Experimental Study Of The Expression And Role Of DKK1 In Regulating Formation And Development Of Malignant Melanoma

PART ONE EXPRESSION AND SIGNIFICANCE OF DKK1 ANDβ-CATENIN IN HUMAN MALIGNANT MELANOMAObjectiveTo study the expression of DKK1 andβ-catenin in human malignant melanoma in situ tissues, malignant melanoma with lymph node metestases tissues and normal skin tissues. To evaluate the role of DKK1 in melanoma formation and development and to figure out the relationship between DKK1 and canonical Wnt signaling pathway.MethodsImmunohistochemistry was used to detect the expression of DKK1 andβ-catenin in human malignant melanoma in situ tissues, malignant melanoma with lymph node metestases tissues and normal skin tissues.Results(1) Malignant melanoma tissues displayed lower DKK1 expression than normal skin tissues. (2) Malignant melanoma with lymph node metestases tissues displayed lower DKK1 expression than malignant melanoma in situ tissues.(3) Malignant melanoma tissues displayed higherβ-catenin expression than normal skin tissues.(4) Malignant melanoma with lymph node metestases tissues displayed higherβ-catenin expression than malignant melanoma in situ tissues.Conclusion(1) The expression of DKK1 was high elevated in normal skin. However, DKK1 expression was low in malignant melanoma, so we surpose that DKK1 may effect the malignant melanoma formation.(2) DKK1 was lower expressed in malignant melanoma with lymph node metestases tissues than malignant melanoma in situ tissues, so we guess that it might have negative relationship between DKK1 and malignant degree in malignant melanoma.(3) The expression ofβ-catenin was high elevated in malignant melanoma. However,β-catenin expression was low in normal skin, so we surpose that DKK1 may modulateβ-catenin to effect malignant melanoma formation.(4)β-catenin was higher expressed in malignant melanoma with lymph node metestases tissues than malignant melanoma in situ tissues, so we guess that it might have positive relationship betweenβ-catenin and malignant degree in malignant melanoma.PART TWO CONSTRUCTION OF THE RECOMBINANT ADENOVIRUS AD-DKK1ObjectiveTo construct the recombinant adenovirus plasmid Ad-DKK1 overexpressing DKK1, then transfect the plasmid into the package cell HEK293 and generate the high titer recombinant adenovirus.MethodsFirstly, we amplify the target gene DKK1 containing the linker through the PCR, then intergrate the target gene into the shuttle vector of pAdtrack-TO4 by restrict ligation. Secondly, recombine the plasmid in the competent E coli BJ-Adeasy containing the adenovirus skeleton plasmid Adeasy-1 to get Ad-DKK1 plasmid. Finally, we transfect the recombinant plasmid Ad-DKK1 into the package cell HEK293 and generate the high titre adenovirusResults(1) The recombinant plasmid was identified by the restriction enzyme Pac I, and DNA electrophoresis showed that there exist band: 4.5kb, in coincidence with the anticipated results, which means the Ad-DKK1 was constructed successfully.(2) The human melanoma A375 cells illuminate the green fluorescence after infected by Ad-DKK1 adenovirus under the fluroresence microscope.(3) Through the repeated infection, collect the high titer Ad-DKK1 adenovirus.(4) RT-PCR detect the expression of DKK1 mRNA, and the expression level of DKK1 mRNA in human melanoma A375 cell infected by Ad-DKK1 adenovirus was much higher than in the control group(P<0.05).(5) Western-blot detect the expression of DKK1 protein, and the expression level of DKK1 protein in human melanoma A375 cell infected by Ad-DKK1 adenovirus was much higher than in the control group(P<0.05).Conclusion(1) After the endoclease and sequencing identification, the recombinant adenovirus Ad-DKK1 expressing the DKK1 was made successfully.(2) The human melanoma A375 cells illuminate the green fluorescence after infected by Ad-DKK1 adenovirus under the fluroresence microscope.(3) The overexpression of DKK1 was identified in the A375 cells infected by the recombinant adenovirus Ad-DKK1 through the measurement of RT-PCR and Western-blot.PART THREE EFFECT OF DKK1 ON BIOLOGICAL BEHAVIOR OF HUMAN MELANOMA CELLSObjectiveTo investigate the effect of the proliferation, invasion and adhesion abilty of A375 cells in the condition of the differentiate expression of DKK1, and study whether the DKK1 play a role in A375 cells through regulating the classic Wnt/β-catenin signaling.Methods(1) Investigate the proliferation ability of A375 cells in the condition of the differentiate expression of DKK1 through the cell counting, cloning formation assay and MTT test.(2) Investigate the invasion ability of A375 cells in the condition of the differentiate expression of DKK1 through the Transwell chamber assay and scratching test. (3) Investigate the adhesion ability of A375 cells in the condition of the differentiate expression of DKK1 through the adhesion test between the tumor cells and extracellular matrix.(4) Investigate theβ-catenin mRNA expression level of A375 cells in the condition of the differentiate expression of DKK1 through the PCR.Results(1) The number of cell counting,the rate of colone formation assay and the absorbance of MTT in A375 cells overexpressing DKK1 all were lower than the control group(P<0.05), but the number of cell counting,the rate of colone formation assay and the absorbance of MTT in A375 cells silencing DKK1 all were higher than the control group(P<0.05).(2) The number of invasion cells of A375 cells overexpressiong DKK1 in Transwell chamber assay was less than the control group(P<0.05), and the distance of scratch assay in A375 cells overexpressiong DKK1 after 36h was longer than the control group, but the number of invasion cells of A375 cells silencing DKK1 in Transwell chamber assay was more than the control group(P<0.05), and the distance of scratch assay in A375 cells silencing DKK1 after 36h was shorter than the control group.(3) The adhesion ability of A375 cells in the DKK1 overexpression group was lower than in the control group, and the DKK1 silencing group was higher than in the control group.(4) Theβ-catenin mRNA expression level of A375 cells in the DKK1 overexpression group was lower than in the control group(P<0.05), and the DKK1 silencing group was higher than in the control group (P<0.05).Conclusion(1) DKK1 overexpression could inhibit the proliferation of A375 cells, and DKK1 silencing could promote the proliferation.(2) DKK1 overexpression could inhibit the invasion and migration of A375 cells, and DKK1 silencing could promote the invasion and migration.(3) DKK1 overexpression could inhibit the adhesion of A375 cells, and DKK1 silencing could promote the adhesion.(4) DKK1 effect A375 cells through prohibiting the classic Wnt/β-catenin signaling.PART FOUR EFFECT OF DKK1 ON TUMOR GROWTH OF HUMAN MELANOMA CELL XENOGRAFT IN NUDE MICEObjectiveInvestigate the effect of the growth of the subcutaneous melanoma xenograft in the condtion of the differentiate expression of DKK1 in A375 cells.MethodsThe differentiate DKK1 expresion A375 cells, the control group A375 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 DKK1 andβ-catenin expression.Results(1) The tumor size, weight and growth rate of A375 cells in the DKK1 overexpression group were obviously lower than in the control group(P<0.05).(2) The tumor size, weight and growth rate of A375 cells in the DKK1 silencing group were obviously higher than in the control group(P<0.05).(3) Immunohistochemical staining of DKK1 in the DKK1 overexpression group was stronger than in the control group, immunohistochemical staining of DKK1 in the DKK1 silencing group was weaker than in the control group.(4) Immunohistochemical staining ofβ-catenin in the DKK1 overexpression group was weaker than in the control group, immunohistochemical staining ofβ-catenin in the DKK1 silencing group was stronger than in the control group.Conclusion(1) DKK1 overexpression could inhibit the growth of A375 cells inoculated subcutaneously in nude mice.(2) DKK1 silencing could promote the growth of A375 cells inoculated subcutaneously in nude mice.(3) DKK1 may effect A375 cells inoculated subcutaneously in nude mice, which perhaps was related with theβ-catenin of the Wnt signaling.