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Objectives: Mouse CD99 antigen-like 2 (mCD99L2) was confirmed to be expressed on murine B lymphoma (A20) cells by our group previously.The present study was aimed to establish a mCD99L2 down-regulated A20 cell line and to investigate the effect of shRNA targeting mCD99L2 on A20 cells in vitro and in vivo.Methods: Four pLenti6/mCD99L2 expression vectors containing the mCD99L2 shRNA-expressing cassette were constructed,transfected into A20 ceils and stable mCD99L2 down-regulated A20 subclones,named A20-mCD99L2-cells,was established and identified by quantitative PCR and western blot.Light microscopy,transmission electron microscopy,MTT assay,flow cytometry and immuno-fluorenscence labeling were used to observe the morphological,biological and phenotypic characteristics in vitro.Results: Part of A20-mCD99L2-cells exhibited H/RS like morphology,which was also exhibiting decreased proliferating ability,prolonged G2 phase,and increased CD30 and CD15 expression.When cells were injected into nude or immunocompetent BALB/c mice,tumor genesis,tumor growth,morphology and phenotypes in vivo were observed.A20-mCD99L2-cells induced tumors in nude and BALB/c mice,but with less potency in the latter than the controls.Similar morphological,biological and phenotypic characteristics were observed in A20-mCD99L2-cells-induced tumors as those in vitro.Several cytokines including CD30T,IL-12p40/p70,IL-3,IFN-γ,CXCL16,MIP-1α and CD40 were up-regulated after mCD99L2 being down-regulated when detected by antibody arrays.Western blot results indicated regulating mCD99L2 may involve activated nuclear factor-κ,B pathway in the murine B lymphoma cells as the related pathway was concerned.Conclusion: The present work confirmed that silencing mCD99L2 in B cell lymphoma by RNAi induces H/RS-like cells in vitro and in vivo,which provides data for further research into the mCD99L2 gene in lymphomas.