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