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URI regulates tumorigenicity and chemotherapeutic resistance of multiple myeloma by modulating IL-6 transcription.

Fan JL, Zhang J, Dong LW, Fu WJ, Du J, Shi HG, Jiang H, Ye F, Xi H, Zhang CY, Hou J, Wang HY - Cell Death Dis (2014)

Bottom Line: URI inhibition significantly attenuated the proliferation of MM cells and decreased colony formation compared with the control cells.Furthermore, URI knockdown markedly reduced the abundance of SP in MM cell lines and enhanced the chemotherapeutic sensitivity of MM towards bortezomib.In conclusion, URI may have an important role in the development of MM and chemotherapeutic resistance through activating the IL-6/STAT3 pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, The Myeloma and Lymphoma Center, Changzheng Hospital, The Second Military Medical University, Shanghai, China.

ABSTRACT
Unconventional prefoldin RPB5 interactor (URI), which acts as an oncoprotein in solid tumors, is associated with RNA polymerase II subunit 5. However, its impact on multiple myeloma (MM) has not been determined. We demonstrate here that URI is overexpressed in MM compared with plasma cells derived from healthy volunteers. Side population (SP) cells sorted from MM cells showed a much higher level of URI than non-SP cells. Using lentivirus-delivered shRNA, we established stable URI knockdown MM cell lines. URI inhibition significantly attenuated the proliferation of MM cells and decreased colony formation compared with the control cells. Tumor growth assays in NOD/SCID mice further confirmed the promotion role of URI during MM development in vivo. Furthermore, URI knockdown markedly reduced the abundance of SP in MM cell lines and enhanced the chemotherapeutic sensitivity of MM towards bortezomib. Mechanically, URI appears to be critically involved in modulating STAT3 activity through regulating interleukin (IL)-6 transcription via interaction with NFκBp65. In conclusion, URI may have an important role in the development of MM and chemotherapeutic resistance through activating the IL-6/STAT3 pathway.

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Related in: MedlinePlus

Knockdown of URI inhibits MM cell growth in vitro and in vivo. (a and b) URI expression was analyzed by real-time PCR and immunoblotting in URI knockdown (shURI) and control cells (NC). The URI protein levels were quantified relative to the loading control. (c) Cell proliferation was detected in control (NC) and shURI MM cells.The MM cells (2 × 103 cells per well) were seeded in 96-well plates and cultured overnight at 37 °C. Cell proliferation was detected by a CCK-8 assay at various time points according to the manufacturer's instructions. (d) Flow cytometric analysis of the proportion of cells in G2/M phase in shURI MM and control cells (NC). Representative results are shown. (e) Quantification of methylcellulose colony formation of NCI-H929 and LP-1 after URI knockdown (shURI) of three independent experiments. Myeloma cells were adjusted to a concentration of 1 × 103 per ml cells using human methylcellulose-based medium according to the manufacturer's instructions and were then plated in 35-mm2 culture dishes. Colonies were counted by microscopy 10 days after plating. (f and g) The tumors excised from NOD/SCID mice after being inoculated subcutaneously with shURI MM cells and their control cells (NC) for 15 days. Tumor size was measured once every 3 days, and tumor volume was calculated by the formula: (width)2 × length/2. Error bars represent the S.E.M. *P<0.05
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fig2: Knockdown of URI inhibits MM cell growth in vitro and in vivo. (a and b) URI expression was analyzed by real-time PCR and immunoblotting in URI knockdown (shURI) and control cells (NC). The URI protein levels were quantified relative to the loading control. (c) Cell proliferation was detected in control (NC) and shURI MM cells.The MM cells (2 × 103 cells per well) were seeded in 96-well plates and cultured overnight at 37 °C. Cell proliferation was detected by a CCK-8 assay at various time points according to the manufacturer's instructions. (d) Flow cytometric analysis of the proportion of cells in G2/M phase in shURI MM and control cells (NC). Representative results are shown. (e) Quantification of methylcellulose colony formation of NCI-H929 and LP-1 after URI knockdown (shURI) of three independent experiments. Myeloma cells were adjusted to a concentration of 1 × 103 per ml cells using human methylcellulose-based medium according to the manufacturer's instructions and were then plated in 35-mm2 culture dishes. Colonies were counted by microscopy 10 days after plating. (f and g) The tumors excised from NOD/SCID mice after being inoculated subcutaneously with shURI MM cells and their control cells (NC) for 15 days. Tumor size was measured once every 3 days, and tumor volume was calculated by the formula: (width)2 × length/2. Error bars represent the S.E.M. *P<0.05

Mentions: To observe the function of URI in MM, the expression of URI was knocked down in MM cell lines. As shown in Figures 2a and b, lentivirus-delivered shRNA significantly inhibited the mRNA and protein levels of URI in NCI-H929 and LP-1 cells. In the stably transfected cell lines, the viable cell numbers were determined by the CCK-8 assay (Figure 2c). Suppression of URI resulted in a significant inhibition of MM cell line growth in normal culture conditions. Interestingly, growth inhibition was associated with lower numbers of URI-shRNA cells in the G2/M stage than in the control cells (Figure 2d). Next, we determined whether depletion of URI interfered with the clonogenic growth and survival of MM cells. As shown in Figure 2e, shRNA-URI MM cells suppressed colony formation compared with control cells. The effect of URI on the tumorigenic potential of MM cells was evaluated in vivo. NCI-H929 and LP-1 cells were transduced with a lentivirus expressing an shRNA targeting URI or the empty vector. These cells were inoculated in NOD/SCID mice, and tumor formation was monitored (Figure 2f). As shown in Figure 2g, knocked down URI markedly reduced the growth of MM xenografts compared with the control group in vivo.


URI regulates tumorigenicity and chemotherapeutic resistance of multiple myeloma by modulating IL-6 transcription.

Fan JL, Zhang J, Dong LW, Fu WJ, Du J, Shi HG, Jiang H, Ye F, Xi H, Zhang CY, Hou J, Wang HY - Cell Death Dis (2014)

Knockdown of URI inhibits MM cell growth in vitro and in vivo. (a and b) URI expression was analyzed by real-time PCR and immunoblotting in URI knockdown (shURI) and control cells (NC). The URI protein levels were quantified relative to the loading control. (c) Cell proliferation was detected in control (NC) and shURI MM cells.The MM cells (2 × 103 cells per well) were seeded in 96-well plates and cultured overnight at 37 °C. Cell proliferation was detected by a CCK-8 assay at various time points according to the manufacturer's instructions. (d) Flow cytometric analysis of the proportion of cells in G2/M phase in shURI MM and control cells (NC). Representative results are shown. (e) Quantification of methylcellulose colony formation of NCI-H929 and LP-1 after URI knockdown (shURI) of three independent experiments. Myeloma cells were adjusted to a concentration of 1 × 103 per ml cells using human methylcellulose-based medium according to the manufacturer's instructions and were then plated in 35-mm2 culture dishes. Colonies were counted by microscopy 10 days after plating. (f and g) The tumors excised from NOD/SCID mice after being inoculated subcutaneously with shURI MM cells and their control cells (NC) for 15 days. Tumor size was measured once every 3 days, and tumor volume was calculated by the formula: (width)2 × length/2. Error bars represent the S.E.M. *P<0.05
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Related In: Results  -  Collection

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fig2: Knockdown of URI inhibits MM cell growth in vitro and in vivo. (a and b) URI expression was analyzed by real-time PCR and immunoblotting in URI knockdown (shURI) and control cells (NC). The URI protein levels were quantified relative to the loading control. (c) Cell proliferation was detected in control (NC) and shURI MM cells.The MM cells (2 × 103 cells per well) were seeded in 96-well plates and cultured overnight at 37 °C. Cell proliferation was detected by a CCK-8 assay at various time points according to the manufacturer's instructions. (d) Flow cytometric analysis of the proportion of cells in G2/M phase in shURI MM and control cells (NC). Representative results are shown. (e) Quantification of methylcellulose colony formation of NCI-H929 and LP-1 after URI knockdown (shURI) of three independent experiments. Myeloma cells were adjusted to a concentration of 1 × 103 per ml cells using human methylcellulose-based medium according to the manufacturer's instructions and were then plated in 35-mm2 culture dishes. Colonies were counted by microscopy 10 days after plating. (f and g) The tumors excised from NOD/SCID mice after being inoculated subcutaneously with shURI MM cells and their control cells (NC) for 15 days. Tumor size was measured once every 3 days, and tumor volume was calculated by the formula: (width)2 × length/2. Error bars represent the S.E.M. *P<0.05
Mentions: To observe the function of URI in MM, the expression of URI was knocked down in MM cell lines. As shown in Figures 2a and b, lentivirus-delivered shRNA significantly inhibited the mRNA and protein levels of URI in NCI-H929 and LP-1 cells. In the stably transfected cell lines, the viable cell numbers were determined by the CCK-8 assay (Figure 2c). Suppression of URI resulted in a significant inhibition of MM cell line growth in normal culture conditions. Interestingly, growth inhibition was associated with lower numbers of URI-shRNA cells in the G2/M stage than in the control cells (Figure 2d). Next, we determined whether depletion of URI interfered with the clonogenic growth and survival of MM cells. As shown in Figure 2e, shRNA-URI MM cells suppressed colony formation compared with control cells. The effect of URI on the tumorigenic potential of MM cells was evaluated in vivo. NCI-H929 and LP-1 cells were transduced with a lentivirus expressing an shRNA targeting URI or the empty vector. These cells were inoculated in NOD/SCID mice, and tumor formation was monitored (Figure 2f). As shown in Figure 2g, knocked down URI markedly reduced the growth of MM xenografts compared with the control group in vivo.

Bottom Line: URI inhibition significantly attenuated the proliferation of MM cells and decreased colony formation compared with the control cells.Furthermore, URI knockdown markedly reduced the abundance of SP in MM cell lines and enhanced the chemotherapeutic sensitivity of MM towards bortezomib.In conclusion, URI may have an important role in the development of MM and chemotherapeutic resistance through activating the IL-6/STAT3 pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, The Myeloma and Lymphoma Center, Changzheng Hospital, The Second Military Medical University, Shanghai, China.

ABSTRACT
Unconventional prefoldin RPB5 interactor (URI), which acts as an oncoprotein in solid tumors, is associated with RNA polymerase II subunit 5. However, its impact on multiple myeloma (MM) has not been determined. We demonstrate here that URI is overexpressed in MM compared with plasma cells derived from healthy volunteers. Side population (SP) cells sorted from MM cells showed a much higher level of URI than non-SP cells. Using lentivirus-delivered shRNA, we established stable URI knockdown MM cell lines. URI inhibition significantly attenuated the proliferation of MM cells and decreased colony formation compared with the control cells. Tumor growth assays in NOD/SCID mice further confirmed the promotion role of URI during MM development in vivo. Furthermore, URI knockdown markedly reduced the abundance of SP in MM cell lines and enhanced the chemotherapeutic sensitivity of MM towards bortezomib. Mechanically, URI appears to be critically involved in modulating STAT3 activity through regulating interleukin (IL)-6 transcription via interaction with NFκBp65. In conclusion, URI may have an important role in the development of MM and chemotherapeutic resistance through activating the IL-6/STAT3 pathway.

Show MeSH
Related in: MedlinePlus