LncRNA CANT1 suppresses retinoblastoma progression by repellinghistone methyltransferase in PI3Kγ promoter

Abstract

Retinoblastoma (RB) is the most common malignant intraocular tumor of childhood. Recent studies have shown that long noncoding RNAs (lncRNAs), which are longer than 200 bp and without protein-coding ability, are key regulators of tumorigenesis. However, the role of lncRNAs in retinoblastoma remains to be elucidated. In this study, we found that the expression of lncRNA CASC15-New-Transcript 1 (CANT1) was significantly downregulated in RB. Notably, overexpression of CANT1 significantly inhibited RB growth both in vitro and in vivo. Furthermore, lncRNA CANT1, which was mainly located in the nucleus, occupied the promoter of phosphoinositide 3-kinase gamma (PI3Kγ) and blocked histone methyltransferase hSET1 from binding to the PI3Kγ promoter, thus abolishing hSET1-mediated histone H3K4 trimethylation of the PI3Kγ promoter and inhibiting PI3Kγ expression. Furthermore, we found that silencing PI3Kγ either by lncRNA CANT1 overexpression or by PI3Kγ siRNA, reduced the activity of PI3K/Akt signaling and suppressed RB tumorigenesis. In summary, lncRNA CANT1 acts as a suppressor of RB progression by blocking gene-specific histone methyltransferase recruitment. These findings outline a new CANT1 modulation mechanism and provide an alternative option for the RB treatment.

Fig. 1. Identification of CANT1 lncRNA.

a RNA-sequence analysis was performed to evaluate the transcriptome of three retinoblastoma samples and normal retina samples. Red chart: the exons of lncRNA CANT1. Green chart: normal expression of the SOX4 gene in Chr6p22.3. b Expression of CANT1 and CASC15 in three RB cell lines: Y79, Weri-Rb1, and RB44. ARPE19 cells were used as normal control. GAPDH was used as the internal control; M: marker. c Real-time PCR was performed to show CANT1 expression level in RB cells; data are presented as the mean ± SEM. *P < 0.05 compared with ARPE19. d Real-time PCR examination of CANT1 expression in RB tissue. Normal tissues were used as a control. The relative values were normalized to the GAPDH expression level and are presented as mean ± SEM. ***P < 0.001. e Real-time PCR showing overexpression of CANT1 in RB cell lines Y79 and Weri-Rb1. Wild-type tumor cells were used as control. Mock, an empty vector. Data are presented as the mean ± SEM. *P < 0.05 compared with the respective control. f A colony formation assay was performed to assess the tumor growth of RB cells. g, h Colony numbers of Y79 (g) and Weri-Rb1 (h) cells were counted in three independent plates. All of the data are presented as the mean ± SEM. *P < 0.05: compared with the control.

Fig. 2. Functional roles of CANT1 lncRNA in RB.

a, b A CCK8 assay was performed to measure the cell growth rate of CANT1-overexpressing Y79 and Weri-Rb1 cells. The experiments were performed in triplicate, and the absorbance at day 1 was set as 100%. *P < 0.05 compared with the control and mock. c, d A soft agar assay was used to assess the tumor formation ability in vitro. c Small colonies were observed and counted under the microscope. d Colony count statistics showed a significant reduction in colonies formed by CANT1-overexpressing cells. Colony numbers were counted in three independent soft agar plates. All of the data are presented as the mean ± SEM. *P < 0.05 compared with the control and mock. Scale bar: 600 μm. e, f Eye weight of the orthotropic xenograft formed by Weri-Rb1 cells injected into the vitreous cavity with or without CANT1 overexpression at 40 days after implantation; n = 7, *P < 0.05 compared with the mock. f Representative images of H&E staining for the evaluation of tumor formation in vivo. Scale bar: 200 μm.

Fig. 3. Regulatory targets of CANT1 lncRNA in RB.

a The overlapping genes presenting differential expression between the two RB cell lines are shown. Twelve genes were co-upregulated, and 454 genes were co-downregulated (fold change ≥ 1.5, FDR < 0.05). b KEGG analysis for all genes with differential expression. c Heatmap of genes in the PI3K/Akt signaling pathway. d Real-time PCR was performed to measure the PI3Kγ mRNA level in tumor and normal cells. All of the data are presented as the mean ± SEM. *P < 0.05: compared with the control and mock. e Western blot showing the protein levels of PI3Kγ, p-AKT, and pan-AKT in tumor and normal cells. GAPDH was used as internal control. f Immunohistochemical staining of PI3Kγ in RB and normal tissues. PI3Kγ expression in tumor sections was higher than normal tissues. Scale bar: 200 μm. g Two siRNAs were used to silence PI3Kγ, as examined by Western blot.

Fig. 4. Oncogenic role of PI3Kγ in RB.

a, b CCK8 assay showed that the cell growth rate decreased after the silencing of PI3Kγ. All of the data are presented as the mean ± SEM. *P < 0.05: compared with the control and NC. c Colony formation assay was used to assess the cell growth rate of tumor cells treated with siRNA. d Small colonies on each plate were counted. All of the data are presented as the mean ± SEM. *P < 0.05: compared with control. e A soft agar assay was performed to estimate the in vitro tumor formation ability of Y79 and Weri-Rb1 cells treated with siRNA. f Quantification of visible colonies on each plate. All of the experiments were performed in triplicate, and the number of colonies formed is shown as the mean ± SEM. *P < 0.05.

Fig. 5. CANT1 binds to the promoter of its targets.

a The location of mature CANT1. CANT1 is mainly located in the nucleus. U2 RNA was used as a positive control for nuclear RNA, and GAPDH served as a positive control for the cytoplasmic RNA. b Representative RNA FISH images showed that the CANT1 signal overlapped with DAPI staining in ARPE19 cells. The scale bars represent 10 μm. c RNA-FISH was performed with CANT1 oligos on clinical retinoblastoma samples and normal control samples. Scale bar: 20 μm. d Schematic diagram of lncRNA CANT1 and the PI3Kγ promoter region. CANT1 oligo-1 and oligo-2 indicate the biotinylated antisense oligonucleotides targeting lncRNA CANT1. Random oligo indicates the scrambled oligonucleotide used as a negative control in the ChOP assay. e, f PCR examination of the binding of CANT1 to the PI3Kγ promoter in the ChOP assay. Site a: CANT1 interacts with the PI3Kγ promoter. Site b: a negative control locus. Input: Total RNA was reverse transcribed before incubation with labeled CANT1 fragments and amplified with primers for site a and site b. g, h Quantification of the binding of CANT1 to the PI3Kγ promoter in the ChOP assay by real-time qPCR. The data are presented as mean ± SEM. *P < 0.05.

Fig. 6. CANT1 modulates PI3Kγ transcription by abolishing H3K4 methylation.

a Schematic diagram of the PI3Kγ and the GAPDH promoter regions. Arrow: transcriptional direction; sites X–Z: different sites used in this assay. b–d PCR examination of histone H3K4 trimethylation changes in the PI3Kγ promoter (c) and GAPDH promoter (d) upon CANT1 overexpression in Y79 and Weri-Rb1 cells. IgG was used as a negative control. Input: total RNA was reverse transcribed before incubation and amplified with primers; M: marker. e, f Real-time qPCR examination of histone H3K4 trimethylation changes in the PI3Kγ promoter. Site X is 6 kb upstream of the PI3Kγ TSS. All data are presented as the means ± SEM. *P < 0.05: compared with the control and mock.

Fig. 7. CANT1 competes with hSET1 at the PI3Kγ promoter in vitro.

a, b ChIP assays demonstrated that CANT1 blocks the recruitment of hSET1 to the PI3Kγ promoter. IgG was used as a negative control. Sites X, Y: ChIP detection sites. Input: total RNA was reverse transcribed before incubation and amplified with primers. M: marker. c, d Real-time qPCR examination of hSET1 changes in the PI3Kγ promoter. All data are presented as the means ± SEM. *P < 0.05: compared with the control and mock. e Model of CANT1 regulation in tumorigenesis. In parent cancer cells, CANT1 lncRNA is inactivated and the hSET1 methyltransferase freely modifies the PI3Kγ promoter, providing histone H3K4 methylation to induce PI3Kγ expression; however, in CANT1-overexpressing cells, CANT1 occupies the PI3Kγ promoter and blocks the hSET1 interaction with the PI3Kγ promoter. Then, free hSET1 fails to methylate the PI3Kγ promoter and silences PI3Kγ expression, thus decreasing PI3K/Akt signaling and inhibiting tumor growth.