HS1 Regulates High-grade "Double-hit" DLBCL Cells Differentiation Towards Plasmacytoid Cells

Background and Objective WHO Classification of Tumours of the Haematopoietic and Lymphoid Tissues(Revised 4th Ed,2017)defines the invasive mature B-cell lymphoma with rearrangements of myc and bcl-2 and/or bcl-6 as high-grade B cell lymphoma(HGBL),with myc and bcl-2 and/or bcl-6 rearrangements known as "double-hit" lymphoma(DHL),"double-hit" DLBCL is a sub-category of DLBCL,In our previous study,our group found that Hematopoietic cell-specific Lyn substrate 1(HS1)was highly expressed in DLBCL and played an important role in the differentiation of B cells.Our study is going to clarify the relationship between double-expression and "double-hit",explore double-expression predicting double-hit IHC positive cut-off values which provide reference for immunohistochemistry and FISH testing for clinical pathology.Exploring the regulation and regulation mechanism of the differentiation-related gene HS1 on plasmacytoid differentiation of the "double-hit" DLBCL from the cellular level and the global level,providing a theoretical basis for inducing differentiation treatment.Methods FISH detected myc,bcl-2 and bcl-6 rearrangements of clinical tissue samples and cell lines of DLBCL.IHC,ICC and Western Blot detected the expression of C-Myc,BCL-2 and Bcl-6 protein.HE detected cell morphology.Constructing lentiviral vector,infecting "double-hit" DLBCL cell lines,down-regulating HS1 expression,selecting stably expressing cell lines by puromycin,observing the efficiency of infection by fluorescence microscope and flow cytometry,detecting the down-regulation efficiency of HS1 by Western blot;Bivariate correlation analysis and Pearso1 χ2 test was used to analyze the relationship between double-expression and"double-hit";Kaplan-Meier analysis was used to analyze the relationship between the expression of C-Myc,BCL-2,BCL-6 and prognosis.Flow cytometry were used to detect the plasmacytoid differentiation markers after that the HS1 expression of double-hit DLBCL cell lines were down-regulated;Transmission electron microscopy detecting organelles and autophagy;constructing the animal model of subcutaneous tumors and a liver metastasis model by splenic capsule injection,observing tumors and metastases by live animal imaging,fluorescence microscope observation was used to confirm the tumor formation and double-hit DLBCL cells morphology before and after HS1 down-regulation,Western blot detecting HS1 expression,the changes of plasmacytoid differentiation markers in double-hit DLBCL control group and down-regulation HS1 group were verified by immunohistochemistry and scoring;FISH detecting whether there were rearrangements of myc,bcl-2 and bcl-6 after down-regulation of HS1 in subcutaneous tumor model tissue;The GO database was used to analyze the role of HS1,C-Myc,BCL2,and BCL-6 in differentiation and possible signaling pathways.Western blot was used to detect the expression of HS1 when NF-kappaB signaling pathway was inhibited and the expression of NF-kB signaling pathway family member proteins after down-regulation of HS1,transwell migration assay detecting the migration ability of cells,and comprehensively analyzing the effects of blockade of NF-κB signaling and down-regulation of HS1 on differentiation of "double-hit" DLBCL cells.Results myc and bcl-2/bcl-6 "double-hit" and C-Myc and BCL-2/BCL-6 double-expression is not absolutely related.From the perspective of clinical prognosis and diagnostic cost,when C-Myc(35%),BCL-2(65%),and BCL-6(5%)are expressed as positive cutoff values,it is better to perform a FISH testing "double-hit".The exclusion rate was 78.8%.Ki-67≥85%was also a reference indicator for FISH screening "double-hit".In vitro experiments,subcutaneous tumor models,and liver metastasis models constructed by splenic capsule injection were used to confirm that HS1 can regulate plasmacytoid differentiation of "double-hit"DLBCL cells,and the effect was more obvious on of "double-hit" DLBCL cells derived from Non-germinal centers,and it didn’t change the rearrangement nature of"double-hit"DLBCL cells.Our study also showed that the downregulation of autophagy and blockade of NF-κB signaling pathway were related to HS1 regulating the plasmacytoid differentiation,but it needs further verification.Conclusion IHC expression of C-Myc(≥35%),BCL-2(≥65%),BCL-6(≥5%),Ki-67(≥85%)can be used as a reference index for clinical FISH screening double-hit.Down-regulation of HS1 can open the high-grade "double-hit" DLBCL cells plasmacytoid differentiation switch protein and promote the differentiation process of "double-hit" DLBCL cells into plasmacytoid cells.