Mouse anti-Human CD49c Monoclonal antibody, clone Q2C6

Purpose: Eliminating contamination by corneal stromal cells is critical when preparing cultured human corneal endothelial cells (CECs) transplantation. We investigated markers for the purification of cultured human CECs and markers for excluding cultured human corneal stromal myofibroblasts (CSMFs) from cultured human CECs. Materials and methods: CECs and CSMFs were obtained from human donor corneas by culturing separately in serum-containing medium. Candidate markers of CECs and CSMFs were screened with microarray analysis in the fourth passaged CECs and CSMFs. Then, selected factors were evaluated in reverse transcription polymerase chain reaction (RT-PCR), western blot, immunocytochemistry, and flow cytometry to investigate differential markers for each cell. Results: Among the genes identified by microarray analysis, cultured human CECs, but not CSMFs, expressed integrin alpha 3 (ITGA3 and CD49c) protein according to immunocytochemistry and western blotting. Iroquois homeobox 2 (IRX2) gene was a marker that distinguished CSMFs from cultured human CECs by RT-PCR. The IRX2 gene can be used as a marker of CSMFs contaminating cultured CECs. Conclusion: These molecules could be important markers for the production of highly purified cultured CECs for regenerative medicine.

As a member of the integrin family, integrin alpha 3 beta 1 (ITGA3) has been linked to intercellular communication and serves an important role in the signaling among cells and the extracellular matrix. MicroRNA (miR)-199a-5p has been demonstrated to be related to the pathogenesis and progression of multiple malignant diseases. However, the biological functions of miR-199a-5p and ITGA3 in colorectal cancer (CRC) have rarely been reported. The aim of the present study was to explore the roles of miR-199a-5p and ITGA3 in CRC. Immunohistochemistry staining and western blotting were applied to detect the protein expression of ITGA3 in CRC tissues and cells. Reverse transcription-quantitative PCR was performed to investigate the expression of miR-199a-5p and ITGA3 mRNA. HCT-116 cells were transfected with miR-199a-5p mimics, mimics control, short hairpin RNA targeting ITGA3, or pcDNA-ITGA3 for the functional experiments. Dual luciferase reporter assay was applied to confirm whether miR-199a-5p targeted the 3 ' untranslated region (3 ' UTR) of ITGA3. The MTT, Transwell and wound healing assays were used to evaluate the proliferation, invasion and migration of CRC cells. Immunofluorescence assay was used to monitor the epithelial-mesenchymal transition (EMT) biomarker expression. The results demonstrated downregulation of miR-199a-5p and upregulation of ITGA3 in CRC tissues and cell lines. miR-199a-5p mimics and knockdown of ITGA3 suppressed the proliferation, invasion and migration of CRC cells. Bioinformatics analysis and luciferase reporter assay indicated that miR-199a-5p targeted the 3 ' UTR of the ITGA3 transcript, and overexpression of ITGA3 reversed the tumor-suppressive effects of miR-199a-5p elevation. In addition, the immunofluorescence assay suggested that miR-199a-5p mimics suppressed the EMT of CRC cells, whereas the overexpression of ITGA3 restored this effect. In conclusion, miR-199a-5p may act as a tumor suppressor by targeting and negatively regulating ITGA3 in CRC.