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Lentivirus mediated silencing of ubiquitin specific peptidase 39 inhibits cell proliferation of human hepatocellular carcinoma cells in vitro.
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Crystal violet staining indicated that colony numbers and sizes were both reduced after knock-down of USP39.In addition, Annexin V showed that downregulation of USP39 significantly increased the population of apoptotic cells.All our results suggest that USP39 is important for HCC cell proliferation and is a potential target for molecular therapy of HCC.
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PubMed Central - PubMed
Affiliation: The Third Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, 200438, Shanghai, China. zeya_pan@yeah.net.
ABSTRACT
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Background: Ubiquitin Specific Peptidase 39 (USP39) is a 65 kDa SR-related protein involved in RNA splicing. Previous studies showed that USP39 is related with tumorigenesis of human breast cancer cells. Results: In the present study, we investigated the functions of USP39 in human hepatocellular carcinoma (HCC) cell line SMMC-7721. We knocked down the expression of USP39 through lentivirus mediated RNA interference. The results of qRT-PCR and western blotting assay showed that both the mRNA and protein levels were suppressed efficiently after USP39 specific shRNA was delivered into SMMC-7721 cells. Cell growth was significantly inhibited as determined by MTT assay. Crystal violet staining indicated that colony numbers and sizes were both reduced after knock-down of USP39. Furthermore, suppression of USP39 arrested cell cycle progression at G2/M phase in SMMC-7721cells. In addition, Annexin V showed that downregulation of USP39 significantly increased the population of apoptotic cells. Conclusions: All our results suggest that USP39 is important for HCC cell proliferation and is a potential target for molecular therapy of HCC. Related in: MedlinePlus |
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Fig1: Expression of USP39 is suppressed efficiently in SMMC-7721 cells after Lv-shUSP39 infection. (A) Representative images of Con, Lv-shCon and Lv-shUSP39 infected SMMC-7721 cells under fluorescence microscope. Left, bright field; right, GFP. Scale bar, 10 μm. (B) qRT-PCR analyzed mRNA levels of USP39 in Con, Lv-shCon and Lv-shUSP39 infected SMMC-7721 cells. Actin was used as control gene. **, P < 0.01. (C) Western blotting analysis of protein levels of USP39 in in Con, Lv-shCon and Lv-shUSP39 infected SMMC-7721 cells. GAPDH was used as control protein. Mentions: To investigate the potential functions of USP39 in HCC, we knocked down USP39 in SMMC-7721 cells using lentivirus-mediated gene transfection. As shown in Figure 1A, most SMMC-7721 cells presented GFP-positive signals after infected by lentivirus recombined with shRNA targeting USP39 (Lv-shUSP39) or control scrambled shRNA (Lv-shCon), indicating that the recombinant lentivirus we got could infect SMMC-7721 cells with high efficiency. Further Real-time PCR and western-blot analysis suggested that the mRNA and protein levels of USP39 were both down-regulated significantly in Lv-shUSP39 infected SMMC-7721 cells (Figure 1B and C). The mRNA of USP39 was only 27% of that in control or Lv-shCon infected SMMC-7721 cells. No USP39 protein band was detected in Lv-shUSP39 infected cells. The above results indicated that recombinant lentivirus taking shUSP39 could effectively suppress the expression of endogenous USP39 in HCC cells.Figure 1 |
View Article: PubMed Central - PubMed
Affiliation: The Third Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, 200438, Shanghai, China. zeya_pan@yeah.net.
Background: Ubiquitin Specific Peptidase 39 (USP39) is a 65 kDa SR-related protein involved in RNA splicing. Previous studies showed that USP39 is related with tumorigenesis of human breast cancer cells.
Results: In the present study, we investigated the functions of USP39 in human hepatocellular carcinoma (HCC) cell line SMMC-7721. We knocked down the expression of USP39 through lentivirus mediated RNA interference. The results of qRT-PCR and western blotting assay showed that both the mRNA and protein levels were suppressed efficiently after USP39 specific shRNA was delivered into SMMC-7721 cells. Cell growth was significantly inhibited as determined by MTT assay. Crystal violet staining indicated that colony numbers and sizes were both reduced after knock-down of USP39. Furthermore, suppression of USP39 arrested cell cycle progression at G2/M phase in SMMC-7721cells. In addition, Annexin V showed that downregulation of USP39 significantly increased the population of apoptotic cells.
Conclusions: All our results suggest that USP39 is important for HCC cell proliferation and is a potential target for molecular therapy of HCC.
