ufl1  (Proteintech)

Journal: Advanced Science

Article Title: RPL6 Interacts with HMGCS1 to Stabilize HIF‐1α by Promoting Cholesterol Production in Hepatocellular Carcinoma

doi: 10.1002/advs.202501373

Figure Lengend Snippet: The identification of RPL6 as a crucial gene closely linked to HCC metastasis. a) A flow chart illustrating the process for creating an orthotopic liver cancer mouse model with pulmonary metastasis using MHCC97H‐GFP cells. b) The representative bioluminescence images showing liver tumors and pulmonary metastases at four time points in the orthotopic xenograft MHCC97H mouse model. c) Temporal trend clustering of gene expression captured by R package Tcseq. d) Cytoscape visualization of the expression profiles and PPI interaction networks between the DNB genes and DNB‐Neighbor DEGs at four time points. A color gradient from green to red is applied to represent gene expression levels, ranging from low to high. e) The analysis includes correlation studies between DNB genes and metastasis‐specific DNB‐Neighbor DEGs, as well as GSVA to assess the correlation of DNB genes with metastasis‐associated KEGG pathways (left). The temporal trends in the expression of the corresponding DNB genes are presented (right). The top 10 most significant DNB genes are highlighted. f) Global view of the expression patterns associated with RPL6‐mediated metastasis pathways. g) Dynamic changes of expression levels of RPL6 and its 84 metastasis‐neighbor DEGs at four time points. DNB, dynamic network biomarker.

Article Snippet: Primary antibodies against RPL6 (NBP2‐20216, Novus Biologicals, 1:400), HMGCS1 (53‐9003‐82, Invitrogen, 1:400), and HIF‐1α (53‐9003‐82, Invitrogen, 1:400) were used in this experiment.

Techniques: Gene Expression, Expressing, Biomarker Discovery

Journal: Advanced Science

Article Title: RPL6 Interacts with HMGCS1 to Stabilize HIF‐1α by Promoting Cholesterol Production in Hepatocellular Carcinoma

doi: 10.1002/advs.202501373

Figure Lengend Snippet: RPL6 is up‐regulated and correlated with HCC patient prognosis. a) The mRNA level of RPL6 in HCC tissues (n = 371) and non‐tumor liver tissues (n = 50) based on the TCGA‐LIHC database. b) The Kaplan‐Meier plot illustrates overall survival within the TCGA‐LIHC cohort, stratified based on the median expression level of RPL6. c) The mRNA level of RPL6 in HCC metastasis group (TNM II to IVB, n = 189) and HCC non‐metastasis group (TNM I, n = 182) based on the TCGA‐LIHC database. d,e, The mRNA level of RPL6 was analyzed by qRT‐PCR in 48 paired HCC tissues and their corresponding adjacent tissues. f) The mRNA level of RPL6 in primary HCC tissues with EHMH and MFH was assessed by qRT‐PCR analysis. g) The Kaplan‐Meier plots illustrating the correlation between the RPL6 mRNA level and overall survival in 48 HCC patients. h) The protein level of RPL6 in primary HCC tissues with EHMH (n = 24) and MFH (n = 24) was assessed by western blot analysis. Band intensity was quantified using image J. i) The representative images demonstrated that RPL6 expression in primary HCC of the EHMH and MFH groups was assessed using multiple immunofluorescence (mIFC) staining. Scar bar, 50 µm. j) The forest plot displayed the results of the multivariate analysis of factors associated with overall survival (OS). Data are shown as mean ± SD. Statistical significance was determined by two‐tailed paired Student's t‐test (d,f,h), two‐tailed unpaired Student's t‐test or log‐rank test (a,c). Multivariate Cox regression analysis was used to assess HRs and the associated 95% CIs (j). * P < 0.05; ** P < 0.01; *** P < 0.001. NT, non‐tumor; HCC, hepatocellular carcinoma; TNM, Tumor‐Node‐Metastasis classification; MFH, HCC tissues with metastasis‐free; EHMH, HCC tissues with extrahepatic metastasis; N, non‐tumor; T, tumor.

Article Snippet: Primary antibodies against RPL6 (NBP2‐20216, Novus Biologicals, 1:400), HMGCS1 (53‐9003‐82, Invitrogen, 1:400), and HIF‐1α (53‐9003‐82, Invitrogen, 1:400) were used in this experiment.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: RPL6 Interacts with HMGCS1 to Stabilize HIF‐1α by Promoting Cholesterol Production in Hepatocellular Carcinoma

doi: 10.1002/advs.202501373

Figure Lengend Snippet: Knockout of RPL6 inhibits metastasis of HCC cells in vitro and in vivo. a,b, Migratory (a) and invasive (b) properties of MHCC97H and HLE HCC cells with RPL6 knockout compared to non‐target control. The relative numbers of migrated or invaded cells were expressed as percentage of the control group. Cell counts were counted from 6 images. c,d, The effect of RPL6 knockout on invadopodia function in MHCC97H (c) and HLE (d) cells was evaluated using a gelatin degradation assay. Cells were seeded on Cy3‐gelatin matrix (red) and cultured for 48 h. After fixing the cells and staining with FITC‐phalloidin (green) and DAPI (blue), confocal images were captures and representative images are displayed. Degraded areas were quantified using Image J from at least six fields. Scar bar, 25 µm. e,f, The effect of RPL6 knockout on lamellipodia function was assessed by F‐actin staining in MHCC97H (e) and HLE (f) cells. Representative images were presented, and the percentage of cells exhibiting lamellipodia formation was counted from at least six fields. Scar bar: 25 µm. g,h, A DEN/CCl4‐induced HCC model was established in hepatocyte‐specific RPL6 knockout mice (RPL6 HKO ) and corresponding control mice (RPL6 fl/fl ). Liver and lung tissues collected at 10 months post DEN treatment were analyzed, n = 25‐30 per group. g) Representative macroscopic images and H&E staining of tumor‐bearing livers in hepatocyte‐specific RPL6 knockout mice (RPL6 HKO , n = 25) and corresponding control mice (RPL6 fl/fl , n = 30). Tumor number and tumor size were examined. Scar bar, 200 µm. h) Representative macroscopic images and H&E staining of tumor‐metastasis lung in RPL6 HKO and RPL6 fl/fl mice. The number of metastatic nodules and foci were counted. Scar bar, 2 mm. Data are shown as mean ± SD of at least three independent experiments (a‐f). Statistical significance was determined by two‐tailed unpaired Student's t‐test. * P < 0.05; *** P < 0.001.

Article Snippet: Primary antibodies against RPL6 (NBP2‐20216, Novus Biologicals, 1:400), HMGCS1 (53‐9003‐82, Invitrogen, 1:400), and HIF‐1α (53‐9003‐82, Invitrogen, 1:400) were used in this experiment.

Techniques: Knock-Out, In Vitro, In Vivo, Control, Degradation Assay, Cell Culture, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: RPL6 Interacts with HMGCS1 to Stabilize HIF‐1α by Promoting Cholesterol Production in Hepatocellular Carcinoma

doi: 10.1002/advs.202501373

Figure Lengend Snippet: RPL6 interacts with HMGCS1 mRNA in HCC cells. a) An UpSet plot illustrated both the RPL6‐associated mRNAs identified by RIP‐seq and RPL6‐regulated downstream mRNAs determined by RNA‐seq. b) Association analysis of KEGG pathway enrichment was performed between the Orthotopic xenograft model RNA‐seq and HCC cell RIP‐seq and RNA‐seq (left), showing differential expression of key genes in the cholesterol biosynthesis pathway (right). c) Differential expression of key genes in the cholesterol biosynthesis pathway in RPL6‐knockout MHCC97H and HLE cells. d) qRT‐PCR analysis of HMGCS1 mRNA half‐life in RPL6‐knockout MHCC97H and HLE cells after actinomycin D treatment. e) qRT‐PCR analysis of HMGCS1 mRNA half‐life in RPL6‐overexpressing Huh7 and PLC/PRF/5 cells after actinomycin D treatment. f) The enrichment of HMGCS1 mRNA on RPL6 was detected by RIP assay (upper). Western blot was performed to confirm that 3xFlag‐RPL6 immunoprecipitated in the RIP experiments (lower). g) RIP experiments detected the binding between RPL6 truncation mutants and HMGCS1 mRNA (upper). The diagrams illustrate the full length of Flag‐tagged RPL6 (FL, 1–288aa) and its Flag‐tagged truncations, including the N‐terminal domain (T1, 1–95aa), the middle intervening sequence (T2, 95–136aa), and the C‐terminal KOW domain (T3, 136–288aa). h) qRT‐PCR analysis measured the HMGCS1 mRNA half‐life in Huh7 HCC cells overexpressing RPL6 truncation mutants after treatment with actinomycin D. i) An RNA Electrophoretic mobility shift (REMSA) assay was used to determine the interaction between purified RPL6 protein and a biotin‐labeled probe of HMGCS1 truncations (5′UTR, CDS, and 3′UTR). j,k) REMSA assays were conducted to determine the interaction between the RPL6 protein and biotin‐labeled probes of specified truncations of HMGCS1 3′UTR. Data are shown as mean ± SD of at least three independent experiments (c‐h). Statistical significance was determined by two‐tailed unpaired Student's t‐test (c–g) or one‐way analysis of variance (ANOVA) (h). * P < 0.05; ** P < 0.01; *** P < 0.001. Act.D, actinomycin D; T, truncation; FL, full length; UTR, untranslated regions; CDS, coding sequences; WT, wild type; Mut, mutation.

Article Snippet: Primary antibodies against RPL6 (NBP2‐20216, Novus Biologicals, 1:400), HMGCS1 (53‐9003‐82, Invitrogen, 1:400), and HIF‐1α (53‐9003‐82, Invitrogen, 1:400) were used in this experiment.

Techniques: RNA Sequencing, Quantitative Proteomics, Knock-Out, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Purification, Labeling, Two Tailed Test, Mutagenesis

Journal: Advanced Science

Article Title: RPL6 Interacts with HMGCS1 to Stabilize HIF‐1α by Promoting Cholesterol Production in Hepatocellular Carcinoma

doi: 10.1002/advs.202501373

Figure Lengend Snippet: RPL6‐upregulated HMGCS1 expression promotes HCC metastasis by increasing intracellular cholesterol level. a) The effect of HMGCS1 overexpression on the migration and invasion of RPL6 knockout cells was assessed through transwell assays. The cells were counted from 6 images. b) The effect of HMGCS1 overexpression on the lamellipodia formation of RPL6 knockout cells was tested by F‐actin staining assay. Representative images were acquired by confocal, and the percentage of cells with lamellipodia formation were counted from at least six fields. Scar bar, 25 µm. c,d, 10‐month‐old DEN‐treated RPL6 HKO or RPL6 fl/fl mice were injected via the tail vein with AAV8‐GFP or AAV8‐HMGCS1. The liver and lung were harvested at 8 weeks post‐injection for H&E staining. n = 12‐14 per group. c) Representative macroscopic images and H&E staining of the tumor‐bearing livers in these mice were provided. The tumor number and tumor size were examined. Scar bar, 200 µm. d) Representative macroscopic images and H&E staining of the lungs with tumor metastatic nodules and foci were calculated. Scar bar, 2 mm. e) Heatmap of lipid metabolomics in RPL6 knockout versus non‐target control MHCC97H cells (n = 4). f) Levels of total cholesterol, free cholesterol, and cholesterol ester in RPL6‐knockout MHCC97H cells. g) Isotopologue distribution of cholesterol from 13 C glucose in RPL6 knockout versus non‐target control MHCC97H cells (n = 4). h) Levels of total, free, and esterified cholesterol in tumor tissues of RPL6 HKO and RPL6 fl/fl mice (n = 10). i) Liver tumor tissue of RPL6 HKO mouse (indicated with dashed lines) and the adjacent normal tissues were stained with HE and Filipin III. Scale bar, 200 µm. j) The total intracellular cholesterol content was quantified in HMGCS1 overexpression of RPL6‐knockout cells. k) Correlation analysis between RPL6 and HMGCS1 mRNA expression levels and the content of cholesterol esters (ChEs) with varying fatty acid chain lengths in the pulmonary metastasis HCC mouse model. Data are shown as mean ± SD of at least three independent experiments (a, b f, h, and j). Statistical significance was determined by two‐tailed unpaired Student's t‐test (e‐h) or one‐way analysis of variance (ANOVA) (a‐d and j). * P < 0.05; ** P < 0.01. ChE, cholesteryl ester; H&E, hematoxylin and eosin.

Article Snippet: Primary antibodies against RPL6 (NBP2‐20216, Novus Biologicals, 1:400), HMGCS1 (53‐9003‐82, Invitrogen, 1:400), and HIF‐1α (53‐9003‐82, Invitrogen, 1:400) were used in this experiment.

Techniques: Expressing, Over Expression, Migration, Knock-Out, Staining, Injection, Control, Two Tailed Test

Journal: Advanced Science

Article Title: RPL6 Interacts with HMGCS1 to Stabilize HIF‐1α by Promoting Cholesterol Production in Hepatocellular Carcinoma

doi: 10.1002/advs.202501373

Figure Lengend Snippet: Cholesterol is involved in RPL6‐mediated HCC metastasis. a) The effect of cholesterol (10 µM) and the addition of methyl‐b‐cyclodextrin (MβCD) on invadopodia function in Huh7 cells was tested by gelatin degradation assay. Images were obtained by confocal microscopy and the degraded areas of at least six fields were quantified using image J software. Scale bars, 25 µm. b) The effect of cholesterol (10 µM) and the addition of methyl‐b‐cyclodextrin (MβCD) on lamellipodia formation in Huh7 cells was evaluated through F‐actin staining. Representative images were shown and the percentage of cells with lamellipodia formation was counted from at least six fields. Scale bar, 25 µm. c,d) Huh7 cells were orthotopically injected into the left lobe liver of nude mice, which were then subjected a normal diet (ND), high cholesterol diet (HCD) or HCD+ MβCD (10 mg/kg of mouse, i.p.), to establish a lung metastasis model. Liver and lung tissues collected at 8 weeks post injection were used for detection. n = 12–14 per group. c) Representative macroscopic images and H&E staining of the tumor‐bearing liver from mice on normal diet (ND), high cholesterol diet (HCD) or HCD+ MβCD mice. The tumor number and tumor size were examined. Scar bar, 200 µm. d) Representative macroscopic images and H&E staining of tumor‐metastasis lungs from these mice. The number of metastatic nodules and metastatic foci were examined. Scar bar, 2 mm. e–g) The effect of cholesterol on migration and invasion, invadopodia function, and lamellipodia formation in RPL6‐knockout MHCC97H cells was determined by transwell assays e), gelatin degradation assays f) and F‐actin staining assays g), respectively. h‐j, RPL6‐knockout MHCC97H cells and non‐target control cells were orthotopically injected into the left lobe liver of nude mice, which were then subjected to either an ND or HCD to establish a lung metastasis model. Liver and lung tissues collected at 8 weeks post injection were used for detection. n = 12‐14 per group. h) Representative macroscopic images and H&E staining of tumor‐bearing liver in these mice. The tumor number and tumor size were examined. Scar bar, 200 µm. i) Representative macroscopic images and H&E staining of tumor‐metastasis lungs from these mice. The number of metastatic nodules and metastatic foci were examined. Scar bar, 2 mm. j) Statistical analysis for lung metastasis events of these mice. Data are shown as mean ± SD of at least three independent experiments. Statistical significance was determined by two‐tailed unpaired Student's t‐test (a‐d) or one‐way analysis of variance (ANOVA) (e‐j). * P < 0.05. Cont, control; Cho, cholesterol; MβCD, methyl‐β‐cyclodextrin.

Article Snippet: Primary antibodies against RPL6 (NBP2‐20216, Novus Biologicals, 1:400), HMGCS1 (53‐9003‐82, Invitrogen, 1:400), and HIF‐1α (53‐9003‐82, Invitrogen, 1:400) were used in this experiment.

Techniques: Degradation Assay, Confocal Microscopy, Software, Staining, Injection, Migration, Knock-Out, Control, Two Tailed Test

Journal: Advanced Science

Article Title: RPL6 Interacts with HMGCS1 to Stabilize HIF‐1α by Promoting Cholesterol Production in Hepatocellular Carcinoma

doi: 10.1002/advs.202501373

Figure Lengend Snippet: Cholesterol stabilizes HIF‐1α through inhibiting proteasome‐mediated degradation in HCC. a) KEGG analysis of differentially expressed proteins in control and cholesterol‐treated (10 µM) Huh7 cells was performed by proteomic data. b) The network of differentially expressed proteins was visualized using STRING database. The size of circles presented the degree of linkage between proteins. c) Huh7 and PLC/RPF/5 cells treated with cholesterol (10 µM) were exposed to hypoxia for 6 h and harvested at the indicated times. The impact of cholesterol treatment on HIF‐1α protein levels was determined by western blot. d) Microscale thermophoresis (MST) was used to investigate the binding of cholesterol with HIF‐1α. His‐tagged HIF‐1α (20 nM) was mixed with 10 µL of cholesterol (10 µM). Different concentrations of cholesterol were mixed with His‐tagged HIF‐1α to analyze Binding Affinity analysis, and the dissociation constant (Kd) was calculated. e) Microscale thermophoresis (MST) was used to investigate the binding of cholesterol with HIF‐1α‐MUT (Q352A/K388A). f) Cells treated with cholesterol (10 µM) were exposed to hypoxia for 6 h (upper), or cells transfected with HIF‐1α‐MUT plasmids for 24 h (lower) then incubated with 10 µg/mL cycloheximide at the indicated times. g) Cells treated with cholesterol (10 µM) and subjected to hypoxia for 6 h, were exposed to the proteasome inhibitor MG132 (10 µM) for 6 h. Subsequently, HIF‐1α protein levels were analyzed by western blotting. h) Following 6‐hour hypoxia treatment with 10 µM cholesterol, cells were treated with PYR‐41 (50 µM) for another 6 hours. HIF‐1α protein levels were analyzed by western blotting. i) Huh7 cells transfected with HA‐Ub plasmid for 24 h and treated with cholesterol (10 µM) under hypoxia for 6 h (left) or transfected with HIF‐1α‐MUT plasmid for 24 h (right), then exposed to MG132 (10 µM) for 6 h. Whole‐cell extracts were immunoprecipitated with anti‐HIF‐1α antibody, and ubiquitinated‐HIF‐1α was detected using an anti‐ubiquitin antibody. j) The impact of cholesterol addition on HIF‐1α protein levels in RPL6‐knockout cells was determined by western blot. RPL6‐knockout cells treated with cholesterol (10 µM) were exposed to hypoxia for 6 h and harvested at the indicated times. k) The transcriptional activity of HIF‐1α in RPL6‐knockout cells with or without cholesterol was assessed using dual‐luciferase reporter assays. Cells were co‐transfected with 4 × HRE‐luc and Renilla plasmids, incubated with cholesterol for 24 h, and then exposed to hypoxia for 6 h. Luciferase activity was detected and normalized to Renilla activity. l) The mRNA levels of HIF‐1α targeted genes in RPL6‐knockout cells with or without cholesterol treatment were evaluated. HCC cells were exposed to hypoxia for 6 h. m H&E staining and representative images of RPL6 (green), HMGCS1 (red), and HIF‐1α (purple) protein expression in MFH and EHMH tissues by multiple immunofluorescence (mIFC) staining. Scale bar, 50 µm. Data are shown as mean ± SD of at least three independent experiments (c‐l). Statistical significance was determined by two‐tailed unpaired Student's t‐test (k,l). * P <0.05; ** P <0.01. CHX, cycloheximide.

Article Snippet: Primary antibodies against RPL6 (NBP2‐20216, Novus Biologicals, 1:400), HMGCS1 (53‐9003‐82, Invitrogen, 1:400), and HIF‐1α (53‐9003‐82, Invitrogen, 1:400) were used in this experiment.

Techniques: Control, Western Blot, Microscale Thermophoresis, Binding Assay, Transfection, Incubation, Plasmid Preparation, Immunoprecipitation, Ubiquitin Proteomics, Knock-Out, Activity Assay, Luciferase, Staining, Expressing, Immunofluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: RPL6 Interacts with HMGCS1 to Stabilize HIF‐1α by Promoting Cholesterol Production in Hepatocellular Carcinoma

doi: 10.1002/advs.202501373

Figure Lengend Snippet: The anti‐tumor effect of PLGA‐siRPL6/NPs in HCC. a) Scheme indicating the step‐by‐step synthesis of PLGA‐siRPL6/NPs and the subsequent experiment design for treatments in vivo. b) Representative TEM images of PLGA‐siRPL6/NPs. Scale bar, 0.2 µm (left). Particle size distribution (middle) and Zeta potential values (right) of PLGA‐siRPL6/NPs were determined by Dynamic Light Scattering. c) The cellular uptake of HCC cells incubated with Cy5‐siRNA/NPs, blank NPs, free siRNA, or PBS control at 37 °C for 2 h was evaluated by fluorescence assay. The siRNA was labeled with Cy5 (red), and cell nuclei were stained with DAPI (blue). Scar bars, 25 µm. d,e, MHCC97H cells were orthotopically injected into the left lobe liver of nude mice to establish a lung metastasis model, followed by the indicated treatments. Liver and lung tissues collected at 8 weeks post‐injection were used for detection. n = 12 per group. d) Representative macroscopic pictures and H&E staining of tumor‐bearing liver in these mice are shown. The tumor number and tumor size were examined. Scar bar, 200 µm. e) Representative macroscopic images and H&E staining of the lungs with tumor metastasis in these mice are shown. The number of metastatic nodules and metastatic foci was examined. Scar bar, 2 mm. f) Illustration of the therapeutic study based on the HCC patient‐derived xenograft (PDX) model. Tumor tissues from patients were implanted into the liver of mice, which then injected with PLGA‐siNC/NPs or PLGA‐siRPL6/NPs every 2 days for 6 weeks. n = 5 per group. g) The effect of siRPL6 on the PDX model (left), and the tumor size and metastatic nodules were examined (right). h) Western blot analysis of HIF‐1α, HMGCS1 and RPL6 expression in PDX tumors. i) Mechanistic model of RPL6‐mediated HMGCS1 mRNA stability to promote HCC migration. In HCC cells, RPL6 expression level was significantly upregulated, and RPL6 enhanced HMGCS1 mRNA stability by binding to HMGCS1 3′UTR that increases cholesterol level. Elevated cholesterol protects HIF‐1α from degradation by ubiquitin proteasome, then stable HIF‐1α further enhances the expression of downstream metastasis‐associated genes, ultimately promoting HCC metastasis. Data are shown as mean ± SD (d,e,g). Statistical significance was determined by two‐tailed unpaired Student's t‐test (g) or one‐way analysis of variance (ANOVA) (d,e). * P < 0.05; *** P < 0.001. PDX, patient‐derived xenograft; NPs, nanoparticles.

Article Snippet: Primary antibodies against RPL6 (NBP2‐20216, Novus Biologicals, 1:400), HMGCS1 (53‐9003‐82, Invitrogen, 1:400), and HIF‐1α (53‐9003‐82, Invitrogen, 1:400) were used in this experiment.

Techniques: In Vivo, Zeta Potential Analyzer, Incubation, Control, Fluorescence, Labeling, Staining, Injection, Derivative Assay, Western Blot, Expressing, Migration, Binding Assay, Ubiquitin Proteomics, Two Tailed Test