Establishment Of Mesenchymal Subline With Highly Metastatic Potential Based On Microfluidic System And Evaluation Of EMT-related MicroRNAs

Objective:Malignant tumors have been proved to threaten human health and life severely.Metastasis is widely accepted to be the leading cause of tumor mortality.Epithelial-to-mesenchymal transition(EMT)is one of the most significant processes during metastasis,by which tumor cells gain increasing invasion and metastatic potential.Thus,EMT is important to delineate metastasis mechanism,and establishment of EMT model will help to discover new metastasis-suppressing therapeutic targets and to screen anticancer drugs.There are two maint strategies in EMT investigation.One is the induction of EMT by some special factors,such as exposing cells to TGF-?,stable transfection of EMT transcription factors.The other is the selection of mesenchymal subline from heterogeneous cell populations.The rationale for the selection strategy is based on the existence of mesenchymal subpopulation within tumors and cell lines.The selection can be performed by in vivo and in vitro selection.In in vivo models,mesenchymal subline with highly metastatic potential can be established from metastatic foci after multiple rounds of selection.In vivo selection model has the superiority to present sophisticated in vivo microenvironment but can be easily influenced by hosts due to individual differences.Additionally,it is often laborious and time consuming.Accordingly,in vitro selection emerged as another important and simpler strategy to select mesenchymal sublines.For example,mesenchymal cells can be obtained from a Transwell chamber based on their enhanced matrix-degrading and invasion ability via secretion of higher amounts of degrading enzymes.However,such models often need more selection cycles to obtain sublines.Thus,a more effective and rapid approach to obtain mesenchymal subline is highly desirable.Microfluidic chips,also known as lab-on-a-chip,have been widely applied in biological,medical and nanotechnology fields.The advantages of excellent microenvironment simulation,liquid handing and low time and labor costs enable them to be used in investigation of tumor invasion and metastasis conveniently.Previously,we designed a microfluidic device with the ability to generate dynamic chemotactic factor concentration gradients that could efficiently resolve the invasiveness of different types of cells.Here,we modified the selection strategy by relatively high and stepwise-increasing Matrigel concentration for mesenchymal subline screening by the microfluidic system.MicroRNAs(miRNA)are endogenous non-coding RNAs of 18~25 nucleotides that mediate post-transcriptional repression by binding 3'UTR sequence of their target genes directly.They often result in a translation inhibition or degradation of target mRNA.Many miRNAs have been found to have functions on cell differentiation,proliferation,migration,drug-resistance and apoptosis,half of which are closely related with tumors.The regulatory mechanisms of miRNAs are extremely sophisticated.For example,an individual miRNA can regulate several target mRNA simultaneously and one mRNA can also be regulated by many different miRNAs.So it is of great importance to investigate the network of miRNAs regulation thoroughly.Based on the obtained mesenchymal subline with highly metastatic potential and its parental cell line,we intended to perform miRNA expression profiling for screening EMT related miRNAs or even other useful EMT and metastasis related biomarkers.Methods: 1.Fabrication of microfluidic system for mesenchymal subline selection.(1)The microfluidic device was produced by SU-8 photoresist.(2)The microfluidic subline screening system was fabricated by connecting with syringe pump,Petri dish and culture medium.2.Establishment of highly metastatic subline of lung cancer cell.(1)Highly invasive subline cells were harvested throuth the opened collection channel and cultured.(2)Evaluation of in vitro proliferation ability was performed by MTT and soft agar colony formation assay.(3)Evaluation of in vitro migration and invasion abilities were performed by wound healing assay,Transwell migration and invasion assay.(4)Evaluation of in vivo proliferation and metastasis abilities were performed by tumorigenicity assays.(5)Molecular analysis was performed by Western blot.3.Identification and functional verification of EMT and metastasis related miRNAs.(1)Differentiated expressed miRNAs were screened and analyzed by miRNA expression profiling.(2)Expression of metastasis related miRNAs was confirmed by qRT-PCR.(3)Transfection efficiency of miRNAs was confirmed by qRT-PCR.(4)In vitro proliferation ability of transfected cells were evaluated by MTT assay.(5)In vitro migration and invasion ability of transfected cells were evaluated by Transwell assays.(6)Target gene prediction was analyzed by Miranda,MiRBase and Targetscan databases.(7)Expression of target protein was evaluated by Western blot.Results: 1.Fabrication of microfluidic system for subline screening.(1)The microfluidic device was produced by PDMS and glass slide.The microfluidic device was composed of four indirect connected microchannels which are pump channel,cell culture channel,matrigel channel and opened collection channel.(2)Under the flow rate of 3?l/hour,a stable fluorescence intensity gradient was formed from medium channel to opened channel and maintained for 3 days by use of FITC/PBS solution.(3)The microfluidic subline screening system was fabricated by connected with syringe pump,Petri dish and culture medium.The matrigel loading microfluidic device was placed in the Petri dish containing complete culture medium and then the medium with 2% FBS was pumped into the device by a single syringe pump.Cells were driven to invade through the concentrated Matrigel barrier.2.Establishment of highly metastatic subline of lung cancer.(1)Mesenchymal subline A3 cells were obtained by only three cycles.(2)There was significant enhanced colony formation ability and anchorage independent growth ability of subline by evaluation of soft agar assay.(3)The subline cells revealed a significant enhanced in vitro migration and invasion ability by wound healing assay,Transwell migration and invasion assay.(4)The subline cells had an enhanced metastatic ability by in vivo tumorigenicity assays.(5)Expression of E-cadherin and ZO-1 was down-regulated while ZEB1,fibronectin,vimentin and MMP-9 were up-regulated in subline cells by Western blot.3.Identification and functional verification of EMT and metastasis related miRNAs.(1)There were 436 differential expressed miRNAs of which were 259 were up-regulated and 177 were down-regulated in subline cells.EMT regulating pathways including Wnt?TGF-??PI3K-Akt were enriched in subline A3 cells.(2)Metastasis and EMT related miRNAs(miR-200b-3p and miR-194-5p)expression were confirmed down-regulated in subline cells by qRT-PCR.(3)In vitro proliferation ability of transfected cells was not changed.(4)In vitro migration and invasion ability were inhibited in cells transfected with miRNA mimics.(5)A total of 194 target genes of miR-200b-3p and 48 target genes of miR-194-5p were predicted by Miranda,MiRBase and Targetscan databases.ZEB1 was the most possible target gene of miR-200b-3p by the highest binding score.(6)Expression of ZEB1 was down-regulated by miR-200b-3p mimics transfection and up-regulated by inhibitor transfection.The expression of E-cadherin was negatively correlated with that of ZEB1.Conclusion: 1.Based on the relative high and stepwise-increasing Matrigel concentration within the microfluidic system,we established a mesenchymal subline with highly-metastatic potential named A3 from heterogeneous A549 cell line in high efficiency.2.Differential expression of miRNAs was successfully performed by use of our established subline.EMT related miRNAs(miR-200b-3p and miR-194-5p)were also proved to be related with cell migration and invasion.Our established microfluidic system shed the light for screening mesenchymal subline and more tumor related biomarkers such as miRNAs.