Study Of The Relationship Between OPN Protein And The Ability Of Breast Cancer Cells To Form Vasculogenic Mimicry In Vitro

Purpose：Osteopontin(OPN) is overexpressed in human breast cancer, and is likelyassociated with the formation of vasculogenic mimicry(VM) in some tumor cells. Inthis study, we investigated whether OPN plays a role in the formation of VM in breastcancer,and explored the relationship between OPN and VM-associated proteinMMP-9and VEGF.Method：1. Lipotransfected MDA-MB-231cells with recombinant plasmids: miR-opn1,miR-opn2, miR-opn3and miR-opn4, and selected the highest transfection efficiencyone for the next steps by Western Blot.2. Experimental groups: normal group, transfection reagent control group(LipLTX), negative vector control group (miR-neg) and recombinant vector group(miR-opn2), transfected MDA-MB-231cells for30h and then collected cells for3-dimensional culture in vitro for24h, at last observed the number of VM underinverted microscope and proved the presence of VM by PAS staining.3. After24h, extracted the3-dimensional culture cells in each experimentalgroup, RT-PCR and Western Blot were used to detect the mRNA and protein level ofOPN, MMP-9and VEGF.Result：1. miR-opn1, miR-opn2, miR-opn3and miR-opn4were transfected into humanbreast cancer cell line MDA-MB-231, the fluorescence microscope and WB testshowed that: compared to the other three groups, the efficiency of gene silencing ofmiR-opn2was the highest.2. MDA-MB-231cells which were transfected recombinant vector miR-opn2form less VM than the normal group, transfection reagent control group and negativevector control group(P<0.05), and there was no significant difference among thenormal group, transfection reagent control group, negative vector control group. These results indicated that OPN involves in the formation of the VM ofMDA-MB-231cell line.3. OPN, MMP-9and VEGF mRNA and protein levels of recombinant vectorgroup (miR-opn2) of the MDA-MB-231cells were less than the normal group,transfection reagent control group, negative vector control group(P<0.05), and therewas no significant difference among the normal group, transfection reagent controlgroup, negative vector control group.These results indicated that OPN involves in theformation of the VM via MMP-9and VEGF.Conclusion：1. Human breast cancer cell line MDA-MB-231can form VM in three-dimensional cultures in vitro, which is demostrated by PAS positive staining.2. After koncking down OPN protein, the number of the VM of human breastcancer cells MDA-MB-231significantly reduced, suggesting that OPN plays a role inthe formation of VM in human breast cancer.3. These results indicated that OPN plays an important role in the formation ofVM,MMP-9and VEGF may play a role in the formation of VM.