MiR-218-5p Target CD44-ROCK Pathway To Inhibit The Invasion Of Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma(OSCC)is one of the most common malignant tumors in the oral and maxillofacial region.Lymph-node metastasis occurs early in OSCC and distant metastasis often involves lung,liver and bone.The patients' five-year survival rate is about 50%,and the early metastasis and relapse contribute to the failure of treatment.Recent study demonstrated that early metastasis occur at the tumour–host interface named invasive front,where the deepest and presumably most aggressive cells reside.It is now well documented that several significant molecular events of tumour spread,such as secretion of proteolytic enzymes,increased cell proliferation,initiation of angiogenesis and changes of adhesion molecules take place at the invasive front of OSCC.Previous studies focused on observing EMT related makers by immunohistochemical staining using tumour tissues.It is difficult to isolate the cells at invasion front.Microfluidic chip is a novel technology,especially in operating cells in vitro.Objectives We aim to isolate OSCC cells at the invasion front based on microfluidic chips and further study the mechanism of OSCC invasion.Materials and methodsA microfluidic chip developed by our group was used in this study.An OSCC cell line,UM-SCC6,was induced to invaded into matrix on the microfluidic chip.The cells at the invasive front were isolated and subculture in dishes.Scratch assay was used to evaluate cell migration ability.The microfluidic chip and traditional transwell assay were used to evaluate cell invasion ability.Immunofluorescence staining and western blot were performed to detect protein expression.CCK8 assay was used to measure the proliferation ability of cells.Total RNA was extracted from cells and sent to Novogene to perform database construction,quality control and sequencing analysis.After basic analysis of data quality,the differential expression of miRNAs between UM-SCC6 and UM-SCC6-M was analyzed.Mi Randa was used to predict the target genes of miRNAs and KEGG pathway enrichment was performed.Realtime PCR and western blot,and miRNA realtime PCR was used to detect the expression level of miRNAs.Exogenous miRNA mimic and inhibitor were used to interfere with the miRNA level in cells.Y27632,an inhibitor of ROCK,was used to downregulate cell invasion.Results Based on the microfluidic chip,the UM-SCC6 cells at invasion front,named as UM-SCC6-M,were isolated and subcultured.UM-SCC6-M had morphological characteristics of mesenchymal cells.Scratch assay demonstrated that UM-SCC6-M cells showed stronger migration ability than that of UM-SCC6.Both the microfluidic chip invasion assay and Transwell invasion assay indicated that UM-SCC6-M cells showed higher invasion ability than that of UM-SCC6.Immunofluorescence staining and western blot showed that the expression of E-cadherin reduced in UM-SCC6-M,compared to UM-SCC6.And the expression of Vim and N-cadherin increased significantly in UM-SCC6-M,compared to UM-SCC6.CCK8 assay showed that UM-SCC6-M and UM-SCC6 had similar proliferation ability.Overall,UM-SCC6-M presented mesenchymal characteristics regarding as cellular morphology,migration and invasion ability,and biomarker expression.Small RNA sequencing found 44 miRNAs expressed differently between UM-SCC6 and UM-SCC6-M,in which 27 miRNAs expression decreased in UM-SCC6-M.We analyzed the target genes of these miRNAs and got many tumor related pathways in KEGG enrichment analysis.Among them,“Proteoglycans in cancer” was the highest enriched pathway.Then,we focused our study on CD44 pathway that is a part of Proteoglycans in cancer and has been reported influencing tumor invasion and metastasis greatly.Increased expression of CD44 protein was confirmed in UM-SCC6-M using western blot,compared to UM-SCC6.We picked out the miRNAs potently targeting CD44-ROCK pathway,and miR-218-5p was selected after detection of miRNA level using realtime PCR.Luciferase report system confirmed that miR-218-5p had a direct binding site on the 3' UTR region of CD44 m RNA and could downregulated CD44 protein expression.We synthesized miR-218-5p mimic and inhibitor,and determined their influence on the level of miR-218-5p.Then they were used to explore the biological function of miR-218-5p.Western blot proved the influence of mimic and inhibitor of miR-218-5p on the protein expression of CD44.Furthermore,both the microfluidic invasion assay and Transwell assay demonstrated that miR-218-5p mimic significantly inhibit the invasion of UM-SCC6-M,while the miR-218-5p inhibitor significantly promoted UM-SCC6 invasion.In addition,the invasion promoting effect of miR-218-5p inhibitor can be reversed by ROCK inhibitor that is downstream of CD44.These results suggest that miR-218-5p could regulate CD44-ROCK pathway.Conclusions We developed a method to isolated cancer cells at invasion front based on microfluidic technology.OSCC cells at invasion front,UM-SCC6-M,were demonstrated to have the characteristics of mesenchymal cells with high invasion ability.miR-218-5p was proved to inhibit OSCC invasion by downregulating CD44-ROCK pathway.