Mechanism Of SMAD5-AS1/miR-135b-5p/APC Axis In The Progression Of Diffuse Large B-cell Lymphoma

Diffuse large B-cell lymphoma(DLBCL)is the highest incidence of adult non-Hodgkin's lymphoma(NHL),accounting for approximately 30% to 35% of NHL.DLBCL is a malignant invasive disease.With the application of R-CHOP in the clinical treatment of DLBCL,about 50% of patients can be cured,but recurrence and drug resistance still occure in about one third of patients.At present,the pathogenesis of DLBCL is still not very clear.An in-depth understanding of the pathogenesis of DLBCL will provide new ideas and theoretical basis for the treatment of DLBCL.Long-chain non-coding RNAs(lncRNAs)are nucleotides that are more than 200 nt in length but without protein encoding ability.LncRNAs can bind to proteins,DNA and RNA and play a wide range of regulatory roles at epigenetic levels,transcription levels,and post-transcription levels.With the wide application of DNA microarray technology and high-throughput sequencing technology in tumor researches,more and more evidence indicates that lncRNAs are widely disregulated in tumors and participate in the occurrence and development of tumors.Therefore,lncRNAs have attracted more and more attention as new biomarkers and important regulators of tumors.The screening of important lncRNAs in DLBCL and its functional and molecular mechanisms will further deepen the understanding of the pathogenesis of DLBCL and provide a new target for the development of DLBCL therapeutic drugs.In this study,we first screened 11 lncRNAs differentially expressed in DLBCL tissues using microarray technology.And the down-regulation of SMAD5-AS1 was one of the most significant.The relusts of real time quantitative PCR and in situ hybridization further confirmed that SMAD5-AS1 was significantly down-regulatedin DCLBCL.Due to the lack of relevant research on the function and mechanism of action of SMAD5-AS1,we performed functional gain and functional loss experiments in vitro and in vivo to further investigate the biological functions of SMAD5-AS1.We found that SMAD5-AS1 functions as a "tumor suppressor gene" in DLBCL.SMAD5-AS1 knockdown can promote the proliferation of DCLBCL cells and inhibit apoptosis.The overexpression of SMAD5-AS1 can significantly inhibit the proliferation of DLBCL cells,cause cell cycle arrest and promote cell apoptosis.To further clarify its mechanism of action,we predicted the potential mechanism of lncRNA competitive endogenous RNA of SMAD5-AS1 by bioinformatics analysis and found that SMAD5-AS1 may bind to miR-135b-5p.Furthermore,the results of dual luciferase reporter systems,RNA-binding protein immunoprecipitation experiments,and RNA-pulldown assays confirmed the binding of SMAD5-AS1 to miR-135b-5p.Moreover,down-regulation of SMAD5-AS1 in DCLBCL cells can increase the expression of miR-135b-5p;conversely,down-regulation of SMAD5-AS1 can decrease the expression of miR-135b-5p.In addition,the "rescue experiment" also confirmed that SMAD5-AS1 mainly functions through miR-135b-5p.To find the target gene of miR-135b-5p,we used the "targetscan","miRDB",and "miRTarBase" prediction platforms to find that the 3'-UTR of the adenomatous colon polyp(APC)disease gene may be related to miR-135b-5p.To confirm the bindng fo is binding of miR-135b-5 to 3'-UTR of APC,we used and the dual luciferase reporter assay and rescue experiments and prove that the APC gene was the target gene of miR-135b-5p,and APC gene was regulated by SMAD5-AS1.The APC gene was known to regulate Wnt/beta-catenin.To confirm the regulation of SMAD5-AS1/miR-135b-5p/APC axis in DLBCL,we used the TOPFlash / FOPFlash reporter system to measure intracellular Wnt/beta-catenin-mediated transcriptional activity.The experiment confirmed that SMAD5-AS1 affected the activity of this pathway,and the "rescue experiment" further confirmed that SMAD5-AS1 affected the expression of miR-135b-5p,which in turn affectsed APC-mediated Wnt/beta-catenin,thereby exerting a tumor suppressing effect.Taken together,SMAD5-AS1 acts as a "molecular sponge" to down-regulate miR-135b-5p,regulateAPC expression and inactivate the canonical Wnt/beta-catenin pathway,thereby inhibiting tumor proliferation.This suggests that SMAD5-AS1 can serve as a potential biomarker and therapeutic target for DLBCL.