Long Noncoding RNA Expression Profiling By Microarray in Diffuse Large B-Cell Lymphoma and Preliminary Bioinformatics Study

Objective: Application of high-throughput genomics technology to analysis the differences in lncRNA/mRNA expression profiles between the patients with diffuse large B cell lymphoma (DLBCL) and reactive hyperplasia of lymph node (RLN), and between different disease states of DLBCL, in order to explore the role of the aberrantly expressed lncRNAs in the pathogenesis of DLBCL.

Materials and methods: Tumor tissue samples from five newly diagnosed DLBCL, 3 relapsed DLBCL patients and 4 samples of RLN were collected. Agilent Human lncRNA/mRNA Microarray (4×180K) was used to profile differentially expression of lncRNA/mRNAs. Bioinformatics methods were performed for data analysis, including screening of differentially expressed lncRNA/mRNAs, gene ontology (GO) and KEGG pathway enrichment analysis, and lncRNA/mRNA co-expression network construction. Portion of dysregulated lncRNAs from the microarray data analysis was selected for validation using quantitative real-time polymerase chain reaction (qRT-PCR).

Results: (1) In patients with newly diagnosed DLBCL, compared to RLN, a total of 5,806 dysregulated lncRNAs were identified (P<0.05, FC≥2-fold). Among these, 2,650 lncRNAs were significantly upregulated, while 3,156 lncRNAs were significantly downregulated. From the mRNA expression profiling data, 5,628 differently expressed mRNAs were found (P<0.05, FC≥2-fold). Among them, 2,045 were significantly upregulated and 3,583 downregulated. The most enriched GO term and KEGG pathway annotation associated with DLBCL was cell cycle regulation. The coding and non-coding gene expression network construction showed the significantly correlation between lnc-FAM27B-22:1 and mRNAs, including CDCA7L, CEBPB, CHRDL1 and SMIM9. We speculated that lnc-FAM27B-22:1 might have a critical function in pathogenesis of DLBCL.

(2) In patients with primary refractory DLBCL, compared with chemo-sensitive patients, a total of 425 dysregulated lncRNAs were identified(P<0.05, FC≥2-fold). Among these, 124 lncRNAs were significantly upregulated, while 301 lncRNAs were significantly downregulated. From the mRNA expression profiling data, 251 differently expressed mRNAs were found(P<0.05, FC≥2-fold). Among them, 90 were significantly upregulated and 106 downregulated. The most enriched GO terms targeted by dysregulated transcripts were involved in a variety of functions. KEGG pathway analysis showed that 25 pathways corresponded to dysregulated transcripts and the most enriched networks correlated with tumor were Wnt and P53 signaling pathway.

(3) In patients with relapsed DLBCL, compared with newly diagnosed patients, a total of 605 dysregulated lncRNAs were identified(P<0.05, FC≥2-fold). Among these, 169 lncRNAs were significantly upregulated, while 436 lncRNAs were significantly downregulated. From the mRNA expression profiling data, 365 differently expressed mRNAs were found(P<0.05, FC≥2-fold). Among them, 201 were significantly upregulated and 164 downregulated. The most enriched GO terms targeted by dysregulated transcripts were involved in a variety of functions. KEGG pathway analysis showed that 24 pathways corresponded to dysregulated transcripts and the most enriched network correlated with tumor was JAK-STAT signaling pathway.

(4) Fourteen dysregulated lncRNAs expressions were analyzed in the same sample series using qRT-PCR to validate the microarray analysis results. In all but one lncRNA, consistent trend of expression changes was observed in two methods.

Conclusions: In patients with newly diagnosed DLBCL versus RLN, and between different disease states of DLBCL, the lncRNA/mRNA expression profiles were significantly altered. Differentially expressed mRNAs are involved in a number of biological functions and signaling pathways. It prompt lncRNA may play an important role in the mechanism of occurrence, drug resistance and relapse of DLBCL. The above results provide the theory basis for the further study on the role of differentially expressed lncRNA in the development of DLBCL and its potential application as the possible biomarkers of early diagnosis and prognosis.