Research Paper Volume 13, Issue 15 pp 19317—19338

The deubiquitinase OTUD3 stabilizes ACTN4 to drive growth and metastasis of hepatocellular carcinoma

OTUD3 knockdown suppresses HCC cell growth in vitro and in vivo. (A) qRT-PCR analysis of OTUD3 mRNA level in normal liver cell HL7702 and HCC cell lines. (B, C) Western blot showing the protein expression of OTUD3 in normal liver cell HL7702 and HCC cell lines. (D) EdU assay evaluating the proliferation ability for HCCLM3 cells transfected with control shRNA or shRNA targeting OTUD3. Right panel is quantification of the results of the EdU assay. (E) CCK8 examining the effect of OTUD3 knockdown on the proliferation of HCCLM3 cell. (F) Luciferase intensity of the nude mice (n=6 per group) injected with luciferase-expressing HCCLM3 cells stably transfected with shNC or shRNA targeting OTUD3 were detected by IVIS, Representative images obtained are shown. (G, H) Tumour sizes and tumour weights of HCCLM3-shNC or HCCLM3-shOTUD3 group of nude mice were measured and corresponding tumour growth curves were obtained. (I) Immunofluorescence staining of Ki67 in subcutaneously tumour of nude mice injected with HCCLM3 cells stably transfected shOTUD3 or shNC (Scale bar: 100μm; Magnification: 200X). *PP

Figure 2. OTUD3 knockdown suppresses HCC cell growth in vitro and in vivo. (A) qRT-PCR analysis of OTUD3 mRNA level in normal liver cell HL7702 and HCC cell lines. (B, C) Western blot showing the protein expression of OTUD3 in normal liver cell HL7702 and HCC cell lines. (D) EdU assay evaluating the proliferation ability for HCCLM3 cells transfected with control shRNA or shRNA targeting OTUD3. Right panel is quantification of the results of the EdU assay. (E) CCK8 examining the effect of OTUD3 knockdown on the proliferation of HCCLM3 cell. (F) Luciferase intensity of the nude mice (n=6 per group) injected with luciferase-expressing HCCLM3 cells stably transfected with shNC or shRNA targeting OTUD3 were detected by IVIS, Representative images obtained are shown. (G, H) Tumour sizes and tumour weights of HCCLM3-shNC or HCCLM3-shOTUD3 group of nude mice were measured and corresponding tumour growth curves were obtained. (I) Immunofluorescence staining of Ki67 in subcutaneously tumour of nude mice injected with HCCLM3 cells stably transfected shOTUD3 or shNC (Scale bar: 100μm; Magnification: 200X). *P< 0.05, **P<0.01 by t-tests.