Experimental Study Of The Role Of CXCL12/CXCR7 In Regulating Invasion And Angiogenesis Of Human Liver Cancer Cells

PART ONE EXPRESSION AND SIGNIFICANCE OF CHEMOKINE RECEPTOR CXCR7 IN HCC AND LIVER CANER CELL LINESObjectiveTo study the expression of CXCR7 in hepatocellular carcinoma tissues, matched adjacent non-neoplastic tissues , normal liver tissues and liver cancer cells. To evaluate the role of CXCR7 in HCC development.MethodsImmunohistochemistry was used to detect the expression of CXCR7 in human hepatocellular carcinoma tissues, matched adjacent non-neoplastic tissues and normal liver tissues. RT-PCR and Western-blot was used to evaluate CXCR7mRNA and CXCR4mRNA and protein levels in liver cancer cell lines(HepG2, Hep3B, SMMC-7721, MHCC97L, MHCC97H ,HCCLM6)and HUVECs.Results(1)HCC tissues displayed higher CXCR7 expression than matched adjacent non-neoplastic tissues and normal liver tissues.(2)Human liver cancer cells displayed higher CXCR7 expression than HUVECs.( 3 ) High aggressive liver cancer cells displayed higher CXCR7 expression than low aggressive liver cancer cells.(4)The expression of CXCR4 was detected in six liver cancer cells and HUVECs.Conclusion①The expression of CXCR7 was high elevated in HCC. However, CXCR7 expression was low in matched adjacent non-neoplastic tissues and normal liver tissues.②CXCR7 was overexpressed in high aggressive liver cancer cells. CXCR7 might play a role in invasion of liver cancer cells.PART TWO CONSTRUCTION AND IDENTIFICATION OF CXCR7shRNA EUKARYOTIC EXPRESSION VECTORObjectiveTo construct siRNA eukaryotic expression vectors targeting to CXCR7 gene.To specifically silence CXCR7 expression through transfecting recombinant plasmid of CXCR7shRNA into SMMC-7721 cells. To obtain SMMC-7721 cells stably expressing recombinant plasmid of CXCR7shRNA through G418 selection. MethodsTranscription template sequence targeting to CXCR7 was designed and synthesized. The recombinant plasmid of CXCR7shRNA was constructed and further identified by enzyme digestion and sequencing. The recombinant plasmid of CXCR7shRNA was transfected into SMMC-7721 cells and further selected by G418. Transfection efficiency was evaluated by fluorescent microscope. CXCR7mRNA levels in SMMC-7721 cells transfected with CXCR7shRNA were detected by RT-PCR. The protein levels of CXCR7 in SMMC-7721 cells transfected with CXCR7shRNA were detected by Western-blot.Results(1)The positive recombinant plasmid was cut by BamH I but not Pst I after recombinant plasmid was digested.(2) The recombinant plasmid of CXCR7shRNA was successfully transfected into SMMC-7721 cells. After G418 selection, a great quantity of cells transfected with recombinant plasmid displayed green fluorescence.(3)After G418 selection, CXCR7mRNA levels in SMMC-7721 cells were detected by RT-PCR. The cells transfected with CXCR7shRNA exhibited lower CXCR7mRNA levels compared with the control cells(P<0. 05).(4)After G418 selection, protein levels of CXCR7 in SMMC-7721 cells were detected by Western-blot. Expression of CXCR7 in cells transfected with CXCR7shRNA was significantly downregulated compared with the control cells(P<0. 05).Conclusion①The recombinant plasmid of CXCR7shRNA was successfully constructed and identified by enzyme digestion and sequencing.②Under fluorescent microscope, SMMC-7721 cells stably transfected with recombinant plasmid of CXCR7shRNA displayed a great quantity of green fluorescence. Transfection efficiency was approximate 80%-90%。③Expression of CXCR7 in cells stably transfected with recombinant plasmid of CXCR7shRNA was efficiently silenced through RT-PC R and Western-blot assay.PART THREE EFFECT OF CXCL12/CXCR7 ON BIOLOGICAL BEHAVIOR OF HUMAN LIVER CANCER CELLSObjectiveTo investigate the effect of CXCR7 silencing on invasion, adhesion, VEGF secretion and angiogenesis of SMMC-7721 cells. To evaluate the effect of VEGF stimulation on CXCR7 expression and invasive ability of SMMC-7721 cells.Methods①Invasive ability of SMMC-7721 cells induced by CXCL12 was evaluated by Transwell chamber assay.②Effect of CXCR7 silencing on invasive ability of SMMC-7721 cells was evaluated by Transwell chamber assay.③Effect of CXCR7 silencing on adhesive ability of SMMC-7721 cells was evaluated by tumor cell- Extra Cellular Matrix adhesion assay.④Effect of CXCR7 silencing on VEGF secretion in SMMC-7721 cells was investigated by ELISA assay.⑤Effect of CXCR7 silencing on SMMC-7721 cells induced-angiogenesis was detected by tumor cell- HUVECs coculture system.⑥Effect of VEGF stimulation on CXCR7 expression in SMMC-7721 cells and HUVECs was evaluated by RT-PCR and Western-blot assay.⑥Effect of VEGF stimulation on invasive ability of SMMC-7721 was evaluated by Transwell chamber assay.Results(1) We found that CXCL12(0, 10,100 ng/ml) induced a significant and dose-dependent increase of invasion in SMMC-7721 cells.(2) CXCL12(100 ng/ml) induced invasion in SMMC-7721 cells was decreased after silencing of CXCR7.(3)CXCL12(100 ng/ml) induced a significant increase of cell-Extra Cellular Matrix (FN and LN) adhesion. Adhesion of SMMC-7721 cells to LN was greater than adhesion to FN.(4)CXCL12(100 ng/ml)induced adhesion of SMMC-7721 cells to LN and FN was decreased after silencing of CXCR7.(5)CXCL12(100 ng/ml)could induce VEGF secretion in SMMC-7721 cells.(6)VEGF secretion induced by CXCL12(100 ng/ml)was decreased after silencing of CXCR7.(7)In vitro tube formation induced by SMMC-7721 cells was decreased after silencing of CXCR7.(8)VEGF(50 ng/ml )stimulation induced a time-dependent increase of CXCR7mRNA levels in SMMC-7721 cells and HUVECs.(9)VEGF(50 ng/ml )stimulation induced a time-dependent increase of protein levels of CXCR7 in SMMC-7721 cells and HUVECs.(10)Invasive ability of SMMC-7721 cells was enhanced after VEGF(50 ng/ml )stimulation.Conclusion①CXCL12 could induce invasion, adhesion, VEGF secretion in SMMC-7721 cells.②Invasion, adhesion and VEGF secretion was decreased after silencing of CXCR7. Also, in vitro tube formation induced by SMMC-7721 cells was decreased after silencing of CXCR7. These results demonstrated that CXCL12/ CXCR7 could regulate invasion and adhesion of SMMC-7721 cells. CXCL12/ CXCR7 axis might play an important role in metastasis of SMMC-7721 cells.③Up-regulation of CXCR7 expression by VEGF stimulation was functional. PART FOUR EFFECT OF CXCR7 SILENCING ON TUMOR GROWTH OF HUMAN LIVER CANCER CELL XENOGRAFT IN NUDE MOUSEObjectiveTo study the effect of CXCR7 silencing on tumor growth of SMMC-7721 cells xenograft in nude mice.MethodsThe control, negative shRNA and stable CXCR7shRNA transfected SMMC-7721 cells were inoculated subcutaneously into the back of nude mice. The tumor size was measured and the growth curve of tumor was drawed. The tumors were completely separated and weighed after the mice were sacrificed. Subsequently, the tumors were fixed in 4% polyformaldehyde and embedded with paraffin. The tumor sections were excised for immunohistochemical analysis of CD31 and calculation of MVD. Metastasis was evaluated in lung and liver of nude mice.Results(1) The cells stably transfected with CXCR7shRNA showed smaller tumor size compared with control cells(P<0.05). However, no significant difference was observed between control and negative shRNA group(P>0.05).(2) The cells stably transfected with CXCR7shRNA showed significantly reduced tumor weight compared with control cells(P<0.05). However, no significant difference of tumor weight was observed between control and negative shRNA group(P>0.05). (3) The cells stably transfected with CXCR7shRNA showed significantly reduced expression of CD31 and decreased MVD compared with control cells. Statistic differences were obtained between control and CXCR7shRNA group(P<0.05). However, no significant difference of tumor angiogenesis was observed between control and negative shRNA group(P>0.05).Conclusion①Tumor growth of SMMC-7721 cells xenograft in nude mice could be regulated by silencing of CXCR7.②Silencing of CXCR7 substantially suppressed angiogenesis and subsequently regulated the tumor growth..③We did not find metastasis of xenograft in nude mice after silencing of CXCR7.However, we could not conclude that CXCR7 was unable to regulate metastasis of transplanted tumor in nude mice. Orthotopic transplanted tumor model is a better way to further evaluate the role of CXCR7 in regulating tumor metastasis.