Wuping Yang1,2,3,4, Kenan Zhang1,2,3,4, Lei Li1,2,3,4, Yawei Xu1,2,3,4, Kaifang Ma1,2,3,4, Haibiao Xie1,2,3,4, Jingcheng Zhou1,2,3,4, Lin Cai1,2,3,4, Yanqing Gong5,6,7,8, Kan Gong9,10,11,12. 1. Department of Urology, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing, 100034, China. 2. Hereditary Kidney Cancer Research Center, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing, 100034, China. 3. Institute of Urology, Peking University, Beijing, 100034, P. R. China. 4. National Urological Cancer Center, Beijing, 100034, P. R. China. 5. Department of Urology, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing, 100034, China. yqgong@bjmu.edu.cn. 6. Hereditary Kidney Cancer Research Center, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing, 100034, China. yqgong@bjmu.edu.cn. 7. Institute of Urology, Peking University, Beijing, 100034, P. R. China. yqgong@bjmu.edu.cn. 8. National Urological Cancer Center, Beijing, 100034, P. R. China. yqgong@bjmu.edu.cn. 9. Department of Urology, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing, 100034, China. gongkan_pku@126.com. 10. Hereditary Kidney Cancer Research Center, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing, 100034, China. gongkan_pku@126.com. 11. Institute of Urology, Peking University, Beijing, 100034, P. R. China. gongkan_pku@126.com. 12. National Urological Cancer Center, Beijing, 100034, P. R. China. gongkan_pku@126.com.
Abstract
BACKGROUND: Emerging evidence confirms that lncRNAs (long non-coding RNAs) are potential biomarkers that play vital roles in tumors. ZNF582-AS1 is a novel lncRNA that serves as a potential prognostic marker of cancers. However, the specific clinical significance and molecular mechanism of ZNF582-AS1 in ccRCC (clear cell renal cell carcinoma) are unclear. METHODS: Expression level and clinical significance of ZNF582-AS1 were determined by TCGA-KIRC data and qRT-PCR results of 62 ccRCCs. DNA methylation status of ZNF582-AS1 promoter was examined by MSP, MassARRAY methylation and demethylation analysis. Gain-of-function experiments were conducted to investigate the biological roles of ZNF582-AS1 in the phenotype of ccRCC. The subcellular localization of ZNF582-AS1 was detected by RNA FISH. iTRAQ, RNA pull-down and RIP-qRT-PCR were used to identify the downstream targets of ZNF582-AS1. rRNA MeRIP-seq and MeRIP-qRT-PCR were utilized to examine the N(6)-methyladenosine modification status. Western blot and immunohistochemistry assays were used to determine the protein expression level. RESULTS: ZNF582-AS1 was downregulated in ccRCC, and decreased ZNF582-AS1 expression was significantly correlated with advanced tumor stage, higher pathological stage, distant metastasis and poor prognosis. Decreased ZNF582-AS1 expression was caused by DNA methylation at the CpG islands within its promoter. ZNF582-AS1 overexpression inhibited cell proliferative, migratory and invasive ability, and increased cell apoptotic rate in vitro and in vivo. Mechanistically, we found that ZNF582-AS1 overexpression suppressed the N(6)-methyladenosine modification of MT-RNR1 by reducing rRNA adenine N(6)-methyltransferase A8K0B9 protein level, resulting in the decrease of MT-RNR1 expression, followed by the inhibition of MT-CO2 protein expression. Furthermore, MT-RNR1 overexpression reversed the decreased MT-CO2 expression and phenotype inhibition of ccRCC induced by increased ZNF582-AS1 expression. CONCLUSIONS: This study demonstrates for the first time that ZNF582-AS1 functions as a tumor suppressor gene in ccRCC and ZNF582-AS1 may serve as a potential biomarker and therapeutic target of ccRCC.
BACKGROUND: Emerging evidence confirms that lncRNAs (long non-coding RNAs) are potential biomarkers that play vital roles in tumors. ZNF582-AS1 is a novel lncRNA that serves as a potential prognostic marker of cancers. However, the specific clinical significance and molecular mechanism of ZNF582-AS1 in ccRCC (clear cell renal cell carcinoma) are unclear. METHODS: Expression level and clinical significance of ZNF582-AS1 were determined by TCGA-KIRC data and qRT-PCR results of 62 ccRCCs. DNA methylation status of ZNF582-AS1 promoter was examined by MSP, MassARRAY methylation and demethylation analysis. Gain-of-function experiments were conducted to investigate the biological roles of ZNF582-AS1 in the phenotype of ccRCC. The subcellular localization of ZNF582-AS1 was detected by RNA FISH. iTRAQ, RNA pull-down and RIP-qRT-PCR were used to identify the downstream targets of ZNF582-AS1. rRNA MeRIP-seq and MeRIP-qRT-PCR were utilized to examine the N(6)-methyladenosine modification status. Western blot and immunohistochemistry assays were used to determine the protein expression level. RESULTS:ZNF582-AS1 was downregulated in ccRCC, and decreased ZNF582-AS1 expression was significantly correlated with advanced tumor stage, higher pathological stage, distant metastasis and poor prognosis. Decreased ZNF582-AS1 expression was caused by DNA methylation at the CpG islands within its promoter. ZNF582-AS1 overexpression inhibited cell proliferative, migratory and invasive ability, and increased cell apoptotic rate in vitro and in vivo. Mechanistically, we found that ZNF582-AS1 overexpression suppressed the N(6)-methyladenosine modification of MT-RNR1 by reducing rRNA adenine N(6)-methyltransferase A8K0B9 protein level, resulting in the decrease of MT-RNR1 expression, followed by the inhibition of MT-CO2 protein expression. Furthermore, MT-RNR1 overexpression reversed the decreased MT-CO2 expression and phenotype inhibition of ccRCC induced by increased ZNF582-AS1 expression. CONCLUSIONS: This study demonstrates for the first time that ZNF582-AS1 functions as a tumor suppressor gene in ccRCC and ZNF582-AS1 may serve as a potential biomarker and therapeutic target of ccRCC.
Entities:
Keywords:
DNA methylation; MT-RNR1; N(6)-methyladenosine modification; ZNF582-AS1; ccRCC
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