)(t = 18.973,P < 0.001;t = 19.011,P <0.001);In addition to the cancer cell supernatant control group in the above group,the three groups of cancer cell supernatants stimulated HUVECs(named as control group,pCMV3-CRIM1 group,and pCMV3-CRIM1+U0126 group).The experimental results show that in the pCMV3-CRIM1 group,the endothelial proliferation ability and the migration ability was increased(t = 8.749,P < 0.001;t = 4.745,P < 0.01).The ability of their tube formation was also increased(t = 8.5,P<0.05).The proliferation,migration and tabulation ability of the U0126+pCMV3-CRIM1 group was notably decreased compared with the pCMV3-CRIM1 group(t = 8.198,P < 0.001;t = 2.786,P <0.05;t = 9.827,P < 0.001).4.The expression level of CRIM1,p-FAK,p-ERK and VEGF in pCMV3-CRIM1 group of cancer cells(0.315 ± 0.016,1.462 ± 0.278,0.416 ± 0.057 and 0.135 ± 0.022)was clearly increased in comparison with the expression level of each protein in the control group of cancer cells(0.162 ± 0.030,0.294 ± 0.169,0.124± 0.016 and 0.086 ± 0.010)(t = 7.833,P < 0.01;t = 6.203,P < 0.001;t = 8.475,P <0.01;t = 3.423,P < 0.05).However,compared with pCMV3-CRIM1 group,the expression levels of p-FAK and p-ERK protein in the combined treatment group(Y15+pCMV3-CRIM1)(0.951 ± 0.054,0.291 ± 0.041)was decreased(t = 3.119,P <0.05;t = 3.068,P < 0.05),and correspondingly,the VEGF protein level(0.060 ±0.006)was also significantly attenuated(t = 5.564,P < 0.01),while CRIM1 protein level showed no significant change(t = 1.124,P > 0.05).5.Human recombinant protein CRIM1 was used to directly treat cervical squamous carcinoma cells,and there was no significant change in VEGF protein expression and VEGF content in the supernatant(P > 0.05).Conclusion:1.CRIM1 is highly expressed in cervical squamous cell carcinoma and itsexpression is associated with the cancer microvessel density(MVD).2.The high expression of endogenous CRIM1 promotes angiogenesis of cervical squamous cell carcinoma.The molecular mechanism may be related to CRIM1-mediated activation of the FAK-ERK1/2 signaling pathway to up-regulate VEGF expression.3.CRIM1 doesn’t function as an exocrine protein to affect the angiogenesis inducing capacity of cervical squamous cell carcinoma. |
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