RNA Interference CXCR4 And CXCR7 Influences Human Endometrial Cancer Xenografts Growth In Nude Mice

Objective:1.To establish nude mice models of human endometrial cancer;2.To evaluate the effect of CXCR4-siRNA and/or CXCR7-siRNA on the growth of endometrial cancer xenografts in nude mice.Methods:1.To establish animal models,about 1×107 Ishikawa cells in o.2 ml of PBS were subcutaneously injected into the right flank of each mouse.2.Chemically modified CXCR4-siRNA,CXCR7-siRNA and Negative-siRNA was designed and validated.All tumor-bearing mice were randomly assigned to five groups(six nude mice in each group)when tumors reached 5~7mm in diameter,and received intra-tumor injections in 50 μl of CXCR4-siRNA(5nmol/mouse),CXCR7-siRNA(5nmol/mouse),CXCR4-siRNA(5nmol/mouse)plus CXCR7-siRNA(5nmol/mouse),Negative-siRNA(5nmol/mouse),and normal saline,respectively.An intra-tumor injection was given once every three days for six cycles.3.The longest(a)and shortest(b)diameters of tumors were measured with a caliper every three days and the volumes were calculated as the formula V=a×b2/2 and plotted against time.4.At the end of treatment,the nude mice were sacrificed,tumor tissues were dissected and their weights and volumes were measured,and tumor growth control rate(%)was calculated.5.CXCR4 and CXCR7 expression in tumor xenografts were detected by quantitative reverse transcription polymerase chain reaction(QRT-PCR),Western Blotting and Immunohistochemical staining.The proliferation rate of tumor cells was investigated by immunohistochemical assessment and probed with anti-PCNA antibody.The terminal deoxynucleotid transferase-mediated d UTP nick end labeling(TUNEL)assay was used to investigate the level of cell apoptosis.Results:1.We established available Ishikawa-derived xenografts nude mice models of human endometrial carcinoma.2.During the treatment period,the tumors in Ne-si and NS group grew rapidly,and the difference between these two groups has no statistical significance(P>0.05).The growth speed of tumor xenografts in the three treatment groups was significantly delayed compared with those in control groups(P<0.01).Compared whith NS group,tumor growth control rate of the three treatment groups were 58.77%,56.61% and 55.85%,respectively,but which in the Ne-si group was only 9.24%.3.The results of QRT-PCR,Western Blotting and Immunohistochemical staining showed CXCR4 and CXCR7 gene expression could be down regulated by siRNA(P<0.01).4.We also observed that CXCR4-siRNA andCXCR7-siRNA reduced the cell proliferation rate(P<0.01),but there is no statistical difference among these three treatment groups(P>0.05).However,there is no statistical difference in cell apoptosis among the five groups(P>0.05).Conclusion:Knocking down CXCR4 and/or CXCR7 gene can delayed the growth speed of endometrial cancer xenografts,also significantly inhibit the proliferation ability of tumor cells.These results indicated that CXCR4 and CXCR7 are alternative potential targets for gene therapy in endometrial cancer.