Effect Of CD82 On Invasion Of Breast Cancer Cells Based On The Microfludic Platform

Objective: In this experiment,we study the effect of CD82 on the invasion of breast cancer cells by comparing the fine and real-time invasive dynamic information of wild-type breast cancer cells(MDA-MB-231)and CD82 overexpressing breast cancer cells(CD82-231)on microfluidic chip and the protein and gene expression of Wnt/?-catenin pathway of the two groups.Methods: This experiment is a combination of methodology and traditional biology.MDA-MBA-231 cells were stably overexpressing CD82 by lentiviral transfection technology,and CD82-231 cell line was established.Then we designed a microfluidic chip to mimic the 3D microenvironment of the human mammary extracellular matrix,and monitor the cell invasion real time,including cell morphology,cell invasion area and other information,so as to compare the difference between MDA-MB-231 and CD82-231 on the chip.Furthermore,we studied how CD82 regulated the Wnt/?-catenin pathway and effected the invasion of breast cancer cells by immunofluorescence technology,real-time quantitative PCR technology and western blot experiments.Results: 1.The transfection efficiency of CD82 was 95.34%.2.The longer culture time of breast cancer cells on the chip,the larger difference of the invasion area between??the two groups.However,the invasion speed of the CD82-231 cell group was always slower than that of the MDA-MB-231 cell group under the same conditions.And the greater the collagen concentration,the slower the cell invasion.It can be clearly seen that the cells are more inclined to invade in the form of cell clusters.According to theinvasion position of the cell clusters,it can also be judged that the invasion rate of the CD82-231 cell group is slower.3.The invasion pattern of cells in the three-dimensional environment of the chip can be divided into four types: ? a pseudopod is extended forward;? a pseudopod is extended from the front and the rear;? two pseudo-foots are extended forward;? a plurality of pseudo-foots are extended to the periphery.And the cell length / width ratio in the three-dimensional environment is significantly larger than that in the two-dimensional environment.4.CD82 prevents the downstream genes(C-myc,Survivin,Cyclin-D and F1)transcription by reducing the expression of Fzd-2and increasing E-Cadherin in the Wnt/?-catenin signaling pathway and increasing?-catenin expression from the nucleus to the cytoplasm and cell membrane,which in turn inhibits cell invasion and metastasis.And CD82 and KYA1797 K work together to have a better inhibition effect.Conclusion: We designed a microfluidic chip that can mimic the human mammary extracellular matrix microenvironment and demonstrated that CD82 inhibits breast cancer cell invasion.The protein of E-Cadherin was increased and Fzd-2 was decreased in the Wnt/?-catenin signaling pathway.A large amount of ?-catenin stays in the cytoplasm and cell membrane and cannot enter the nucleus to start the transcription of downstream target genes,thereby inhibiting breast cancer cells invasion in microfluidic chips.And CD82 and KYA1797 K work together to have a better inhibition effect.