Retinol dehydrogenase-10 promotes development and progression of human glioma via the TWEAK-NF-κB axis

Abstract

Retinol dehydrogenase-10 (RDH10) is a member of the short-chain dehydrogenase/reductase family, which plays an important role in retinoic acid (RA) synthesis. Here, we show that RDH10 is highly expressed in human gliomas, and its expression correlates with tumor grade and patient survival times. In vitro, lentivirus-mediated shRNA knockdown of RDH10 suppressed glioma cell proliferation, survival, and invasiveness and cell cycle progression. In vivo, RDH10 knockdown reduced glioma growth in nude mice. Microarray analysis revealed that RDH10 silencing reduces expression of TNFRSF12A (Fn14), TNFSF12 (TWEAK), TRAF3, IKBKB (IKK-β), and BMPR2, while it increases expression of TRAF1, NFKBIA (IκBα), NFKBIE (IκBε), and TNFAIP3. This suggests that RDH10 promotes glioma cell proliferation and survival by regulating the TWEAK-NF-κB axis, and that it could potentially serve as a novel target for human glioma treatment.

Keywords: glioma; nuclear factor kapaB (NF-κB); retinol dehydrogenase 10 (RDH10); the cancer genome atlas (TCGA); tumor necrosis factor-like weak inducer of apoptosis (TWEAK).

Figure 1. RDH10 is over-expressed in human gliomas and predicts a high grade and poor prognosis

(A) Characteristic micrographs of RDH10 immunostaining in gliomas at WHO grade I, II, III, and IV (magnification left, ×100 and right, ×200). (B) Analysis of RDH10 expression from GE-mini(TCGA and GTEx datasets) revealed significant differences between normal brain tissues and glioma. (C) Analysis of RDH10 expression in LGG and GBM. A generalized linear model (GLM) analysis was performed for TCGA RNA-sequencing data of Gliomas. ^**P < 0.01. (D) Survival analysis showed that higher RDH10 expressing gliomas had a poorer prognosis than lower RDH10 expressing gliomas. ^**P < 0.01.

Figure 2. Lentiviral-mediated RDH10 knockdown suppressed human glioma proliferation

(A) Relative RDH10 mRNA levels in glioma cell lines by qPCR. (B) qPCR revealed that RDH10 expression was efficiently reduced in U87 and U251 cells. ^**P < 0.01. (C and D) RDH10 knockdown inhibited U87 (C) and U251 (D) proliferation in vitro. Proliferation detected by CellomicsArrayScan VTI every day for 5d. All experiments were repeated at least three times. Data are shown as mean ± SD, ^**P < 0.01.

Figure 3. RDH10 is essential for glioma cell proliferation, survival and invasion

(A and B) MTT assays indicated that the growth rates decreased in RDH10-silenced U87 (A) and U251 (B) cells. (C and D) RDH10 knockdown induced apoptosis in U87 (C) and U251 (D) cells. (E and F) RDH10 knock- down induces an S and G2/M cell cycle arrest in U87 (E) and U251 (F) cells. (G and H) Transwell Matrigel invasion assays showed that the number of invasive cells in the RDH10 siRNA group was decreased significantly compared with that in the control group for U87 (G) and U251 (H) cells. All experiments were repeated at least three times. Data are shown as mean ± SD, ^**P < 0.01.

Figure 4. in vivo xenograft models confirmed the effect RDH10 on tumorigenicity

(A) Tumor growth curves of RDH10-shRNA U87 cells compared with control lentivirus infected cells. (B) Tumor weights were compared at the end of the experiment. (C) Fluorescence density of tumors were compared at the end of the experiment. ^**P <0.01, ^*P =0.011.

Figure 5. Changes in gene expressions in U-87 cells with RDH10 knockdown by microarray

(A) Heat-map analysis showing 1773 genes that were detected as altered by microarray profiling. corrected P<0.05 and absolute FC >1.5. (B and C) Diseases and functions enrichment of whole-genome expression microarray in RDH10 knockdown U87 cells were analyzed by IPA. (D) Canonical pathways enrichment of whole-genome expression microarray in RDH10 knockdown U87 cells were analyzed by IPA.

Figure 6. TWEAK–NF-κB signaling pathway was significantly inhibited in RDH10 knockdown U87 cells

(A) IPA showing that TWEAK signaling was one of the most significantly inhibited pathway. (B) qPCR was used to validate the mRNA expression of related genes. (C) Western blotting was used to validate the protein expression levels of related genes. (D) Densitometry showed relative protein expression levels of related genes. (E and F) Gene interaction network of RDH10 and related genes in TWEAK–NF-κB signaling pathway. The results show that NFKBIA is located in the central regulatory position. Data are shown as mean ± SD.

Figure 7. NF-κB agonist rescued abnormal cellular apoptosis and proliferation induced by RDH10 knockdown in U-87 cells

(A-B) NF-κB agonist fusicoccin blocked up-regulated cellular apoptosis mediated by RDH10 knockdown in U87 cells. (C) NF-κB agonist Fusicoccin rescued impaired cellular proliferation status mediated by RDH10 knockdown in U-87 cells. cellular proliferation status was analyzed by MTT assay. ^****P <0.0001.