Alternative polyadenylation reprogramming of MORC2 induced by NUDT21 loss promotes KIRC carcinogenesis
Yuqin Tan, … , Ke Li, Ning Na
Yuqin Tan, … , Ke Li, Ning Na
Published September 22, 2023
Citation Information: JCI Insight. 2023;8(18):e162893. https://doi.org/10.1172/jci.insight.162893.
View: Text | PDF
Research Article Oncology

Alternative polyadenylation reprogramming of MORC2 induced by NUDT21 loss promotes KIRC carcinogenesis

Abstract

Alternative polyadenylation (APA), a posttranscriptional mechanism of gene expression via determination of 3′UTR length, has an emerging role in carcinogenesis. Although abundant APA reprogramming is found in kidney renal clear cell carcinoma (KIRC), which is one of the major malignancies, whether APA functions in KIRC remains unknown. Herein, we found that chromatin modifier MORC2 gained oncogenic potential in KIRC among the genes with APA reprogramming, and moreover, its oncogenic potential was enhanced by 3′UTR shortening through stabilization of MORC2 mRNA. MORC2 was found to function in KIRC by downregulating tumor suppressor DAPK1 via DNA methylation. Mechanistically, MORC2 recruited DNMT3A to facilitate hypermethylation of the DAPK1 promoter, which was strengthened by 3′UTR shortening of MORC2. Furthermore, loss of APA regulator NUDT21, which was induced by DNMT3B-mediated promoter methylation, was identified as responsible for 3′UTR shortening of MORC2 in KIRC. Additionally, NUDT21 was confirmed to act as a tumor suppressor mainly depending on downregulation of MORC2. Finally, we designed an antisense oligonucleotide (ASO) to enhance NUDT21 expression and validated its antitumor effect in vivo and in vitro. This study uncovers the DNMT3B/NUDT21/APA/MORC2/DAPK1 regulatory axis in KIRC, disclosing the role of APA in KIRC and the crosstalk between DNA methylation and APA.

Authors

Yuqin Tan, Tong Zheng, Zijun Su, Min Chen, Suxiang Chen, Rui Zhang, Ruojiao Wang, Ke Li, Ning Na

×

Figure 7

Loss of APA regulator NUDT21 induces 3′UTR shortening and upregulation of MORC2 in KIRC.

Options: View larger image (or click on image) Download as PowerPoint
Loss of APA regulator NUDT21 induces 3′UTR shortening and upregulation o...
(A and B) qPCR (A) and immunoblots (B) were performed to evaluate MORC2 expression in Caki-1 cells transfected with indicated plasmid (n = 3). (C and D) qPCR (C) and immunoblots (D) were performed to evaluate MORC2 expression in A498 cells transfected with Flag vector or Flag-NUDT21 plasmid (n = 3). (E) Immunofluorescence staining was performed to evaluate MORC2 expression in Caki-1 cells transfected with Flag-NUDT21 plasmid. Scale bar: 25 μm. (F) qPCR was performed to evaluate the ratio of long 3′UTR MORC2 expression/total MORC2 expression in KIRC cells transfected with Flag vector or Flag-NUDT21 plasmid (n = 3). (G and H) 3′RACE was performed with GSP-1/2 primers (G) to evaluate 3′UTR of MORC2 in Caki-1 cells transfected with Flag vector or Flag-NUDT21 plasmid. (I and J) qPCR was performed to quantitatively analyze half-life of MORC2 in Caki-1 cells transfected with Flag vector or Flag-NUDT21 plasmid (n = 3). (K) Diagram indicated the UGUA motif of MORC2 and the mutant motif. (L) EMSA was performed to explore the binding site of NUDT21 at MORC2 3′UTR. (M and N) qPCR (M) and immunoblots (N) were performed to evaluate DAPK1 expression in WT and MORC2-depleted Caki-1 cells transfected with Flag vector or Flag-NUDT21 plasmid (n = 3). (O) NUDT21 expression in KIRC tissues and normal kidney tissues was analyzed with UALCAN database. (P) Immunoblotting was performed to evaluate NUDT21 expression in HK-2 cells and KIRC cells. (Q) The expression associations between NUDT21 and MORC2/DAPK1, and between MORC2 and DAPK1, were analyzed in KIRC specimens. All data represent the mean ± SD. Two-tailed t test analyses were performed. **P < 0.01; ***P < 0.001.