The β-catenin-LINC00183-miR-371b-5p-Smad2/LEF1 axis promotes adult T-cell lymphoblastic lymphoma progression and chemoresistance

Abstract

Background: High-intensity chemotherapy regimens are often used in adult T-cell lymphoblastic lymphoma (T-LBL) patients. Nevertheless, the response rate remains unsatisfactory due to emergence of chemoresistance. Growing evidence has shown that long non-coding RNAs (lncRNAs) are involved in tumor progression and chemoresistance. Herein, we investigated the potential role of lncRNAs in T-LBLs.

Methods: RNAseq was used to screen and identify candidate lncRNAs associated with T-LBL progression and chemoresistance. Luciferase reporter assay was used to examine the binding of miR-371b-5p to the 3'UTR of Smad2 and LEF1, and the binding of TCF-4/LEF1 to the promoter of LINC00183. Chromatin immunoprecipitation assay was undertaken to analyze the connection between LEF1 and the LINC00183 promoter region. RNA immunoprecipitation assays were used to explore the mechanism whereby LINC00183 regulated miR-371b-5p. MTT and flow cytometry assays were used to measure apoptosis of T-LBL cells.

Results: LINC00183 was upregulated in T-LBL progression and chemoresistant tissues in both the Sun Yat-sen University Cancer Center dataset and the First Affiliated Hospital of Anhui Medical University dataset. High expression of LINC00183 was correlated with poorer overall survival and progression-free survival of T-LBL patients compared to those with low expression of LINC00183. Furthermore, miR-371b-5p was negatively regulated by LINC00183. In vivo and in vitro assays showed that LINC00183-mediated T-LBL chemoresistance depended on miR-371b-5p expression. The direct binding of miR-371b-5p to Smad2 and LEF1 was verified by luciferase assays. It was shown that TCF4/LEF1 could bind to the LINC00183 promoter site and increase its transcript level. Downregulation of miR-371b-5p led to increased expression of Smad2/LEF1, and in turn increased LINC00183 expression. Additionally, phospho-Smad2 promotes nuclear translocation of β-catenin, LINC00183 downregulation decreased chemoresistance induced by β-catenin and TGF-β1 in T-LBL cells.

Conclusion: We unraveled a β-catenin-LINC00183-miR-371b-5p-Smad2/LEF1 feedback loop that promotes T-LBL progression and chemoresistance, indicating that LINC00183 may serve as a potential therapeutic target in T-LBLs.

Keywords: Chemoresistance; LEF1; LINC00183; Smad2; T-LBL; miR-371b-5p.

Fig. 1

LINC00183 promotes T-LBL progression and chemoresistance in vivo and in vitro. A The differential expression of lncRNAs in 3 remission and relapse tissues (left), and in 3 chemosensitivity and chemoresistance tissues (right). B Kaplan-Meier curves of progression-free survival (PFS) and overall survival (OS) stratified by LINC00183 expression in T-LBL patients from the SYSUCC dataset. C Kaplan-Meier curves of PFS and OS stratified by LINC00183 expression in T-LBL patients from the AHAMU dataset. D MTT assays using T-LBL and LINC00183-overexpression T-LBL cells under the treatment of Dox (100ng/ml). E The protein expression of Bax, Bcl-2, caspase-3, and cleaved-caspase-3 in T-LBL and LINC00183-overexpression T-LBL cells treated with Dox (100ng/ml). F Flow cytometric analysis of T-LBL cells and LINC00183-overexpression T-LBL under the treatment of Dox (100ng/ml). G The tumor growth curve of mouse xenografts. The xenografts were formed SUP-T1 or SUP-T1-LINC00183 cells treated with Dox (1 mg/kg/time, 3 times/week). H The tumor size of mouse xenografts. I The protein expression of Bax, Bcl-2, and cleaved-caspase-3 in xenograft tissues. 3 xenografts tissues for each group. Dox, doxorubicin. LINC00183, ectopic LINC00183 expression in T-LBL cells. SUP-T1-LINC00183, SUP-T1 cells transfected with LINC00183. *, P < 0.05

Fig. 2

MiR-371b-5p is regulated by LINC00183. A LINC00183 capable of regulating miRNAs was predicted by bioinformatic tool miRDB. B The relative expression score (miRNAs level/LINC00183 level) was calculated in 9 predicted miRNAs. C The expression of miR-371b-5p in T-LBL cells transfected by LINC00183 and shLINC00183. D Correlation between LINC00183 gene expression and miR-371b-5p level in the SYSUCC and AHAMU dataset. E The predicted binding sites between miR-371b-5p and LINC00183. The mutated site (LINC00183-mut) was used for luciferase reporter assays. The relative luciferase activities were detected in 293T cells transfected by LINC00183-WT and LINC00183-mut. F LINC00183 is abundant in the cytoplasm of Jurkat and SUP-T1 cells. U2 and actin were used as positive control. G Cytoplasmic enrichment of LINC00183 in T-LBL tissues. H RNA-IP was used to identify inhibition of miR-371b-5p by LINC00183. The expression levels of LINC00183 and miR-371b-5p were detected using quantitative RT-PCR. LINC00183, ectopic LINC00183 expression in T-LBL cells. shLINC00183, T-LBL cells were transfected by shRNA targeting LINC00183. Dox, doxorubicin. *, P < 0.05

Fig. 3

miR-371b-5p suppresses T-LBL progression and chemoresistance in vivo and in vitro. A Kaplan-Meier curves of progression-free survival (RFS) and OS stratified by miR-371b-5p expression in patients from the SYSUCC and AHAMU datasets. B MTT assay using T-LBL cells transfected by LINC00183, LINC00183 + miR-371b-5p, shLINC00183, shLINC00183 + anti-miR-371b-5p treated with Dox (100ng/ml). C The protein levels of Bax, Bcl-2 in SUP-T1 cells transfected by LINC00183, and LINC00183 + miR-371b-5p treated with Dox (100ng/ml). D Flow cytometric analysis of T-LBL cells transfected by LINC00183, shLINC00183, LINC00183 + miR-371b-5p, and shLINC00183 + anti-miR-371b-5p treated with Dox (100ng/ml). E The tumor growth curve of mouse xenografts. The xenografts were formed SUP-T1-LINC00183 or SUP-T1-LINC00183 + miR-371b-5p cells treated with Dox (1 mg/kg/time, 3 times/week). F The tumor size of mouse xenografts. G The protein expression of Bax, Bcl-2, and cleaved-caspase-3 in xenograft tissues. 3 xenografts tissues for each group. LINC00183, ectopic LINC00183 expression in T-LBL cells. shLINC00183, T-LBL cells were transfected by shRNA targeting LINC00183. LINC00183 + miR-371b-5p, T-LBL cells were transfected with LINC00183 and miR-371b-5p. shLINC00183 + anti-miR-371b-5p, T-LBL cells were transfected with shLINC00183 and anti-miR-371b-5p. Dox, doxorubicin. *, P < 0.05

Fig. 4

MiR-371b-5p targets Smad2/LEF1 in T-LBL cells. A The predicted target sequence of miR-371b-5p in the 3’UTR of Smad2 (Smad2-3’UTR) and LEF1 (LEF1-3’UTR) and mutant containing three altered nucleotides in the seed sequence of miR-371b-5p (miR-371b-5p-mut). B Smad2/LEF1 mRNA levels in T-LBL cells after transfection with miR-371b-5p mimics and anti-miR-1258. C Luciferase assays of pGL3-Smad2-3’UTR and pGL3-LEF1-3’UTR in the presence of miR-371b-5p mimics, miR-371b-5p -mutant, and anti-miR-371b-5p. D The protein levels of Smad2 and LEF1 after transfection of T-LBL cells with miR-1258 mimics and anti-miR-1258. E MTT assays using T-LBL cells transfected by miR-371b-5p, miR-371b-5p + LEF1, miR-371b-5p + Smad2 treated with Dox (100ng/ml). F Flow cytometric analysis of T-LBL cells transfected with miR-371b-5p, miR-371b-5p + LEF1, miR-371b-5p + Smad2 treated with Dox (100ng/ml). G Smad2/LEF1 mRNA levels after transfection with LINC00183, LINC00183 + anti-miR-371b-5p, shLINC00183 and shLINC00183 + miR-371b-5p. H The protein levels of Smad2 and LEF1 after transfection of T-LBL cells with LINC00183, LINC00183 + anti-miR-371b-5p, shLINC00183 and shLINC00183 + miR-371b-5p. LINC00183, ectopic LINC00183 expression in T-LBL cells. shLINC00183, T-LBL cells were transfected by shRNA targeting LINC00183. LINC00183 + anti-miR-371b-5p, T-LBL cells were transfected with LINC00183 and anti-miR-371b-5p. shLINC00183 + miR-371b-5p, T-LBL cells were transfected with shLINC00183 and miR-371b-5p. Dox, doxorubicin. *, P < 0.05

Fig. 5

LINC01278 is regulated by the Wnt/β-catenin pathway. A The expression of LINC00183 in T-LBL cells transfected with miR-371b-5p and anti-miR-371b-5p. B Identification of TCF-4/LEF-1 binding site in the LINC00183’s promoter sequence. The mutant sequence is designed for luciferase reporter assay. C The relative luciferase activity was detected in 293T cells co-transfected with Binding-WT/Binding-mut and LEF1. D ChIP assay was used to detect the binding of LEF1 to the TRE (TCF responsive element: TTCAAAG) region in the LINC00183 promoter. E The relative expression of LINC00183 in T-LBL cells transfected with β-catenin and siLEF1-4. F The relative expression of LINC00183 in T-LBL cells treated with TGF-β1 (0.5 ng/ml) and siLEF1. G MTT assays of T-LBL cells transfected with β-catenin and shLINC00183 treated with Dox (100ng/ml). H Flow cytometric analysis of apoptotic T-LBL cells transfected with β-catenin and shLINC00183 treated with Dox (100ng/ml). I Schematic illustration of the β-catenin-LINC00183-miR-371b-5p-Smad2/LEF1 axis. The up-regulation of LINC00183 reduce the expression of miR-371b, thereby up-regulate the expression of Smad2 and LEF1 proteins. Smad2 can promote the entry of β-catenin protein into the nucleus, and facilitate the formation of transcription complex between β-catenin, TCF4 and LEF1, and positively feedback the level of transcription LINC0083. The blue arrows indicate downregulated; the red arrows indicate upregulated. LINC00183, ectopic LINC00183 expression in T-LBL cells. shLINC00183, T-LBL cells were transfected by shRNA targeting LINC00183. β-catenin, T-LBL cells were transfected with β-catenin. β-catenin + shLINC00183, T-LBL cells were transfected with shLINC00183 and β-catenin. siLEF1, T-LBL cells were transfected by siRNA targeting LEF1. Dox, doxorubicin. *, P < 0.05