| Objective In this study,CXCL12-CXCR4 axis and CXCL12-CXCR7 axis were selected as the research objects.By detecting the expression of related proteins in cervical cancer,combining with clinical pathological data and prognosis,we analyzed the influence of the above axis on the biological behavior of cervical cancer cell line-SiHa cell line,and under the in vivo experiment of nude mice,we further explored the pathogenesis of cervical cancer,and provided experimental basis for the new treatment methods of cervical cancer.Methods The expression of CXCR4,CXCR7,ERK,p-ERK,Akt and p-Akt in normal cervix uteri,cervical intraepithelial neoplasia and cervical squamous cell carcinoma were detected by immunohistochemistry,and the correlation between CXCR4,CXCR7 and clinicopathological features,and survival prognosis of patients were analyzed.The expression levels of CXCR4 and CXCR7 in cervical squamous cell carcinoma cell line of SiHa,C-33A and CaSki were analyzed by Real-time PCR and Western blotting.Different concentrations of CXCL12 were applied to SiHa cells,and CCK8 method was used to observe the proliferation of SiHa cells,crystal violet staining method to analyze adhesion,Transwell to analyze cell migration,and invasion,flow cytometry to analyze cell cycle and apoptosis,and Western blot method to analyze the activation state of Akt and ERK signal pathway.Then,small interfering RNA was used to interfere the expression of CXCR4 and CXCR7.Real-time PCR and Western blotting were used to analyze the silencing efficiency,and the above methods were used to detect the changes of proliferation,adhesion,migration,invasion,cell cycle and apoptosis of SiHa cells,as well as the activation of Akt and ERK signal pathway.The cervical cancer xenograft model in nude mice was constructed.CXCR4-siRNA and CXCR7-siRNA were injected into the transplanted tumor.The expression level of CXCR4 and CXCR7 in the transplanted tumor were analyzed.The growth state of the transplanted tumor was observed.The tumor inhibition rate was analyzed,and the proliferation of the transplanted tumor cells was detected by CCK8 method.The cell cycle and apoptosis level of the transplanted tumor were detected by flow cytometry.Results The expression of CXCR4 and CXCR7 in cervical intraepithelial neoplasia and cervical squamous cell carcinoma was higher than that in normal cervical tissue(P<0.05).The expression of ERK,p-ERK,Akt and p-Akt was consistent with that of CXCR4(P<0.05).The expression level of CXCR4 was significantly different in diagnosis,disease stage,vascular invasion,lymph node metastasis and tumor volume groups(P<0.05);The expression level of CXCR7 was significantly different in diagnosis,disease stage,lymph node metastasis and tumor volume groups(P<0.05).In normal cervical and cervical intraepithelial neoplasia,the correlation coefficients of CXCR4 and CXCR7 were r=0.081(P>0.05)and R=0.025(P>0.05),respectively;in cervical squamous cell carcinoma,the correlation coefficient was r=0.213<0.4(P<0.05).The DFS(disease free survival)and OS(overall survival)of patients with high expression of CXCR4 and CXCR7 were lower than those of patients with low CXCR4 and CXCR7 expression(P<0.05).In vitro,the proliferation,adhesion,migration and invasion of SiHa cells increased with the increase of CXCL12 dose(P<0.05).After CXCR4-siRNA and CXCR7-siRNA were transfected into SiHa cells alone or jointly,the cell proliferation was significantly inhibited,but there was no significant difference among the three intervention groups(P>0.05).Compared with the control groups,the adhesion,migration and invasiveness of SiHa cell lines in three intervention groups significantly decreased(P<0.05).In addition,compared with siCXCR7 group,the adhesion,migration and invasiveness of siCXCR4 group and common intervention group significantly decreased(P<0.05),but there was no significant difference between siCXCR4 group and common intervention group(P>0.05).Compared with the control groups,the S-phase block rate of three intervention groups was significantly higher(P<0.05).CXCL12(150 ng/ml)can activate Akt and ERK signaling pathway,and the phosphorylation level of Akt reached the highest value at 5 min and 10 min;the phosphorylation level of ERK2 protein reached the peak at 30 min(P<0.05);compared with the control groups,three intervention groups significantly inhibited the expression level of p-Akt and p-ERK2(P<0.05).In addition,compared with siCXCR7 group,the relative expression level of p-Akt in siCXCR4 group and common intervention group was significantly lower(P<0.05);but there was no significant difference in the expression level of p-Akt between siCXCR4 group and common intervention group(P>0.05);there was no significant difference in the relative expression level of p-ERK2 between three intervention groups(P>0.05).Compared with the negative control group,the growth rate of transplanted tumor volume of CXCR4-siRNA and CXCR7-siRNA alone or simultaneously decreased significantly from the 12th day(P<0.05).But there was no significant difference among the three interference groups(P>0.05).The weight of transplanted tumor in siCXCR4 group(0.846 ± 0.049 g),siCXCR7 group(0.923± 0.11 g)and co-intervention group(0.863 ± 0.09 g)was significantly lower than that in Normal-saline group(1.593±0.082 g),Negative-vector group(1.605±0.094 g)and Negative-siRNA group(1.518 ±0.151g)(P<0.05).But there was no significant difference in weight between the three interference groups(P>0.05).Compared with the negative control groups,the tumor inhibition rate of each intervention group was significantly higher than that of the control groups(P<0.05).In addition,there was no significant difference in tumor inhibition rate between the three intervention groups(P>0.05).After CXCR4-siRNA and CXCR7-siRNA treatment alone or simultaneously,the proliferation of transplanted tumor cells was significantly inhibited compared with the control groups(P<0.05).Compared with the control groups,the S-phase block rate of transplanted tumor cells in each intervention group was significantly higher(P<0.05).However,there was no significant difference in each intervention group on the apoptosis of transplanted tumor cells(P>0.05)Conclusion The high expression of CXCR4 and CXCR7 suggests that the prognosis of patients is poor,and the correlation between the expression of CXCR4 and CXCR7 in cervical squamous cell carcinoma is poor.CXCL12CXCR4 axis and CXCL12-CXCR7 axis can enhance the proliferation,adhesion,migration and invasion of cervical squamous cell carcinoma SiHa cells in vitro,but CXCL12-CXCR4 axis plays a major role in the migration and invasion of cervical squamous cell carcinoma cells.There are interference or overlap between CXCL12-CXCR4 axis and CXCL12-CXCR7 axis in cervical squamous cell carcinoma,and the two axis participate in the regulation mechanism of cell cycle,but do not participate in the apoptosis mechanism of SiHa cells.CXCL12-CXCR4 and CXCL12-CXCR7 axis can activate Akt and ERK signaling pathway,and the activation effect of CXCL12-CXCR4 axis on Akt signaling pathway is more significant than that of CXCL12CXCR7 axis.SiCXCR4 and siCXCR7 groups can significantly inhibit the growth of cervical cancer transplanted tumor.CXCR4 and CXCR7 play the role of oncogene in cervical cancer,providing experimental basis for the new treatment methods of cervical cancer. |
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