Circular RNA circRILPL1 promotes nasopharyngeal carcinoma malignant progression by activating the Hippo-YAP signaling pathway

doi:10.1038/s41418-023-01171-8

. 2023 Jul;30(7):1679-1694.

doi: 10.1038/s41418-023-01171-8. Epub 2023 May 12.

Circular RNA circRILPL1 promotes nasopharyngeal carcinoma malignant progression by activating the Hippo-YAP signaling pathway

Pan Wu^{1

2}, Xiangchan Hou^{1

2}, Miao Peng^{1

2}, Xiangying Deng^{1

2}, Qijia Yan³, Chunmei Fan^{1

2}, Yongzhen Mo^{1

2}, Yumin Wang^{2

4}, Zheng Li², Fuyan Wang², Can Guo², Ming Zhou², Qianjin Liao¹, Hui Wang¹, Zhaoyang Zeng^{1

2}, Weihong Jiang⁴, Guiyuan Li^{1

2}, Wei Xiong^{1

2}, Bo Xiang^{5

6}

Affiliations

¹ NHC Key Laboratory of Carcinogenesis and Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, 410013, China.
² Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Cancer Research Institute, Central South University, Changsha, Hunan, 410078, China.
³ Department of Pathology, Xiangya Hospital, Central South University, Changsha, Hunan, 410078, China.
⁴ Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha, Hunan, 410078, China.
⁵ NHC Key Laboratory of Carcinogenesis and Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, 410013, China. xiangbolin@csu.edu.cn.
⁶ Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Cancer Research Institute, Central South University, Changsha, Hunan, 410078, China. xiangbolin@csu.edu.cn.

PMID: 37173390
PMCID: PMC10307875
DOI: 10.1038/s41418-023-01171-8

Circular RNA circRILPL1 promotes nasopharyngeal carcinoma malignant progression by activating the Hippo-YAP signaling pathway

Pan Wu et al. Cell Death Differ. 2023 Jul.

. 2023 Jul;30(7):1679-1694.

doi: 10.1038/s41418-023-01171-8. Epub 2023 May 12.

Authors

Affiliations

¹ NHC Key Laboratory of Carcinogenesis and Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, 410013, China.
² Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Cancer Research Institute, Central South University, Changsha, Hunan, 410078, China.
³ Department of Pathology, Xiangya Hospital, Central South University, Changsha, Hunan, 410078, China.
⁴ Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha, Hunan, 410078, China.
⁵ NHC Key Laboratory of Carcinogenesis and Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, 410013, China. xiangbolin@csu.edu.cn.
⁶ Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Cancer Research Institute, Central South University, Changsha, Hunan, 410078, China. xiangbolin@csu.edu.cn.

PMID: 37173390
PMCID: PMC10307875
DOI: 10.1038/s41418-023-01171-8

Abstract

Circular RNAs (circRNAs) play an important regulatory role in the pathogenesis and progression of nasopharyngeal carcinoma (NPC), which have not been thoroughly elucidated. In this study, we revealed for the first time that circRILPL1 was upregulated in NPC, weakened adhesion and decreased stiffness of NPC cells, and promoted NPC proliferation and metastasis in vitro and in vivo. Mechanistically, circRILPL1 inhibited the LATS1-YAP kinase cascade by binding to and activating ROCK1, resulting in decrease of YAP phosphorylation. Binding and cooperating with transport receptor IPO7, circRILPL1 promoted the translocation of YAP from the cytoplasm to the nucleus, where YAP enhanced the transcription of cytoskeleton remodeling genes CAPN2 and PXN. By which, circRILPL1 contributed to the pathogenesis of NPC. Our results demonstrated that circRILPL1 promoted the proliferation and metastasis of NPC through activating the Hippo-YAP signaling pathway by binding to both ROCK1 and IPO7. Highly expressed circRILPL1 in NPC may serve as an important biomarker for tumor diagnosis and may also be a potential therapeutic target.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. CircRILPL1 is highly expressed in NPC and associated with poor prognosis of NPC patients.**
A Schematic diagram of the structure of circRILPL1. Sanger sequencing verified that circRILPL1 was formed by reverse splicing of exons 3 and 4 of RILPL1 mRNA. F1, R1, F2, and R2: primer sites for detecting circRILPL1 and RILPL1 mRNA are indicated. B The differential expression of circRILPL1 in 16 chronic rhinitis epithelial tissues and 38 NPC tissues was detected by qRT-PCR. C ISH assays indicated that circRILPL1 was highly expressed and correlated with the prognosis of NPC patients in 99 NPC paraffin tissue sections, as compared with 46 adjacent non-cancerous NPEs. Left, representative images of ISH. Magnification: 200×, Scale bar = 100 μm; Magnification: 400×, Scale bar = 50 μm. Middle, statistical analysis of ISH scores. Right, Kaplan-Meier survival analysis showed that the overall survival rate of NPC patients with high circRILPL1 expression was significantly lower than that of NPC patients with low circRILPL1 expression, p = 0.0009. D The relative expression levels of circRILPL1 and RILPL1 mRNA in NPC cells were detected after treating with RNase R for 30 min. E The intracellular localization of circRILPL1 was detected by RNA cytoplasm/nucleus fractionation assay in NPC cells. U6 was used as the nuclear internal reference, and GAPDH was used as the cytoplasmic internal control. F The intracellular localization of circRILPL1 was detected by FISH in NPC cells. Nuclei were stained with DAPI (blue). Scale bar = 20 μm. Data were presented as the means ± SD. *p < 0.05, ***p < 0.001, ****p < 0.0001.

**Fig. 2. CircRILPL1 promotes NPC cells migration, invasion, proliferation and alters the mechanical properties of NPC cells in vitro.**
A The migration ability of circRILPL1 was examined in HNE2 cells after overexpression or knockdown of circRILPL1 by wound healing assay. Images were acquired at 0 and 24 h. Scale bars = 200 μm. B The invasion ability of circRILPL1 was assessed in NPC cells after overexpression or knockdown of circRILPL1 by transwell assay. Scale bars =100 μm. C The effect of circRILPL1 on proliferation was evaluated in NPC cells after overexpression or knockdown of circRILPL1 by MTT assay. D Representative AFM deflection images (top) and representative three-dimensional height distribution images (bottom) of NPC cells. **E, F** The stiffness (N/m) and adhesion force (nN) of NPC cells were measured and analyzed by using JPK image processing software. All experiments were performed in at least triplicate samples. Data were presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

**Fig. 3. CircRILPL1 promotes the proliferation and metastasis of NPC cells in vivo.**
CNE2 cells (2 × 10⁶) transfected with the empty vector, the circRILPL1 overexpression plasmid, antisense oligonucleotides against circRILPL1 (ASO-circRILPL1), or the scramble negative control were injected subcutaneously, through the tail vein, or footpad of nude mice (n = 6 per group). A Representative images of subcutaneous tumor tissues (n = 6 per group). B The tumor volume growth curves (top) and the tumor weights (bottom) of subcutaneous tumors in nude mice. C Representative images of H&E staining, circRILPL1 staining, and Ki67 staining of subcutaneous tumor sections. Magnification: 200×, scale bar = 100 μm. D Representative images of lung tissues dissected from nude mice (top, n = 6 per group). Representative images of metastatic nodules on the lung surface as indicated by arrows (bottom). E Quantification of the number of metastatic nodes in the lungs. F Representative images of H&E staining of metastatic lung lesions and the expression of circRILPL1 by ISH assay. Magnification: 40×, scale bar = 500 μm; Magnification: 200×, scale bar = 100 μm. G Images of inguinal lymph nodes dissected from nude mice 28 days after injection via footpad (n = 6 per group). H The volume (top) and weight (bottom) of lymph nodes were measured for each group. I Representative images of H&E staining of inguinal lymph nodes showing metastatic tumor cells. Magnification: 100×, scale bar = 200 μm; Magnification: 200×, scale bar = 100 μm; Magnification: 400×, scale bar = 50 μm. All experiments were technically repeated six times. Data were presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

**Fig. 4. CircRILPL1 activates the Hippo-YAP signaling pathway.**
A The effect of circRILPL1 on the expression level of key proteins of the Hippo signaling pathway was examined by western blotting in NPC cells. B The stability of YAP protein was measured in NPC cells after overexpression of circRILPL1. Cells were treated with cycloheximide (CHX, 50 μg/mL) for 0, 4, 8, 12 h and western blotting was used to measure the expression of YAP protein. The experiments were repeated three times. C The effect of circRILPL1 on the ubiquitination level of YAP protein was detected in NPC cells treated with MG132 for 12 h after overexpression of circRILPL1 plasmid for 48 h. Cell lysates were subjected to immunoprecipitation using anti-YAP antibody followed by western blotting using anti-ubiquitin antibody. D The interaction between YAP and 14-3-3 proteins in NPC cells after overexpression of circRILPL1 was detected by immunoprecipitation using anti-14-3-3 antibody, followed by western blotting using YAP antibody. GAPDH was used as a negative control. E The abundance of YAP protein in nucleus and cytoplasm was examined in NPC cells after overexpression or knockdown of circRILPL1. PARP was used as a nuclear marker and β-tubulin as a cytoplasmic marker. F The result of CTGF luciferase reporter assay showed that circRILPL1 increased the transcriptional activity of YAP in NPC cells. Data were presented as the means ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001.

**Fig. 5. CircRILPL1 regulates NPC cells migration, invasion, proliferation and mechanical properties through YAP signaling.**
Wound healing assay (A), transwell assay (B), MTT assay (C), and AFM assay (D–F) were performed to assess the effects of YAP on NPC cells migration, invasion, proliferation and biophysical properties that regulated by circRILPL1. All experiments were performed in at least triplicate samples. Data were presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

**Fig. 6. CircRILPL1 inhibits the LATS1-YAP kinase cascade by binding to ROCK1.**
A The proteins pulled-down by circRILPL1 were separated by the SDS-PAGE, followed by silver staining and identification by LC-MS/MS. Biotin: biotinylated probe targeting the circRILPL1 back-spliced site; Un-biotin: unbiotinylated probe, as control. Bands corresponding to ROCK1 (158 kDa) and IPO7 (119 kDa) were marked. B The interaction between circRILPL1 and ROCK1 protein was detected in NPC cells using RNA pull-down assay with a biotin-labeled circRILPL1 probe. GAPDH was used as a negative control. C The interaction between circRILPL1 and ROCK1 protein was detected in NPC cells by RIP assay using anti-ROCK1 antibody. The fold enrichment of circRILPL1 was relative to that of the control IgG. Data were presented as the means ± SD. *p < 0.05, ***p < 0.001, ****p < 0.0001. D The co-localization between circRILPL1 and ROCK1 protein was detected by IF-FISH. DAPI: blue; ROCK1: green; CircRILPL1: red; Scale bar = 20 μm. E The effects of circRILPL1 on MYPT1 (Thr696) phosphorylation and ROCK1 protein were measured by western blotting in NPC cells after overexpression or knockdown of circRILPL1. F. The expressions of p-MYPT1 (Thr696), ROCK1, p-YAP (Ser127 and Ser397), YAP, p-LATS1, and LATS1 were measured in the circRILPL1 overexpressed NPC cells treated with the ROCK1 inhibitor Y27632 (10 uM) for 2 h by western blotting. G The expressions of p-MYPT1 (Thr696), ROCK1, p-YAP (Ser127 and Ser397), YAP, p-LATS1, and LATS1 were measured in NPC cells after co-transfection of the circRILPL1 overexpression plasmid and ROCK1 siRNA by western blotting.

**Fig. 7. CircRILPL1 promotes YAP nuclear translocation through mediating its binding to IPO7.**
A The binding between circRILPL1 and IPO7 protein was detected in NPC cells by RNA pull-down with biotin-labeled circRILPL1 probe. GAPDH was used as a negative control. B The binding between circRILPL1 and IPO7 protein was examined in NPC cells using anti-IPO7 antibody by RIP assay. The fold enrichment of circRILPL1 is relative to that of the control IgG. C CircRILPL1 promotes the nuclear translocation of IPO7 in NPC cells as examined by cytosolic/nuclear protein fractionation. PARP was used as a nuclear marker and β-tubulin as a cytoplasmic marker. D. Co-localization of YAP and IPO7 in NPC cells after overexpression of circRILPL1. DAPI: blue; YAP: red; IPO7: green; Scale bar = 10 μm. E The effect of circRILPL1 on the interaction between YAP and IPO7 was detected by immunoprecipitation using anti-Flag (IPO7) antibody, followed by western blotting using YAP antibody in NPC cells transfected with Flag-IPO7 plasmid, co-transfected with circRILPL1 overexpression plasmid or empty vector. GAPDH was used as a negative control. F The transcriptional activity of YAP was measured by luciferase reporter assay after overexpression or knockdown of circRILPL1 or IPO7 in NPC cells. G Knockdown of IPO7 blocked the nuclear import of YAP induced by circRILPL1 in NPC cells. PARP was used as a nuclear marker and β-tubulin as a cytoplasmic marker. H The effects of knockdown of IPO7 or ROCK1 on circRILPL1-induced YAP nuclear translocation in HNE2 cells. Data were presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

**Fig. 8. CircRILPL1-YAP signaling promotes the transcription of CAPN2 and PXN.**
A The expression levels of CAPN2 and PXN mRNA were examined by qRT-PCR in NPC cells after overexpression or knockdown of circRILPL1. B The expressions of CAPN2 and PXN protein were examined by western blotting in NPC cells after overexpression or knockdown of circRILPL1. C The effects of circRILPL1 and YAP on the expressions of CAPN2 and PXN mRNA in NPC cells were detected by qRT-PCR. D The effect of circRILPL1 on the enrichment of YAP on CAPN2 and PXN promoters was examined by ChIP assay. E Schematic diagram of the signaling pathways regulated by circRILPL1 in NPC cells. CircRILPL1 inhibits the LATS1 kinase through binding to and activating ROCK1, thereby suppressing the phosphorylation of YAP. In cooperation with the nuclear transport receptor IPO7, circRILPL1 prompts loading of YAP onto IPO7 and thus enhances YAP translocation into nucleus, where YAP activates the transcription of cytoskeleton remodeling genes CAPN2 and PXN. CircRILPL1 activates the Hippo-YAP signaling pathway, ultimately resulting in biomechanical differences, proliferation, and metastasis of NPC cells. Data were presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

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References

1. Chen YP, Chan ATC, Le QT, Blanchard P, Sun Y, Ma J. Nasopharyngeal carcinoma. Lancet. 2019;394:64–80. doi: 10.1016/S0140-6736(19)30956-0. - DOI - PubMed
1. Wong KCW, Hui EP, Lo KW, Lam WKJ, Johnson D, Li L, et al. Nasopharyngeal carcinoma: an evolving paradigm. Nat Rev Clin Oncol. 2021;18:679–95. doi: 10.1038/s41571-021-00524-x. - DOI - PubMed
1. Chua MLK, Wee JTS, Hui EP, Chan ATC. Nasopharyngeal carcinoma. Lancet. 2016;387:1012–24. doi: 10.1016/S0140-6736(15)00055-0. - DOI - PubMed
1. Xiong W, Zeng ZY, Xia JH, Xia K, Shen SR, Li XL, et al. A susceptibility locus at chromosome 3p21 linked to familial nasopharyngeal carcinoma. Cancer Res. 2004;64:1972–4. doi: 10.1158/0008-5472.CAN-03-3253. - DOI - PubMed
1. Kang Y, He W, Ren C, Qiao J, Guo Q, Hu J, et al. Advances in targeted therapy mainly based on signal pathways for nasopharyngeal carcinoma. Signal Transduct Target Ther. 2020;5:245. doi: 10.1038/s41392-020-00340-2. - DOI - PMC - PubMed

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[1] Chen YP, Chan ATC, Le QT, Blanchard P, Sun Y, Ma J. Nasopharyngeal carcinoma. Lancet. 2019;394:64–80. doi: 10.1016/S0140-6736(19)30956-0. - DOI - PubMed

[2] Chen YP, Chan ATC, Le QT, Blanchard P, Sun Y, Ma J. Nasopharyngeal carcinoma. Lancet. 2019;394:64–80. doi: 10.1016/S0140-6736(19)30956-0. - DOI - PubMed

[3] Wong KCW, Hui EP, Lo KW, Lam WKJ, Johnson D, Li L, et al. Nasopharyngeal carcinoma: an evolving paradigm. Nat Rev Clin Oncol. 2021;18:679–95. doi: 10.1038/s41571-021-00524-x. - DOI - PubMed

[4] Wong KCW, Hui EP, Lo KW, Lam WKJ, Johnson D, Li L, et al. Nasopharyngeal carcinoma: an evolving paradigm. Nat Rev Clin Oncol. 2021;18:679–95. doi: 10.1038/s41571-021-00524-x. - DOI - PubMed

[5] Chua MLK, Wee JTS, Hui EP, Chan ATC. Nasopharyngeal carcinoma. Lancet. 2016;387:1012–24. doi: 10.1016/S0140-6736(15)00055-0. - DOI - PubMed

[6] Chua MLK, Wee JTS, Hui EP, Chan ATC. Nasopharyngeal carcinoma. Lancet. 2016;387:1012–24. doi: 10.1016/S0140-6736(15)00055-0. - DOI - PubMed

[7] Xiong W, Zeng ZY, Xia JH, Xia K, Shen SR, Li XL, et al. A susceptibility locus at chromosome 3p21 linked to familial nasopharyngeal carcinoma. Cancer Res. 2004;64:1972–4. doi: 10.1158/0008-5472.CAN-03-3253. - DOI - PubMed

[8] Xiong W, Zeng ZY, Xia JH, Xia K, Shen SR, Li XL, et al. A susceptibility locus at chromosome 3p21 linked to familial nasopharyngeal carcinoma. Cancer Res. 2004;64:1972–4. doi: 10.1158/0008-5472.CAN-03-3253. - DOI - PubMed

[9] Kang Y, He W, Ren C, Qiao J, Guo Q, Hu J, et al. Advances in targeted therapy mainly based on signal pathways for nasopharyngeal carcinoma. Signal Transduct Target Ther. 2020;5:245. doi: 10.1038/s41392-020-00340-2. - DOI - PMC - PubMed

[10] Kang Y, He W, Ren C, Qiao J, Guo Q, Hu J, et al. Advances in targeted therapy mainly based on signal pathways for nasopharyngeal carcinoma. Signal Transduct Target Ther. 2020;5:245. doi: 10.1038/s41392-020-00340-2. - DOI - PMC - PubMed

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Circular RNA circRILPL1 promotes nasopharyngeal carcinoma malignant progression by activating the Hippo-YAP signaling pathway

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Circular RNA circRILPL1 promotes nasopharyngeal carcinoma malignant progression by activating the Hippo-YAP signaling pathway

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