| Background and aim:B-cell non-Hodgkin lymphoma(B-NHL)accounts for about 85-90%of all non-Hodgkin lymphomas,of which the most common type is diffuse large B-cell lymphoma(DLBCL),3 0%-40%of patients in this type are still difficult to achieve long-term survival due to disease progression.It is necessary to predict the prognosis accurately at the time of diagnosis to guide the individualized treatment of these patients and improve their clinical outcomes.Currently,although several gene-based classification methods are available to predict the prognosis of DLBCL,some patients are still unable be classified.Therefore,identifying new DLBCL prognostic markers and exploring the molecular functions and mechanisms is expected to be a potential point in improving the clinical outcomes of BNHL patients.The first part of this study was conducted to identify the potential prognostic markers by performing bioinformatic analysis of gene expression profiles of B-NHL patients in public database.Based on the results of the first part,CHN1 was selected as the target gene of the second part.By stably overexpressing CHN1 in the B-NHL cell line Raji cells,combined with the results of RNA-seq on the stable cell line,we preliminarily explored the role and its molecular mechanism of CHN1 in B-NHL cells.Methods:1.A total of 1850 B-NHL with gene expression profiles were retrieved from Gene Expression Omnibus(GEO)database and Lymphoma/Leukemia Molecular Profiling Project(LLMPP)to identify candidate genes through certain screening criteria.2.Two independent datasets with both clinical information and gene expression profiles of DLBCL patients were used to evaluate the prognostic ability of candidate genes,including survival analysis,univariate and multivariate COX regression analysis.3.Gene set enrichment analysis(GSEA)and CIBERSORT algorithm were used to annotate the potential biological functions of CHN1.4.The B-NHL cell line Raji cells were selected for experiments,and the CHN1overexpressing Raji cell line was constructed by lentivirus infection.5.RNA-seq was performed on CHN1-overexpressing Raji cell line and the empty vector negative control cells to analyze the potential biological function and molecular mechanism of CHN1.6.The effect of overexpression of CHN1 on the cell proliferation ability of B-NHL was determined by cell counting method.7.The molecular mechanism of the effect of CHN1 overexpression on the proliferation of B-NHL cells was preliminarily investigated by Western blot.Results:1.We identified 6 candidate genes related to the prognosis of DLBCL patients,including CHN1,CD3D,CLU,ICOS,KLRB1 and LAT.Unlike the other five genes,the role of CHN1 in B-NHL has not been reported.Therefore,CHN1 was selected as the target gene for the subsequent research.2.CHN1 has prognostic value in important clinical subgroups of DLBCL,high CHN1 expression is associated with a good prognosis in DLBCL patients with germinal center Bcell-like(GCB)subtype,stage Ⅲ-Ⅳ or International Prognostic Index(IPI)score>2.3.Multivariate Cox regression analysis suggested that CHN1 was an independent prognostic factor for DLBCL.4.GSEA and CIBERSORT analysis indicated that CHN1 may be associated with PI3K/Akt/mTOR pathway,cell adhesion and T cell immune infiltration.5.The monoclonal Raji cell line stably overexpressing CHN1 was constructed.6.GSEA analysis was performed on the RNA-seq data of the Raji cell line overexpressing CHN1 and the microarray data in the GSE10846 and GSE117556 datasets.We integrated and analyzed all the results of GSEA,the Akt/mTOR signaling pathway was the common pathway.Therefore,the biological function of CHN1 may be related to the Akt/mTOR signaling pathway,which plays an important role in regulating cell proliferation.7.The role of CHN1 overexpression is to inhibit the proliferation of B-NHL cells.8.Based on the above RNA-seq results,the Akt/mTOR pathway-related proteins were verified by Western blot,and it was confirmed that the phosphorylation levels of Akt and its downstream molecule mTOR were inhibited in the CHN1-overexpressing Raji cell line.In addition,treatment of Raji cells with MK2206,an allosteric inhibitor of Akt,showed that MK2206 inhibited the phosphorylation levels of Akt and mTOR in Raji cells in a dosedependent manner,and MK2206 also inhibited the cell proliferation of B-NHL cells.Conclusions:1.High expression of CHN1 is associated with better clinical outcomes in patients with DLBCL,suggesting that CHN1 may be a potential prognostic marker for DLBCL.2.Overexpression of CHN1 can significantly reduce the proliferation ability of Raji cells,and it can also inhibit the phosphorylation levels of Akt and mTOR.The proliferation of B-NHL cells was also inhibited after treatment with Akt’s allosteric inhibitor MK2206.Therefore,inhibition of Akt/mTOR pathway may be one of the molecular mechanisms involved in CHN1 regulating B-NHL cell proliferation. |
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