Low expression of TRAF3IP2-AS1 promotes progression of NONO-TFE3 translocation renal cell carcinoma by stimulating N6-methyladenosine of PARP1 mRNA and downregulating PTEN

Abstract

Background: NONO-TFE3 translocation renal cell carcinoma (NONO-TFE3 tRCC) is one subtype of RCCs associated with Xp11.2 translocation/TFE3 gene fusions RCC (Xp11.2 tRCCs). Long non-coding RNA (lncRNA) has attracted great attention in cancer research. The function and mechanisms of TRAF3IP2 antisense RNA 1 (TRAF3IP2-AS1), a natural antisense lncRNA, in NONO-TFE3 tRCC remain poorly understood.

Methods: FISH and qRT-PCR were undertaken to study the expression, localization and clinical significance of TRAF3IP2-AS1 in Xp11.2 tRCC tissues and cells. The functions of TRAF3IP2-AS1 in tRCC were investigated by proliferation analysis, EdU staining, colony and sphere formation assay, Transwell assay and apoptosis analysis. The regulatory mechanisms among TRAF3IP2-AS1, PARP1, PTEN and miR-200a-3p/153-3p/141-3p were investigated by luciferase assay, RNA immunoprecipitation, Western blot and immunohistochemistry.

Results: The expression of TRAF3IP2-AS1 was suppressed by NONO-TFE3 fusion in NONO-TFE3 tRCC tissues and cells. Overexpression of TRAF3IP2-AS1 inhibited the proliferation, migration and invasion of UOK109 cells which were derived from cancer tissue of patient with NONO-TFE3 tRCC. Mechanistic studies revealed that TRAF3IP2-AS1 accelerated the decay of PARP1 mRNA by direct binding and recruitment of N⁶-methyladenosie methyltransferase complex. Meanwhile, TRAF3IP2-AS1 competitively bound to miR-200a-3p/153-3p/141-3p and prevented those from decreasing the level of PTEN.

Conclusions: TRAF3IP2-AS1 functions as a tumor suppressor in NONO-TFE3 tRCC progression and may serve as a novel target for NONO-TFE3 tRCC therapy. TRAF3IP2-AS1 expression has the potential to serve as a novel diagnostic and prognostic biomarker for NONO-TFE3 tRCC detection.

Keywords: M6A modification; NONO-TFE3; PARP1; PTEN; TRAF3IP2-AS1.

Fig. 1

TRAF3IP2-AS1 expression is significantly decreased in NONO-TFE3 tRCC. a The RNA level of TRAF3IP2-AS1 (red) was analyzed by FISH assays in NONO-TFE3 tRCC and ccRCC. DAPI-stained nuclei are blue. b–d Kaplan–Meier analysis revealed the disease-free survival (DFS) in KICH, KIRC and KIRP patients based on the relative TRAF3IP2-AS1 expression. e The RNA level of TRAF3IP2-AS1 was analyzed by qRT-PCR assay in ccRCC cell line (786-O), tRCC cell lines (UOK109 and UOK120) and normal cell lines (HK-2 and HEK293T). f–h Analysis of TRAF3IP2-AS1 in KICH, KIRC and KIRP tissues compared with normal tissues were performed using TCGA data. i–j The subcellular distribution of TRAF3IP2-AS1 was analyzed via qRT-PCR in HEK293T, HK-2, 786-O, UOK120 and UOK109 cells. U6 and GAPDH were used as nuclear and cytoplasmic markers, respectively. k The location of TRAF3IP2-AS1 (red) in UOK109 and 786-O cells was determined by FISH assay. U6 and 18s rRNA were used as positive controls for the nuclear and cytoplasmic fractions, respectively. DAPI-stained nuclei are blue. The data are presented as the mean ± SD, *P < 0.05, ***P < 0.001

Fig. 2

TRAF3IP2-AS1 deficiency induces development of NONO-TFE3 tRCC. a Schematic illustration of CRISPR/Cas9-based Synergistic Activation Mediator (SAM) system. b The RNA level of TRAF3IP2-AS1 was analyzed by qRT-PCR assay in UOK109 transfected with dCas9 and guide RNA targeting TRAF3IP2-AS1 promoter (gTRAF3IP2-AS1). c The RNA level of TRAF3IP2-AS1 was analyzed by qRT-PCR assay in 786-O transfected with siRNA and antisense oligonucleotides (ASOs). d–f The effects of TRAF3IP2-AS1 overexpression or knockdown on the proliferation of UOK109 and 786-O cells, respectively, were examined by CCK-8 assay (d), colony formation assays (e) and tumor sphere formation (f). g EdU assays were used to detect the proliferation rate of UOK109 and 786-O cells after transfection for 48 h. h Cell cycle was analyzed using flow cytometry after transfection for 48 h. i Cell apoptosis was analyzed via flow cytometry using an Annexin V/PI Kit after transfection for 48 h. j Migration and invasion assays were performed with transfected cells using Transwell inserts. The data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

NONO-TFE3 inhibits lncRNA TRAF3IP2-AS1 transcription. a ChIP assays showed endogenous NONO-TFE3 binding to the TRAF3IP2-AS1 gene promoter. 786-O cells were transfected with NONO-TFE3-Flag-overexpressing vector for 48 h. The binding of NONO-TFE3 at the TRAF3IP2-AS1 promoter region was detected by a ChIP assay. b Schematic summary of the dCas9-gRNA-guided ChIP (upper); Western blot was performed after dCas9-gRNA-guided ChIP (lower). c–d HEK293T cells were co-transfected with TRAF3IP2-AS1 promoter–luciferase truncations and NONO-TFE3 plasmids, and the luciferase activity was determined using a Dual Luciferase Reporter Assay after 48 h. e Dual luciferase assay of HEK293T cells co-transfected with firefly luciferase constructs containing the wild-type or mutant NONO-TFE3 potential binding sites of TRAF3IP2-AS1 promoter and NONO-TFE3 plasmids were performed. g The protein and mRNA levels of NONO-TFE3 and the TRAF3IP2-AS1 expression levels were detected after transfection with NONO-TFE3 plasmids or shTFE3 for 48 h. h NONO-TFE3 immunohistochemistry was performed in paraffin sections of samples from patients. The data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

TRAF3IP2-AS1 down-regulates PARP1 mRNA by direct binding. a The RNA levels of TRAF3IP2-AS1 and PARP1 were detected after transfection with TRAF3IP2-AS1 plasmids or siTRAF3IP2-AS1 for 48 h. b Level of PARP1 mRNA detected by qRT-PCR after MS2-RIP for GFP in UOK109 cells. AS1, NC, AS1-antisense and AS1-Mut correspond to TRAF3IP2-AS1, empty vector, TRAF3IP2-AS1-antisense and TRAF3IP2-AS1 with mutation of potential binding site to PARP1 mRNA, respectively. c–d Dual Luciferase Reporter Assay used to detect the relative luciferase activity in HEK293 T cells co-transfected with siTRAF3IP2-AS1 and pmirGLO-PARP1 3′-UTR WT/MUT. e The stability of PARP1 mRNA and GAPDH mRNA in UOK109 cells transfected with TRAF3IP2-AS1 or TRAF3IP2-AS1 contained binding site mutation was measured by qRT-PCR relative to 0 h after blocking new RNA synthesis with α-amanitin and normalized to 18 s rRNA. f The RNA levels of NONO-TFE3 and PARP1 were detected after transfection with NONO-TFE3 plasmids or shTFE3 for 48 h. g The stability of PARP1 mRNA and GAPDH mRNA in 786-O cells transfected with siTRAF3IP2-AS1 was measured by qRT-PCR after treatment with α-amanitin. h The protein level of PARP1 was detected after transfection with TRAF3IP2-AS1 plasmids or siTRAF3IP2-AS1

Fig. 5

TRAF3IP2-AS1 accelerates the decay of PARP1 mRNA by recruitment of m⁶A methyltransferase complex. a The location of TRAF3IP2-AS1 binding site and a potential m⁶A site are indicated. Abundance of PARP1 transcript among mRNA immunoprecipitated with anti-m⁶A antibody was measured by qRT-PCR and normalized to IgG. b–c The mRNA level of PARP1 was detected by qRT-PCR after transfected with indicated vectors or siRNAs. d–e METTL3 was immunoprecipitated followed by qRT-PCR for assessing the association of the indicated PARP1 mRNA with METTL3 after overexpression or knockdown of TRAF3IP2-AS1. f–g Abundance of PARP1 among mRNA immunoprecipitated with anti-m⁶A antibody from cells transfected with indicated vectors or siRNA/ASO was measured by qRT-PCR. h PARP1-3′-UTR of the wild-type or containing a m⁶A consensus sequence mutant (A to G) was fused with a luciferase reporter. Luciferase activity of PARP1-3′-UTR was measured and normalized to Renilla luciferase activity. p–k Luciferase activity of PARP1-3′-UTR was measured after co-transfected with TRAF3IP3-AS1 and siMETTL3/siMEETTL14/siWTAP. l–m The stability of PARP1 mRNA in cells co-transfected with indicated vectors or siRNAs after treatment with α-amanitin. n Schematic illustration of targeted RNA methylation system. o–p Abundance of PARP1 among mRNA immunoprecipitated with anti-m⁶A antibody from cells transfected with indicated gRNA was measured by qRT-PCR. q The protein and mRNA level of PARP1 were measured by qRT-PCR and Western blot after transfected with indicated gRNA. r–s The stability of PARP1 and GAPDH mRNA in cells transfected with indicated gRNA after treatment with α-amanitin. The data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

YTHDF2 mediates the decay of PARP1 mRNA to regulate NONO-TFE3 tRCC progression. a YTHDF1/2 was immunoprecipitated followed by qRT-PCR for assessing the association of PARP1 mRNA with YTHDF1/2 after overexpression or knockdown of TRAF3IP2-AS1. b Luciferase activity of PARP1-3′-UTR was measured after co-transfected with TRAF3IP3-AS1 and siYTHDF2. c Schematic illustration of MS2-RIP. d Abundance of m⁶A relative proteins among MS2-RIP with anti-GFP antibody from cells transfected with indicated plasmid was measured by Western blot. e The stability of PARP1 mRNA in cells co-transfected with indicated vectors or siRNAs after treatment with α-amanitin. f Schematic illustration of targeted RNA methylation system. g–h The mRNA (g) and protein (h) level of PARP1 were measured by qRT-PCR and Western blot after transfected with indicated gRNA. i–j The stability of PARP1 and GAPDH mRNA in cells transfected with indicated gRNA after treatment with α-amanitin. The data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

TRAF3IP2-AS1 functions as a ceRNA and sponges miRNAs. a Schematic of the selection for the direct downstream target of TRAF3IP2-AS1. b–c The effect of TRAF3IP2-AS1 on multiple miRNAs expression in UOK109 cells was analyzed by qRT-PCR after overexpression or knockdown of TRAF3IP2-AS1. d–f The RNA levels of multiple miRNAs and TRAF3IP2-AS1 were analyzed via qRT-PCR in UOK109 cells after transfected with miRNA inhibitor, respectively. e–g The RNA levels of multiple miRNAs and TRAF3IP2-AS1 were analyzed via qRT-PCR in 786-O cells after overexpression miRNAs, respectively. h–i Model of AGO2-RIP/MS2-RIP assay. j RIP assays were performed using AGO2 antibody in UOK109 cells, and then, the enrichment ofTRAF3IP2-AS1was detected by qRT-PCR. k MS2-RIP-derived RNA was examined by qRT-PCR. The levels of the qRT-PCR products were normalized relative to IgG control. l HEK293T cells were co-transfected with miRNA mimics, respectively, and wild-type or mutant TRAF3IP2-AS1 luciferase reporter vector, and luciferase reporter activity was detected. m–o Schematic illustration of TRAF3IP2-AS1 wild type and mutation luciferase reporter vectors. The data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

TRAF3IP2-AS1 modulates the expression of PTEN through post-transcriptional regulation of miRNAs. a Schematic of the selection for the direct downstream target of TRAF3IP2-AS1. b–c The effect of multiple miRNAs expression on PTEN mRNA in UOK109 cells was analyzed by qRT-PCR after overexpression or knockdown of miRNAs, respectively. d RIP assays were performed using AGO2 antibody in UOK109 cells, and then, the enrichment of PTEN mRNA was detected by qRT-PCR. e HEK293T cells were co-transfected with miRNA mimics, respectively, and wild-type or mutant PTEN 3′-UTR luciferase reporter vector, and luciferase reporter activity was detected. f–h Schematic illustration of PTEN 3′-UTR wild type and mutation luciferase reporter vectors. i The mRNA levels of NONO-TFE3 and PTEN were detected after transfection with NONO-TFE3 plasmids or shTFE3 for 48 h. j–k The protein level of PTEN was detected after co-transfection with indicated vectors, siRNAs/ASOs or miRNAs inhibitor for 48 h. The data are presented as the mean ± SD, **P < 0.01, ***P < 0.001

Fig. 9

Schematic diagram for the mechanisms of TRAF3IP2-AS1 functioning as both an mRNA decay accelerator and a miRNA sponge to inhibit progression of NONO-TFE3 tRCC