Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Downregulation of lncRNA ZNF582-AS1 due to DNA hypermethylation promotes clear cell renal cell carcinoma growth and metastasis by regulating the N(6)-methyladenosine modification of MT-RNR1

doi: 10.1186/s13046-021-01889-8

Figure Lengend Snippet: ZNF582-AS1 expression was regulated by DNA methylation in ccRCC. a Detection of CpG islands in ZNF582-AS1 promoter and design of MSP primers. The horizontal axis of the curved lines represents the input sequence of ZNF582-AS1, and the vertical axis of the curved lines represents GC percentage. TSS: Transcription Start Sites. b MSP analysis of ZNF582-AS1 promoter DNA methylation status in ccRCC cell lines. c MSP analysis of ZNF582-AS1 promoter DNA methylation status in ccRCC tissues. d Detection of 38 CpG sites in ZNF582-AS1 promoter. e Quantitative detection of DNA methylation level of 38 CpG sites in ZNF582-AS1 promoter using Sequenom MassARRAY quantitative DNA methylation analysis. f and g Comparison of the DNA methylation levels of 38 CpG sites in ccRCC and adjacent normal renal tissues. h Treatment with 5-aza-dC and TSA demethylated ZNF582-AS1 promoter and increased ZNF582-AS1 expression in OSRC2 and Caki-1 cells. T refers to Tumor tissue of ccRCC, N refers to Adjacent normal kidney tissue. M = Methylated, U = Unmethylated

Article Snippet: Approximately 5 × 10 6 ZNF582-AS1-overexpressed, MT-RNR1-overexpressed-ZNF582-AS1-overexpressed and control OSRC2 cells suspended in 100 μL Hank’s Balanced Salt Solution (Thermo Fisher Scientific, USA) were mixed with Matrigel (1:1, Corning Inc., USA).

Techniques: Expressing, DNA Methylation Assay, Sequencing, Comparison, Methylation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Downregulation of lncRNA ZNF582-AS1 due to DNA hypermethylation promotes clear cell renal cell carcinoma growth and metastasis by regulating the N(6)-methyladenosine modification of MT-RNR1

doi: 10.1186/s13046-021-01889-8

Figure Lengend Snippet: ZNF582-AS1 overexpression attenuated cell proliferation and induced cell apoptosis in vitro and in vivo. a and b EdU assay and c CCK-8 assay determined cell proliferation of ZNF582-AS1-overexpressed OSRC2 and Caki-1 cells comparing with their control cells. d and e TUNEL assay and f and g flow cytometry determined cell apoptosis of ZNF582-AS1-overexpressed OSRC2 and Caki-1 cells comparing with their control cells. h Tumors collected from mice are shown. i and j Tumor volume curves and tumor weights of the ZNF582-AS1-overexpressed and control groups were measured and compared. (k and l) The cell proliferation and apoptosis of tumors were examined

Article Snippet: Approximately 5 × 10 6 ZNF582-AS1-overexpressed, MT-RNR1-overexpressed-ZNF582-AS1-overexpressed and control OSRC2 cells suspended in 100 μL Hank’s Balanced Salt Solution (Thermo Fisher Scientific, USA) were mixed with Matrigel (1:1, Corning Inc., USA).

Techniques: Over Expression, In Vitro, In Vivo, EdU Assay, CCK-8 Assay, Control, TUNEL Assay, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Downregulation of lncRNA ZNF582-AS1 due to DNA hypermethylation promotes clear cell renal cell carcinoma growth and metastasis by regulating the N(6)-methyladenosine modification of MT-RNR1

doi: 10.1186/s13046-021-01889-8

Figure Lengend Snippet: ZNF582-AS1 overexpression inhibited cell migratory and invasive ability in vitro and in vivo. a and b Wound healing assay determined the migratory distances of ZNF582-AS1-overexpressed OSRC2 and Caki-1 cells and their control cells. c and d Transwell migration and e and f invasion assays determined the migratory and invasive abilities of ZNF582-AS1-overexpressed OSRC2 and Caki-1 cells and their control cells. g and h The luciferase signals in the ZNF582-AS1-overexpressed group were remarkably lower than those in the control group. i There was no significant difference between the mice weight of ZNF582-AS1-overexpressed and control group. j and k The number and size of pulmonary metastases in the ZNF582-AS1-overexpressed group were significantly reduced compared with those in the control group. l and m ZNF582-AS1 overexpression increased E-cadherin expression and decreased N-cadherin expression in pulmonary metastases

Article Snippet: Approximately 5 × 10 6 ZNF582-AS1-overexpressed, MT-RNR1-overexpressed-ZNF582-AS1-overexpressed and control OSRC2 cells suspended in 100 μL Hank’s Balanced Salt Solution (Thermo Fisher Scientific, USA) were mixed with Matrigel (1:1, Corning Inc., USA).

Techniques: Over Expression, In Vitro, In Vivo, Wound Healing Assay, Control, Migration, Luciferase, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Downregulation of lncRNA ZNF582-AS1 due to DNA hypermethylation promotes clear cell renal cell carcinoma growth and metastasis by regulating the N(6)-methyladenosine modification of MT-RNR1

doi: 10.1186/s13046-021-01889-8

Figure Lengend Snippet: ZNF582-AS1 overexpression decreased rRNA adenine N(6)-methyltransferase A8K0B9 expression. a The overexpression efficiency of ZNF582-AS1 in OSRC2 cells. b iTRAQ results showed that 69 proteins were downregulated and 75 proteins were upregulated in ZNF582-AS1-overexpressed OSRC2 cells compared with control OSRC2 cells. c The expression of these 144 differently expressed proteins in ZNF582-AS1-overexpressed OSRC2 cells and control cells. d Biological Process GO term enrichment analysis of the complete 144 statistically significant proteins. e and f Validation of the presence of A8K0B9 protein. g A8K0B9 expression was decreased in ZNF582-AS1-overexpressed OSRC2 cells compared with control cells. h Changes of A8K0B9 protein level in OSRC2 cells after transient transfection of different doses of ZNF582-AS1 overexpression plasmid. i Estimation of the binding propensity of A8K0B9-ZNF582-AS1 pair. j RNA pull-down assay indicated that A8K0B9 protein bound specifically to ZNF582-AS1

Article Snippet: Approximately 5 × 10 6 ZNF582-AS1-overexpressed, MT-RNR1-overexpressed-ZNF582-AS1-overexpressed and control OSRC2 cells suspended in 100 μL Hank’s Balanced Salt Solution (Thermo Fisher Scientific, USA) were mixed with Matrigel (1:1, Corning Inc., USA).

Techniques: Over Expression, Expressing, Multiplex sample analysis, Control, Biomarker Discovery, Transfection, Plasmid Preparation, Binding Assay, Pull Down Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Downregulation of lncRNA ZNF582-AS1 due to DNA hypermethylation promotes clear cell renal cell carcinoma growth and metastasis by regulating the N(6)-methyladenosine modification of MT-RNR1

doi: 10.1186/s13046-021-01889-8

Figure Lengend Snippet: ZNF582-AS1 regulated the N(6)-methyladenosine modification of MT-RNR1 by modulating A8K0B9. a Negative and positive controls of rRNA MeRIP-seq. b The analysis results of rRNA MeRIP-seq. c The expression of MT-RNR1, MT-RNR2 and RNA28SN5 in ZNF582-AS1-overexpressed and control OSRC2 cells. d and e MT-RNR1 expression was negatively associated with ZNF582-AS1 expression ( n = 530), and lower MT-RNR1 expression was related to longer OS (n = 530) based on TCGA-KIRC data. According to the median cutoff of MT-RNR1 expression, patients were divided into two groups for survival analysis. f The expression of MT-RNR1 in ccRCC cell lines. g Analysis of m6A motif DRACH (D = A, G or U; R = A or G; H = A, U or C) in MT-RNR1 sequence. h MeRIP-qPCR determined the methylation level of MT-RNR1 in ZNF582-AS1-overexpressed OSRC2 cells and control cells. i RIP-RT-qPCR showed that A8K0B9 protein had a certain binding ability with MT-RNR1 in OSRC2 cells

Article Snippet: Approximately 5 × 10 6 ZNF582-AS1-overexpressed, MT-RNR1-overexpressed-ZNF582-AS1-overexpressed and control OSRC2 cells suspended in 100 μL Hank’s Balanced Salt Solution (Thermo Fisher Scientific, USA) were mixed with Matrigel (1:1, Corning Inc., USA).

Techniques: Modification, Expressing, Control, Sequencing, Methylation, Quantitative RT-PCR, Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Downregulation of lncRNA ZNF582-AS1 due to DNA hypermethylation promotes clear cell renal cell carcinoma growth and metastasis by regulating the N(6)-methyladenosine modification of MT-RNR1

doi: 10.1186/s13046-021-01889-8

Figure Lengend Snippet: ZNF582-AS1 overexpression decreased MT-CO2 expression by regulating MT-RNR1. a and b MT-CO2 expression was significantly positively correlated with MT-RNR1 expression (n = 530), and lower MT-CO2 expression was associated with longer OS (n = 530) based on TCGA-KIRC data. According to the median cutoff of MT-CO2 expression, patients were divided into two groups for survival analysis. c Based on the iTRAQ results, the expression level of MT-CO2 (A0A0P0C1B5) protein was decreased in ZNF582-AS1-overexpressed OSRC2 cells compared with control OSRC2 cells. d and e MT-CO2, Bcl-2 and N-cadherin protein expression was decreased and Cleaved Caspase 3 and E-cadherin protein expression was increased in ZNF582-AS1-overexpressed OSRC2 cells compared with control cells. f and g The ROS level in ZNF582-AS1-overexpressed OSRC2 cells was increased. h The overexpression efficiency of MT-RNR1 in ZNF582-AS1-overexpressed OSRC2 cells. i and j MT-CO2, Bcl-2 and N-cadherin protein expression was increased and Cleaved Caspase 3 and E-cadherin protein expression was decreased in MT-RNR1-overexpressed-ZNF582-AS1-overexpressed OSRC2 cells compared with control cells. k and l The ROS level in MT-RNR1-overexpressed-ZNF582-AS1-overexpressed OSRC2 cells was decreased

Article Snippet: Approximately 5 × 10 6 ZNF582-AS1-overexpressed, MT-RNR1-overexpressed-ZNF582-AS1-overexpressed and control OSRC2 cells suspended in 100 μL Hank’s Balanced Salt Solution (Thermo Fisher Scientific, USA) were mixed with Matrigel (1:1, Corning Inc., USA).

Techniques: Over Expression, Expressing, Multiplex sample analysis, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Downregulation of lncRNA ZNF582-AS1 due to DNA hypermethylation promotes clear cell renal cell carcinoma growth and metastasis by regulating the N(6)-methyladenosine modification of MT-RNR1

doi: 10.1186/s13046-021-01889-8

Figure Lengend Snippet: MT-RNR1 overexpression reversed inhibited cell proliferation and increased cell apoptosis caused by ZNF582-AS1 overexpression. a and b EdU assay and c CCK-8 assay determined cell proliferation of MT-RNR1-overexpressed-ZNF582-AS1-overexpressed OSRC2 and Caki-1 cells comparing with their control cells. d and e TUNEL assay and f and g flow cytometry determined cell apoptosis of MT-RNR1-overexpressed-ZNF582-AS1-overexpressed OSRC2 and Caki-1 cells comparing with their control cells. h Tumors collected from mice are shown. i and j Tumor volume curves and tumor weights of the MT-RNR1-overexpressed-ZNF582-AS1-overexpressed and control groups were measured and compared. k and l The cell proliferation and apoptosis of tumor were examined

Article Snippet: Approximately 5 × 10 6 ZNF582-AS1-overexpressed, MT-RNR1-overexpressed-ZNF582-AS1-overexpressed and control OSRC2 cells suspended in 100 μL Hank’s Balanced Salt Solution (Thermo Fisher Scientific, USA) were mixed with Matrigel (1:1, Corning Inc., USA).

Techniques: Over Expression, EdU Assay, CCK-8 Assay, Control, TUNEL Assay, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Downregulation of lncRNA ZNF582-AS1 due to DNA hypermethylation promotes clear cell renal cell carcinoma growth and metastasis by regulating the N(6)-methyladenosine modification of MT-RNR1

doi: 10.1186/s13046-021-01889-8

Figure Lengend Snippet: MT-RNR1 overexpression reversed inhibited cell migratory and invasive ability caused by ZNF582-AS1 overexpression. a and b Wound healing assay determined the migratory distances of MT-RNR1-overexpressed-ZNF582-AS1-overexpressed OSRC2 and Caki-1 cells and their control cells. c and d Transwell migration and e and f invasion assays determined the migratory and invasive abilities of MT-RNR1-overexpressed-ZNF582-AS1-overexpressed OSRC2 and Caki-1 cells and their control cells. g and h The luciferase signals in the MT-RNR1-overexpressed-ZNF582-AS1-overexpressed group were remarkably higher than those in the ZNF582-AS1-overexpressed group. i There was no significant difference between the mouse weight of the two treatment group. j and k The number and size of pulmonary metastases in the MT-RNR1-overexpressed-ZNF582-AS1-overexpressed group were significantly increased compared with those in the ZNF582-AS1-overexpressed group. l and m MT-RNR1 overexpression decreased E-cadherin expression and increased N-cadherin expression in pulmonary metastases

Article Snippet: Approximately 5 × 10 6 ZNF582-AS1-overexpressed, MT-RNR1-overexpressed-ZNF582-AS1-overexpressed and control OSRC2 cells suspended in 100 μL Hank’s Balanced Salt Solution (Thermo Fisher Scientific, USA) were mixed with Matrigel (1:1, Corning Inc., USA).

Techniques: Over Expression, Wound Healing Assay, Control, Migration, Luciferase, Expressing

Journal: Cancer Science

Article Title: HOXD8 suppresses renal cell carcinoma growth by upregulating SHMT1 expression

doi: 10.1111/cas.15982

Figure Lengend Snippet: Serine hydroxymethyltransferase (SHMT)1 expression in renal cell carcinoma (RCC) tissues and correlations to survival. (A) Expression of SHMT1 in RCC patients (KIRC) in different stages analyzed with the GEPIA2 online tool. n = 258 patients. (B) Kaplan–Meier analysis was performed to evaluate the association between SHMT1 level and RCC patients' overall survival by GEPIA2. (C, D) Detection of SHMT1 expression by Western blot in RCC tissues and adjacent tissues from six RCC patients. n = 6 samples. (E, F) Representative immunohistochemistry staining of SHMT1 protein level in RCC tumor tissues and adjacent tissues from 13 RCC patients. n = 13 pair of samples. White color scale bars: 100 μm, black color scale bars: 50 μm. ** p < 0.01, *** p < 0.001.

Article Snippet: BALB/c nude mice aged 6–8 weeks were purchased from Gempharmatech Co., Ltd. OSRC‐2 cells expressing SHMT1, or empty vector were prepared into single cell suspension.

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Cancer Science

Article Title: HOXD8 suppresses renal cell carcinoma growth by upregulating SHMT1 expression

doi: 10.1111/cas.15982

Figure Lengend Snippet: Serine hydroxymethyltransferase (SHMT)1 inhibits cell proliferation and migration in renal cell carcinoma (RCC) cells. (A) SHMT1 expression in OSRC‐2 and ACHN cells transfected with pcDNA3.1‐Flag‐SHMT1(OE‐SHMT1) and vector plasmid (Vector). (B) Cell proliferation assay by CCK8 assay in OSRC‐2 and ACHN cell lines. (C) Scratch assay of SHMT1‐overexpressed OSRC‐2 and ACHN cells. Scale bars: 100 μm. (D) Transwell assay of SHMT1‐overexpressed OSRC‐2 and ACHN cells. Scale bars: 50 μm. (E) Western blot validation of SHMT1 expression by siRNA transfection. (F) Transwell assay of SHMT1 knockdown OSRC‐2 and ACHN cells. Scale bars: 100 μm. (G) Cell proliferation of SHMT1 knockdown OSRC‐2 and ACHN cells. n = 3 samples; all experiments were repeated twice. ** p < 0.01, *** p < 0.001.

Article Snippet: BALB/c nude mice aged 6–8 weeks were purchased from Gempharmatech Co., Ltd. OSRC‐2 cells expressing SHMT1, or empty vector were prepared into single cell suspension.

Techniques: Migration, Expressing, Transfection, Plasmid Preparation, Proliferation Assay, CCK-8 Assay, Wound Healing Assay, Transwell Assay, Western Blot, Biomarker Discovery, Knockdown

Journal: Cancer Science

Article Title: HOXD8 suppresses renal cell carcinoma growth by upregulating SHMT1 expression

doi: 10.1111/cas.15982

Figure Lengend Snippet: Serine hydroxymethyltransferase (SHMT)1 inhibits OSRC‐2 tumor growth in vivo. (A) Tumor volume and tumor weight of tumor in vector ( n = 5) and SHMT1‐overexpression tumor ( n = 5). (B) The captured image of tumors shows the tumor size of two groups. (C) Tumor weight of tumor in vector ( n = 5) and SHMT1‐overexpression tumor ( n = 5). (D) Immunohistochemistry of SHMT1 and Ki67 and quantification. Scale bar: 100 μm. (E) Immunohistochemistry of cleaved caspase 3 (CC3), P21, and phosphorylated CDK1. Scale bar: 50 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BALB/c nude mice aged 6–8 weeks were purchased from Gempharmatech Co., Ltd. OSRC‐2 cells expressing SHMT1, or empty vector were prepared into single cell suspension.

Techniques: In Vivo, Plasmid Preparation, Over Expression, Immunohistochemistry

Journal: Cancer Science

Article Title: HOXD8 suppresses renal cell carcinoma growth by upregulating SHMT1 expression

doi: 10.1111/cas.15982

Figure Lengend Snippet: BXD family analysis of the upstream genes of serine hydroxymethyltransferase (Shmt)1. (A) Bar plots of the SHMT1 expression across the BXD kidney. The x ‐axis shows the BXD strains and F1 strains (D2B6F1) and two parental strains. The y ‐axis indicates the normalized log 2 expression levels of Shmt1. (B) eQTL mapping of the Shmt1 gene in BXD strains kidney. The upper x ‐axis shows the chromosome, the lower x ‐axis shows the location in megabases (Mb), and the y ‐axis indicates the −log(p) score, which shows the linkage between gene expression and genomic region. Gray and red horizontal lines indicate the significant and suggestive threshold of the −log(p) scores for eQTL mapping, respectively. (C) The top 2000 genes correlated with Shmt1 were crossed with genes included in the 1.5 LOD QTL interval. (D) Correlation between Shmt1 and Hoxd8 in a BXD mice family. Shmt1 has a positive correlation with Hoxd8 among 53 BXD strains. The Pearson correlation r‐ and p‐ values are shown. (E) Expression of HOXD8 in KIRC in different stages using the GEPIA2 website. (F) Kaplan–Meier analysis was performed to evaluate the association between HOXD8 level and KIRC patients' overall survival using the GEPIA2 website. n = 258 patients. (G) Correlation between SHMT1 and HOXD8 in KIRC patients' database. SHMT1 has a positive correlation with HOXD8 ( r = 0.22, p < 0.001).

Article Snippet: BALB/c nude mice aged 6–8 weeks were purchased from Gempharmatech Co., Ltd. OSRC‐2 cells expressing SHMT1, or empty vector were prepared into single cell suspension.

Techniques: Expressing, Gene Expression

Journal: Cancer Science

Article Title: HOXD8 suppresses renal cell carcinoma growth by upregulating SHMT1 expression

doi: 10.1111/cas.15982

Figure Lengend Snippet: Knockdown of HOXD8 restores the inhibitory effect of serine hydroxymethyltransferase (SHMT)1 on renal cell carcinoma (RCC) cells. (A) The expression of SHMT1 and HOXD8 in OSRC‐2 and ACHN cell transfected with pcDNA3.1‐Flag‐HOXD8 (OE‐HOXD8) and vector plasmid (Vector). (B) Validation of SHMT1 expression in HOXD8‐knockdown OSRC‐2 and ACHN cells. (C) CCK8 assay showed the cell viability of OE‐SHMT1 and control RCC cells transfected with siHOXD8 or siControl. (D) Scratch assay showed the migration ability of OE‐SHMT1 and control RCC cells transfected with siHOXD8 or siControl. (E) Transwell assays were performed to verify the rescue role of knockdown HOXD8 in OE‐SHMT1 RCC cells. C–E, n = 3 samples; all experiments were repeated twice. Scale: 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BALB/c nude mice aged 6–8 weeks were purchased from Gempharmatech Co., Ltd. OSRC‐2 cells expressing SHMT1, or empty vector were prepared into single cell suspension.

Techniques: Knockdown, Expressing, Transfection, Plasmid Preparation, Biomarker Discovery, CCK-8 Assay, Control, Wound Healing Assay, Migration

Journal: Cancer Science

Article Title: HOXD8 suppresses renal cell carcinoma growth by upregulating SHMT1 expression

doi: 10.1111/cas.15982

Figure Lengend Snippet: HOXD8 serves as a transcription activator of serine hydroxymethyltransferase (SHMT)1. (A) Luciferase reporter assay showed that HOXD8 targeted the SHMT1 gene promoter. 293T cells were transfected with si HOXD8 to knock down the endogenous HOXD8 first, followed by SHMT1 ‐promoter‐luc and pcDNA3.1‐HOXD8 transfection. n = 3 samples. (B) The prediction of binding motif and position of HOXD8 on SHMT1 promoter by JASPAR online analysis ( http://jaspar.genereg.net/ ). (C) SHMT1 promoter analysis of potential binding region to HOXD8. P1 and P2 indicate the designed PCR product. TSS, transcription starting site. (D) ChIP‐qPCR assays were performed to verify the HOXD8 binding regions in SHMT1 promoter. n = 3 samples. ** p < 0.01, *** p < 0.001. (E) The illustration of HOXD8‐induced renal cell carcinoma (RCC) inhibition. The transcription factor HOXD8 binds to the TGGATTAT sequence on SHMT1 promoter and upregulates SHMT1 expression. High level of SHMT1 results in cell cycle arrest through blocking multiple G2/M phase check points, such as p21, pCDC25, CDC20, as well as the CDK1/Cyclin B complex, leading to proliferation defects and finally tumor suppression.

Article Snippet: BALB/c nude mice aged 6–8 weeks were purchased from Gempharmatech Co., Ltd. OSRC‐2 cells expressing SHMT1, or empty vector were prepared into single cell suspension.

Techniques: Luciferase, Reporter Assay, Transfection, Knockdown, Binding Assay, ChIP-qPCR, Inhibition, Sequencing, Expressing, Blocking Assay