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Lentivirus mediated silencing of ubiquitin specific peptidase 39 inhibits cell proliferation of human hepatocellular carcinoma cells in vitro.

Pan Z, Pan H, Zhang J, Yang Y, Liu H, Yang Y, Huang G, Ni J, Huang J, Zhou W - Biol. Res. (2015)

Bottom Line: 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.

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.

ABSTRACT

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.

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Related in: MedlinePlus

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.
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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


Lentivirus mediated silencing of ubiquitin specific peptidase 39 inhibits cell proliferation of human hepatocellular carcinoma cells in vitro.

Pan Z, Pan H, Zhang J, Yang Y, Liu H, Yang Y, Huang G, Ni J, Huang J, Zhou W - Biol. Res. (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4389921&req=5

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

Bottom Line: 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.

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.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus