Fig 1: Inhibition of the candidate genes suppress hepatocellular carcinoma cells proliferation and invasion(A) The siRNAs of LMO1, MYADML2, PLK4 and XAGE1B were used to transfect the HEPG2 and Huh7 cell lines. WB was used to detect the LMO1, MYADML2, PLK4 and XAGE1B protein expression levels, ImageJ was used for quantitative analysis; (B) CCK8 assay was used to detect cell viability at 4h, 24h, 48h and 72h in siNC, siLMO1, siMYADML2, siPLK4 and siXAGE1B groups of HEPG2 and Huh7 cells; (C-D) Transwell assays clearly revealed the invasion cells in siNC, siLMO1, siMYADML2, siPLK4 and siXAGE1B groups of HEPG2 and Huh7 cells at 48 h after vaccination in the wells. The cell invasion numbers were counted and compared. Statistical significance was determined through one-way ANOVA. Data were represented as means ± SD of a representative experiment (of three or five independent experiments). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the siNC group.
Fig 2: These candidate genes were valuable biomarker in clinical HCC samples(A) The LIHC data from TCGA was used to analyze the LMO1, MYADML2, PLK4 and XAGE1B mRNA expression in HCC tumor (371 samples) and paracancerous (50 samples) group; (B) The HCC samples from TCGA was devided into low and high expression group according to the median of mRNA levels. Kaplan-Meier analysis revealed that high expression of LMO1, MYADML2, PLK4 and XAGE1B exhibited worse overall survival according the TCGA dataset; (C) MYADML2 protein showed positive or weak immunostaining in the cytoplasm and membrane of HCC tissues from a tissue microarray; (D) MYADML2 protein level has increased in HCC patient over 60 years (Young group: age<60 years, N = 68; Old group: age≥60 years, N = 12); (E) Overall survival and disease-free survival analysis of MYADML2 protein in HCC tissues (N = 80); (F) The HCC samples from TCGA was devided into young (age≤60) and old expression group (age>60), the differences of LMO1, MYADML2, PLK4 and XAGE1B mRNA were analyzed between the young and old group; (G) The distribution of IC50 score. The abscissa represents different groups of samples, and the ordinate represents the distribution of the IC50 score. Comparisons between groups were analyzed through unpaired Student’s t test or Kaplan-Meier Log Rank test. Data were represented as means ± SD ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. ∗∗∗∗p < 0.0001 compared with the control group.
Fig 3: Analysis of immune cell composition in liver cancer(A-B) TCGA data analyzes the proportion of 15 immune cell types in liver cancer; (C) The boxplot showed the different infiltration levels of 15 immune cell types in the high/low PLK4, MYADML2, XAGE1B and LMO1 expression groups. Comparisons between groups were analyzed through unpaired Student’s t test. Data were represented as means ± SD of a representative experiment. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001, ns: No significance compared with the control group.
Fig 4: USP54 combines with PLK4 and mediates its deubiquitination(A) Microarray analysis of mRNAs extracted from SGC7901-vector and SGC7901-CEP120 cells.(B) Results of the ubiquitination level of PLK4 in HEK293T cells, which were co-transfected with Flag-PLK4, HA-Ub, and one of the three DUBs.(C) WB results of USP54 and PLK4 expression in GC tissues. p value derived from Pearson correlation analysis.(D) WB results of PLK4 and USP54 after USP54 overexpression or knockdown.(E) CoIP analysis of the interaction between PLK4 and USP54 in SGC7901 and BGC823 cells.(F) HEK 293T cells were co-transfected with His-USP54 and Flag-PLK4 full length or Flag-PLK4 (1-740), Flag-PLK4 (1-270), and Flag-PLK4 (270-890) constructs and harvested for WB and IP analysis.(G) HEK 293T cells were co-transfected with His-USP54 and Flag-PLK4 wildtype or Flag-PLK4 lysine residue mutants and harvested for IP analysis.(H) Ubiquitination level of PLK4 after USP54 overexpression or knockdown examined by ubiquitination assay.(I) WB results of PLK4 in SGC7901 cells transfected with Lenti-CEP120 and siUSP54 and BGC823 cells transfected with Lenti-shCEP120 and USP54 plasmid.
Fig 5: CEP120 regulates centrosome amplification and GC progression by controlling PLK4 ubiquitination(A) WB analysis of PLK4 expression after CEP120 overexpression and knockdown.(B) WB results of PLK4 and CEP120 expression in GC tissues.(C) The correlation of PLK4 and CEP120 protein level in GC tissues. p value derived from pearson correlation analysis.(D) The correlation of PLK4 and CEP120 mRNA level in GC tissues. p value derived from Pearson correlation analysis.(E) CHX chase analysis of PLK4 protein half-life after CEP120 overexpression and knockdown.(F) WB analysis of PLK4 expression in BGC823 cells treated with MG132 or CQ after CEP120 knockdown.(G) Results of the ubiquitination level of PLK4 after CEP120 overexpression and knockdown examined by ubiquitination assay.
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Trial Size: 20 ul