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Reduced USP39 expression inhibits malignant proliferation of medullary thyroid carcinoma in vitro.

An Y, Yang S, Guo K, Ma B, Wang Y - World J Surg Oncol (2015)

Bottom Line: Both two shRNA sequences against USP39 could efficiently reduce its expression in TT cells.Knockdown of USP39 significantly decreased cell proliferation and caused cell cycle arrest at G2/M phase.Moreover, G2/M phase-associated proteins, Cyclin B1 and CDK1, were obviously down-regulated in TT cells after USP39 silencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Head & Neck Surgery, Fudan University Shanghai Cancer Center and Department of Oncology, Shanghai Medical College, Fudan University, 6W of No.3 Building, No.270 DongAn Road, Shanghai, 200223, China.

ABSTRACT

Background: Medullary thyroid carcinoma (MTC) constitutes approximately 5 % of all thyroid cancers and carries a worse prognosis than other differentiated thyroid cancers. Targeted therapies are being investigated for systemic treatment of MTC. Ubiquitin-specific peptidase 39 (USP39) functions in pre-mRNA splicing as a component of the U4/U6-U5 tri-snRNP and also participates in spindle checkpoint and cytokinesis. In this study, we aimed to evaluate the potential role in MTC.

Methods: We used lentivirus-delivered short hairpin RNA (shRNA) to silence USP39 expression in one MTC cell line TT. USP39 expression was detected by qPCR and Western blot. For functional analysis, MTT assay was performed to evaluate the proliferation activity, and FACS was used to assess the cell distribution in the cell cycle. Moreover, the expressions of cell cycle-related proteins were examined by Western blot.

Results: Both two shRNA sequences against USP39 could efficiently reduce its expression in TT cells. Knockdown of USP39 significantly decreased cell proliferation and caused cell cycle arrest at G2/M phase. Moreover, G2/M phase-associated proteins, Cyclin B1 and CDK1, were obviously down-regulated in TT cells after USP39 silencing.

Conclusions: Therefore, knockdown of USP39 is likely to provide a novel alternative to targeted therapy of MTC and deserves further investigation.

No MeSH data available.


Related in: MedlinePlus

Lentivirus-delivered shRNA targeting USP39 depleted its endogenous expression in TT cells. a Evaluation of the lentivirus transduction rate, which was more than 80 % as calculated by cellular enumeration using fluorescence and light microscopy. b Quantitative analysis of USP39 knockdown efficiency (S1) in TT cells was assessed by qRT-PCR. β-actin gene was used as an internal control. c Representative immunoblot showing USP39 knockdown efficiency determined in TT cells. GAPDH protein was used as an internal control. d Quantitative analysis of USP39 knockdown efficiency (S2) in TT cells was assessed by qRT-PCR. β-actin gene was used as an internal control. Each point represents the mean ± SD of three independent repeats. The significance was determined by t test. **p < 0.01; scale bar, 10 μm
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Fig1: Lentivirus-delivered shRNA targeting USP39 depleted its endogenous expression in TT cells. a Evaluation of the lentivirus transduction rate, which was more than 80 % as calculated by cellular enumeration using fluorescence and light microscopy. b Quantitative analysis of USP39 knockdown efficiency (S1) in TT cells was assessed by qRT-PCR. β-actin gene was used as an internal control. c Representative immunoblot showing USP39 knockdown efficiency determined in TT cells. GAPDH protein was used as an internal control. d Quantitative analysis of USP39 knockdown efficiency (S2) in TT cells was assessed by qRT-PCR. β-actin gene was used as an internal control. Each point represents the mean ± SD of three independent repeats. The significance was determined by t test. **p < 0.01; scale bar, 10 μm

Mentions: TT cells were transduced with shRNA-expressing lentivirus (shCon or shUSP39(S1)/(S2)). GFP expression was observed by fluorescent microscopy 4 days post-transduction. As depicted in Fig. 1a, over 80 % of cells expressed GFP in shCon, shUSP39(S1), and shUSP39(S2) groups, indicating a successful infection rate. The inhibitory effect of USP39 shRNA on its endogenous expression in TT cells was examined by qRT-PCR and Western blotting. As depicted in Fig. 1b, the mRNA level of USP39 was significantly reduced in TT cells infected with shUSP39(S1) with a knockdown efficiency of 73.9 %, in contrast to cells infected with shCon. Immunoblot further verified the down-regulation of USP39 expression at protein level (Fig. 1c). The mRNA level of USP39 was also significantly reduced in TT cells infected with shUSP39(S2) in contrast to cells infected with shCon (Fig. 1d). Therefore, lentivirus-delivered shRNA could specifically deplete endogenous USP39 expression in TT cells.Fig. 1


Reduced USP39 expression inhibits malignant proliferation of medullary thyroid carcinoma in vitro.

An Y, Yang S, Guo K, Ma B, Wang Y - World J Surg Oncol (2015)

Lentivirus-delivered shRNA targeting USP39 depleted its endogenous expression in TT cells. a Evaluation of the lentivirus transduction rate, which was more than 80 % as calculated by cellular enumeration using fluorescence and light microscopy. b Quantitative analysis of USP39 knockdown efficiency (S1) in TT cells was assessed by qRT-PCR. β-actin gene was used as an internal control. c Representative immunoblot showing USP39 knockdown efficiency determined in TT cells. GAPDH protein was used as an internal control. d Quantitative analysis of USP39 knockdown efficiency (S2) in TT cells was assessed by qRT-PCR. β-actin gene was used as an internal control. Each point represents the mean ± SD of three independent repeats. The significance was determined by t test. **p < 0.01; scale bar, 10 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4549085&req=5

Fig1: Lentivirus-delivered shRNA targeting USP39 depleted its endogenous expression in TT cells. a Evaluation of the lentivirus transduction rate, which was more than 80 % as calculated by cellular enumeration using fluorescence and light microscopy. b Quantitative analysis of USP39 knockdown efficiency (S1) in TT cells was assessed by qRT-PCR. β-actin gene was used as an internal control. c Representative immunoblot showing USP39 knockdown efficiency determined in TT cells. GAPDH protein was used as an internal control. d Quantitative analysis of USP39 knockdown efficiency (S2) in TT cells was assessed by qRT-PCR. β-actin gene was used as an internal control. Each point represents the mean ± SD of three independent repeats. The significance was determined by t test. **p < 0.01; scale bar, 10 μm
Mentions: TT cells were transduced with shRNA-expressing lentivirus (shCon or shUSP39(S1)/(S2)). GFP expression was observed by fluorescent microscopy 4 days post-transduction. As depicted in Fig. 1a, over 80 % of cells expressed GFP in shCon, shUSP39(S1), and shUSP39(S2) groups, indicating a successful infection rate. The inhibitory effect of USP39 shRNA on its endogenous expression in TT cells was examined by qRT-PCR and Western blotting. As depicted in Fig. 1b, the mRNA level of USP39 was significantly reduced in TT cells infected with shUSP39(S1) with a knockdown efficiency of 73.9 %, in contrast to cells infected with shCon. Immunoblot further verified the down-regulation of USP39 expression at protein level (Fig. 1c). The mRNA level of USP39 was also significantly reduced in TT cells infected with shUSP39(S2) in contrast to cells infected with shCon (Fig. 1d). Therefore, lentivirus-delivered shRNA could specifically deplete endogenous USP39 expression in TT cells.Fig. 1

Bottom Line: Both two shRNA sequences against USP39 could efficiently reduce its expression in TT cells.Knockdown of USP39 significantly decreased cell proliferation and caused cell cycle arrest at G2/M phase.Moreover, G2/M phase-associated proteins, Cyclin B1 and CDK1, were obviously down-regulated in TT cells after USP39 silencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Head & Neck Surgery, Fudan University Shanghai Cancer Center and Department of Oncology, Shanghai Medical College, Fudan University, 6W of No.3 Building, No.270 DongAn Road, Shanghai, 200223, China.

ABSTRACT

Background: Medullary thyroid carcinoma (MTC) constitutes approximately 5 % of all thyroid cancers and carries a worse prognosis than other differentiated thyroid cancers. Targeted therapies are being investigated for systemic treatment of MTC. Ubiquitin-specific peptidase 39 (USP39) functions in pre-mRNA splicing as a component of the U4/U6-U5 tri-snRNP and also participates in spindle checkpoint and cytokinesis. In this study, we aimed to evaluate the potential role in MTC.

Methods: We used lentivirus-delivered short hairpin RNA (shRNA) to silence USP39 expression in one MTC cell line TT. USP39 expression was detected by qPCR and Western blot. For functional analysis, MTT assay was performed to evaluate the proliferation activity, and FACS was used to assess the cell distribution in the cell cycle. Moreover, the expressions of cell cycle-related proteins were examined by Western blot.

Results: Both two shRNA sequences against USP39 could efficiently reduce its expression in TT cells. Knockdown of USP39 significantly decreased cell proliferation and caused cell cycle arrest at G2/M phase. Moreover, G2/M phase-associated proteins, Cyclin B1 and CDK1, were obviously down-regulated in TT cells after USP39 silencing.

Conclusions: Therefore, knockdown of USP39 is likely to provide a novel alternative to targeted therapy of MTC and deserves further investigation.

No MeSH data available.


Related in: MedlinePlus