Tetraspanin 3 Regulates RAB11a-mediated Intracellular Recycling Of β1 Integrin And Promotes NSCLC Cell Proliferation

Objective: In this study,we aimed to investigate the expression and clinicopathological significance of TSPAN3 in non-small cell lung cancer,the role of TSPAN3 in intracellular transport,and the regulatory mechanism of TSPAN3 to β1 integrin.Methods: TSPAN3 plasmid and si RNA sequence,LEL domain mutant plasmid of TSPAN3 were constructed and transfected.The interaction between TSPAN3 and β1 integrin and Rab11 a was determined by laser confocal experiments.Western Blot was used to observe the effects of transfection of various mutants,co-transfection of TSPAN3 plasmid and Rab11 a interference sequence on the activities of β1 integrin and downstream FAK-MAPK pathway,as well as the changes of downstream target gene expression.Immunofluorescence and flow cytometry were used to detect the effects of these changes on integrin subcellular localization and β1 integrin level on cell membrane.Results: 1.Kaplan-meier survival analysis showed that patients with high TSPAN3 expression had significantly shorter postoperative survival compared with patients with low TSPAN3 expression.2.Immunoprecipitation assay showed that TSPAN3 could interact with β1 integrin,and this interaction depended on its LEL domain.Western Blot assay showed that TSPAN3 up-regulated the protein level of β1 integrin.Immunohistochemical results showed that TSPAN3 was positively correlated with the expression level of β1 integrin in lung cancer tissues.3.MTT and colony formation experiments showed that TSPAN3 promoted proliferation in NSCLC,and Western Blot showed that TSPAN3 promoted the activation of β1 integrin downstream signaling pathway,which depended on its LEL domain.4.PCR showed that TSPAN3 did not affect the m RNA level of β1 integrin.Flow cytometry and immunofluorescence showed that TSPAN3 was involved in and promoted the recycle of β1 integrin.5.The interaction between TSPAN3 and Rab11 a was confirmed by mass spectrometry,immunoprecipitation and immunofluorescence,the co-localization of TSPAN3 and Rab11 a showed an overlapping pattern with that of the dynamic change of co-localisation between TSPAN3 and β1 integrin after NZ washout in time and space.After downregulate TSPAN3 expression,immunofluorescence showed that the co-localization of β1 integrin and Rab11 a was significantly reduced.Western Blot assay was performed after co-transfection of TSPAN3 overexpressing plasmid and Rab11 a si-RNA.The results showed that the activation of β1 integrin protein level and downstream signaling pathway induced by TSPAN3 could be reversed by knockdown of Rab11 a.Conclusion: 1.TSPAN3 is highly expressed in NSCLC cells and tissues and is associated with poor prognosis.2.TSPAN3 promotes β1 integrin recycling by facilitating β1 integrin sorting into Rab11 a recycling endosomes.3.TSPAN3 promotes the upregulation of the β1 integrin cycle,thereby promoting the proliferation of NSCLC cells.