The Inhibitory Effects Of VEGF-C And COX-2 Antisense Oligodeoxynucleotides Combined On Lymphangiogenesis In Breast Carcinoma

PART ONE:Inhibitory effect of VEGF-C and COX-2 ASODN combined transfection on the expression of VEGF-C in breast carcinoma cell line MDA-MB-231OBJECTIVE: To investigate the inhibition effect of VEGF-C and COX-2 antisense oligonucleotides combined transfection by cationic liposome-mediated on the expression of VEGF-C gene's protein and mRNA in breast carcinoma cell line MDA-MB-231.METHODS: Antisense oligonucleotides of VEGF-C and COX-2 which were targeted their coding regions were synthesized respectively and combinedly transfected by cationic liposome-mediated into breast carcinoma cell line MDA-MB-231.And their sense oligonucleotides were synthesized respectively as their control groups;The optimal effect of cationic liposome-mediated antisense oligonucleotides and their toxicity were determined by MTT assay in breast cancer cell line;The proteinic expression of VEGF-C and COX-2 in breast carcinoma cell was measured by immunocytochemistry staining and confocal laser scanning microscopy ; The mRNA expression of VEGF-C and COX-2 was detected by RT-PCR after VEGF-C and COX-2 antisense oligonucleotides was transfected combinedly.RESULTS: Toxicity and Side effect were not found after ASODN/LIP was transfected with 0.2umol/2.5ml/L. The inhibition ratio of VEGF-C mRNA was 70.37% in the combined ASODN group, but the inhibition ratio of VEGF-C mRNA in the single VEGF-C and COX-2 ASODN group was 48.15% and 42.21% respectively,which was lowerer than that in the combined ASODN group (P<0.01); The average optical density(AOD)of COX-2 and VEGF-C in breast carcinoma cells was respectively 0.304±0.072 and 0.249±0.012; The expression of COX-2 was significantly lower in the COX-2 ASODN group and combined ASODN group than that in the SODN group,but no difference was found in the VEGF-C ASODN group(P>0.05). The AOD of VEGF-C was significantly lower in the combined ASODN group than that in the single ASODN groups(P<0.01).CONCLUSIONS: The gene's expression of VEGF-C and COX-2 can been depressed specificitily by their antisense oligonucleotides sequences. The protein and gene's expression of VEGF-C was declined obviously in the combined ASODN group than that in the duble ASODN groups. COX-2 may participate and induce VEGF-C expression. PART TWO:Effect of VEGF-C and COX-2 ASODN combined transfection on lymphangiogenesis in breast carcinomaOBJECTIVE: The model of xenografts derived from breast carcinoma cell line MDA-MB-231 was established in Balb/C nude mice to investigate the depressant effect on VEGF-C , COX-2 and on lymphangiogenesis in breast carcinoma after ASODN combined transfection.METHODS: The model of xenografts derived from breast carcinoma cell line MDA-MB-231 was established in twenty Balb/C nude mice, then they were randomly divided into control group, VEGF-C ASODN groups, COX-2 ASODN groups and combined groups. The expression of COX-2 and VEGF-C was evaluated in breast carcinoma by immunohistochemical staining SP methods.And the inhibitory rate of COX-2 and VEGF-C mRNA was calculated by RT-PCR after VEGF-C and COX-2 antisense oligonucleotides were transfected combinedly. The lymphatic vessels density in tumor was calculated through the special marker D2-40.RESULTS: The expression of VEGF-C mRNA in Balb/C nude mice breast carcinoma was respectively 0.818±0.114,0.406±0.075,0.442±0.078 and 0.280±0.067 in control group,VEGF-C ASODN group,COX-2 ASODN group and combined groups;And their protein expression of VEGF-C was respectively 0.316±0.079,0.157±0.034,0.164±0.042 and 0.125±0.021;And their LVD was respectively 11.600±2.753,6.400±2.716,7.450±2.183 and 5.000±2.195; The protein and gene expression of VEGF-C could be inhibited by single or combined administer, and lymphangiogenesis could be inhibited too.But the depressant effect of combined administer was obviously higher than that of single administer (P<0.01);The expression of VEGF-C was positively correlated with LVD(r=0.757, p<0.01).CONCLUSIONS: The expression of VEGF-C is declined obviously by ASODN combined transfection, and breast carcinoma lymphangiogenesis can also be inhibited.