Additional file 1 of RNA-binding protein RPS7 promotes hepatocellular carcinoma progression via LOXL2-dependent activation of ITGB1/FAK/SRC signaling
Creators: name: Zhou, Yu-Jiao
name: Yang, Min-Li
name: He, Xin
name: Gu, Hui-Ying
name: Ren, Ji-Hua
name: Cheng, Sheng-Tao
name: Fu, Zhou
name: Zhang, Zhen-Zhen
name: Chen, Juan
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Virology
subject: Computational Biology

description: Additional file 1: Supplementary materials and methods. Supplementary Table 1. Primers of different LOXL2 related fragments. Supplementary Table 2. Primers of qRT-PCR in this study. Supplementary Table 3. Primers of RNA pull-down in this study. Supplementary Table 4. Primers of dual luciferase reporter assay in this study. Supplementary Fig 1. Identification of RPS7 as an important gene closely associated with HCC progression. A. The correlation between expression levels of each gene and overall survival rate in HCC with metastases. B. The differential expression of RPS7 in other frequent cancers except HCC. C. The correlation between RPS7 expression and HCC histological grades and clinical stages, respectively. ***, P &lt; 0.001. Supplementary Fig 2. The expression of RPS7 in HCC tissues and matched normal liver tissues. Western blot was performed to detect RPS7 protein levels in EHMH group and MFH group, respectively. GAPDH was used as loading control. EHMH, HCC tissues with extrahepatic metastasis; MFH, metastasis-free HCC tissues. Supplementary Fig 3. Effect of RPS7 on HCC cell phenotypes in vitro. A and B. Effect of PRS7 knockout on MHCC97H and HLE proliferation was determined by CCK-8 assay (A) and colony formation assays (B). C and D. Effect of RPS7 overexpression on Huh7 and PLC/PRF/5 cells proliferation was determined by CCK-8 assay (C) and colony formation assays (D). E. The wound closure abilities of RPS7-knockout cells were determined by wound healing assay. Representative data are from at least 3 independent experiments. Data are shown as mean ± SD. **, P &lt; 0.01. Supplementary Fig 4. Overexpression of RPS7 promotes HCC cell adhesion, migration and invasion in vitro and metastasis in vivo. Huh7 and PLC/PRF/5 cells, two poorly aggressive HCC cell lines, were used to establish stable RPS7 overexpression cells via lentivirus carrying RPS7 (Lv-RPS7). Cells infected with lentivirus carrying empty vector were correspondingly used as controls (Lv-EV). The cell-matrix adhesion capacity, migration and invasion ability of cells in vitro as well as metastasis in vivo were observed under RPS7 overexpression. A. Overexpression efficiency was determined by western blot assay. B. RPS7-overexpressed HCC cells adhesion to fibronectin, collagen I and collagen IV were detected using cell-matrix adhesion assay. C. The wound closure abilities of RPS7-overexpressed cells were determined by wound healing assay. D. The effect of RPS7-overexpressed on cell migration and invasion were determined by Transwell assay. E. A orthotopic mouse model was constructed using RPS7-overexpressed Huh7 cells and control cells (each group, n=12). The effect of RPS7-overexpressed on tumor size and numbers were evaluated. F and G. The effects of RPS7-overexpressed on lung metastasis were evaluated by orthotopic mouse models and tail vein lung metastasis mouse models, respectively. Representative data are from at least 3 independent experiments. Data are shown as mean ± SD. *, P&lt; 0.05, **, P &lt; 0.01. Supplementary Fig 5. Effect of RPS7 on LOXL2 expression at transcriptional level. A. Effect of RPS7 overexpressing/knockdown on LOXL2 promoter activity was detected using dual luciferase reporter assay, respectively. B. Nascent LOXL2 RNA was measured by qRT-PCR following pull-down of biotin-conjugated, EU-labeled RNA in RPS7 knockdown or overexpressed HCC cells. Representative data are from at least 3 independent experiments. Data are shown as mean ± SD. ns, no significant. Supplementary Fig 6. LOXL2 mediates RPS7-induced cell adhesion, migration and invasion. A. Catalytic activity deletion mutant of LOXL2 (LOXL2-Δ) and catalytically inactive point mutant of LOXL2 (LOXL2-Y689F), as well as wild type LOXL2 (LOXL2-WT) were respectively constructed. B. Such plasmids were transfected into Huh7 cells. Effect of LOXL2 overexpression was determined by western blotting. C. Effect of LOXL2 silencing was determined by western blotting. D. The impact of 20 μM CMMH on LOXL2 enzyme activity was evaluated using a lysyl oxidase assay kit. MHCC97H cells were treated with 20 μM CMMH, a highly selective LOXL2 enzyme inhibitor, the effect of CMMH on cell-matrix adhesion ability (E) and migration and invasion (F) were analyzed. G. Cell-matrix adhesion assay was performed to evaluate the effect of 20 μM CMMH on the adhesion ability of RPS7-overexpressed Huh7 cells. H. Transwell assay was performed to assess the effect of 20 μM CMMH on the migration and invasion abilities of RPS7-overexpressed Huh7 cells. I. Cell-matrix adhesion assay was performed to evaluate the effect of LOXL2 overexpressing on the adhesion ability of RPS7-knockout MHCC97H cells. J. Transwell assay was performed to assess the effect of LOXL2 overexpressing on the migration and invasion abilities of RPS7-knockout MHCC97H cells. Data are shown as mean ± SD. **, P &lt; 0.01. ns, no significant. Supplementary Fig 7. LOXL2 is involved in RPS7-mediated HCC metastasis in vivo. A-D. RPS7-overexpressed Huh7 cells were orthotopically injected into the left lobe of orthotopic liver of the nude mice. Four weeks after inoculation, CMMH (15 mg/kg) was administered twice a week for six weeks through tail vein injection. Expression of LOXL2 and RPS7 in tumor tissues were detected by western blot (A). Tumor size and numbers were evaluated in CMMH-treated mice compared to control group (B). Lung metastasis were evaluated according to detect metastatic nodules and foci (C and D). E-H. Orthotopic HCC models were established by using RPS7-deletion MHCC97H cells. At 2 weeks after implantation, mice were injected with AAV8-LOXL2 (1 × 1011 viral genomes in 100 μL saline) or AAV8-Ctrl (1 × 1011 viral genomes in 100 μL saline) via tail vein. Expression of LOXL2 and RPS7 in tumor tissues were detected by western blot (E). Tumor size and numbers were evaluated between each group (F). Lung metastasis were evaluated according to detect metastatic nodules and foci (G and H). Representative data are from at least 3 independent experiments. Data are shown as mean ± SD. **, P &lt; 0.01. ns, no significant. Supplementary Fig 8. LOXL2 regulates ITGB1 expression at protein level. A. Membrane and cytosolic fractionations was performed to explore whether LOXL2 alterations affects the levels of membrane-associated Paxillin. B. qRT-PCR was performed to detect the influence of LOXL2 on ITGB1 mRNA level. C. Co-immunoprecipitation (co-IP) assay was performed in HLE cells to evaluate the interact between LOXL2 and ITGB1. D. Whole-cell extracts of LOXL2 overexpressed PLC/PRF/5 cells were immunoprecipitated with anti-ITGB1 antibody, and ubiquitinated ITGB1 was detected with anti-ubiquitin antibody. E. The immunoprecipitation of 20S and ITGB1 was detected in PLC/PRF/5 cells transfected with empty vector or LOXL2-WT plasmids. Data are shown as mean ± SD. ns, no significant. Supplementary Fig 9. ITGB1 is involved in LOXL2-mediated malignant phenotypes of HCC cells. A. The effect of ITGB1 silencing in Huh7 cells was determined by western blotting. B-E. Functional cell experiments were performed to evaluate the effect of ITGB1 on LOXL2-overexpressed Huh7 cells adhesion capacity (B), wound closure ability (C), migration and invasion abilities (D) and focal adhesion formation (E). Representative data are from at least 3 independent experiments. Data are shown as mean ± SD. **, P &lt; 0.01. ns, no significant. Supplementary Fig 10. FAK/SRC signaling is involved in LOXL2-mediated invasive phenotype of HCC cells. LOXL2-overexpressed Huh7 cells were treated with Defactinib, a FAK inhibitor and PP2, a SRC inhibitor, for 48h, respectively. Functional cell experiments were performed to evaluate the effect of such inhibitors on cells adhesion capacity (A), wound closure ability (B), migration and invasion abilities (C). Representative data are from at least 3 independent experiments. Data are shown as mean ± SD. **, P &lt; 0.01. ns, no significant. Supplementary Fig 11.ITGB1 is involved in RPS7-mediated invasive phenotype of HCC cells. A. The effect of RPS7 knockout on signal activity of ITGB1/FAK/SRC pathway was determined by western blotting. B-E. Functional cell experiments were performed to evaluate the effect of ITGB1 on RPS7-knockout MHCC97H cells adhesion capacity (B), wound closure ability (C), migration and invasion abilities (D) and focal adhesion formation (E). Representative data are from at least 3 independent experiments. Data are shown as mean ± SD. *, P &lt; 0.05, **, P &lt; 0.01.