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LARP7 suppresses P-TEFb activity to inhibit breast cancer progression and metastasis.

Ji X, Lu H, Zhou Q, Luo K - Elife (2014)

Bottom Line: The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating Pol II and antagonizing negative elongation factors.A reservoir of P-TEFb is sequestered in the inactive 7SK snRNP where 7SK snRNA and the La-related protein LARP7 are required for the integrity of this complex.Decreased levels of LARP7 and 7SK snRNA redistribute P-TEFb to the transcriptionally active super elongation complex, resulting in P-TEFb activation and increased transcription of EMT transcription factors, including Slug, FOXC2, ZEB2, and Twist1, to promote breast cancer EMT, invasion, and metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

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Silencing LARP7 induces EMT in MCF10A cells.(A) Upper panel, the levels of 7SK snRNA and LARP7 protein in MCF10A parental cells and cells stably expressing scramble control, shLARP7-1, or shLARP7-2 were examined by Northern blotting (NB) and Western blotting (WB), respectively. Tubulin was used as a loading control. Lower panel, phase-contrast images of control and two shLARP7 pools. (B) Wound healing assay. Confluent cell monolayers were wounded, and wound closure was monitored at 0 hr and 16 hr. (C) Migration assay. MCF10A control or shLARP7 cells were subjected to a Transwell migration assay. The migrated cells were stained and counted. Data were collected from five fields in three independent experiments. (D) Immunofluorescence staining of E-cadherin (green) and actin stress fibers (red). (E) qRT-PCR analysis of N-cadherin expression in control and two shLARP7 pools. (F and G) The levels of LARP7 protein (F) and mRNA (G) as well as 7SK snRNA level (G) in control, shLARP7-2 cells and rescue cells (expressing an shRNA-resistant WT LARP7 cDNA) were examined by Western blotting and qRT-PCR, respectively. (H) Phase contrast pictures of control, shLARP7-2, and rescue cells. (I) The wound healing assay. (J) The Transwell migration assay. For C, E, G, and J panels: data are presented as mean ± SD. *: the p values (p<0.05) were determined by the Student's t test.DOI:http://dx.doi.org/10.7554/eLife.02907.006
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fig2: Silencing LARP7 induces EMT in MCF10A cells.(A) Upper panel, the levels of 7SK snRNA and LARP7 protein in MCF10A parental cells and cells stably expressing scramble control, shLARP7-1, or shLARP7-2 were examined by Northern blotting (NB) and Western blotting (WB), respectively. Tubulin was used as a loading control. Lower panel, phase-contrast images of control and two shLARP7 pools. (B) Wound healing assay. Confluent cell monolayers were wounded, and wound closure was monitored at 0 hr and 16 hr. (C) Migration assay. MCF10A control or shLARP7 cells were subjected to a Transwell migration assay. The migrated cells were stained and counted. Data were collected from five fields in three independent experiments. (D) Immunofluorescence staining of E-cadherin (green) and actin stress fibers (red). (E) qRT-PCR analysis of N-cadherin expression in control and two shLARP7 pools. (F and G) The levels of LARP7 protein (F) and mRNA (G) as well as 7SK snRNA level (G) in control, shLARP7-2 cells and rescue cells (expressing an shRNA-resistant WT LARP7 cDNA) were examined by Western blotting and qRT-PCR, respectively. (H) Phase contrast pictures of control, shLARP7-2, and rescue cells. (I) The wound healing assay. (J) The Transwell migration assay. For C, E, G, and J panels: data are presented as mean ± SD. *: the p values (p<0.05) were determined by the Student's t test.DOI:http://dx.doi.org/10.7554/eLife.02907.006

Mentions: To determine the precise role of LARP7 in breast cancer development, we first knocked down the expression of LARP7 in the untransformed MCF10A cells using short hairpin RNAs (shRNAs). Two independent LARP7-targeting sequences (shLARP7-1 and shLARP7-2), when introduced separately into MCF10A cells, markedly decreased the levels of LARP7 protein and 7SK snRNA (Figure 2A, upper panel). We have previously reported that these KD cells are partially transformed as evidenced by the disruption of epithelial polarity and morphological differentiation in the 3D IrECM (He et al., 2008). During the culture of these cells, we noticed a change in cell morphology from the cobble stone-like shape typical of epithelial cells to a more spindle-like and scattered appearance (Figure 2A, lower panel), indicating these cells may be undergoing EMT.10.7554/eLife.02907.006Figure 2.Silencing LARP7 induces EMT in MCF10A cells.


LARP7 suppresses P-TEFb activity to inhibit breast cancer progression and metastasis.

Ji X, Lu H, Zhou Q, Luo K - Elife (2014)

Silencing LARP7 induces EMT in MCF10A cells.(A) Upper panel, the levels of 7SK snRNA and LARP7 protein in MCF10A parental cells and cells stably expressing scramble control, shLARP7-1, or shLARP7-2 were examined by Northern blotting (NB) and Western blotting (WB), respectively. Tubulin was used as a loading control. Lower panel, phase-contrast images of control and two shLARP7 pools. (B) Wound healing assay. Confluent cell monolayers were wounded, and wound closure was monitored at 0 hr and 16 hr. (C) Migration assay. MCF10A control or shLARP7 cells were subjected to a Transwell migration assay. The migrated cells were stained and counted. Data were collected from five fields in three independent experiments. (D) Immunofluorescence staining of E-cadherin (green) and actin stress fibers (red). (E) qRT-PCR analysis of N-cadherin expression in control and two shLARP7 pools. (F and G) The levels of LARP7 protein (F) and mRNA (G) as well as 7SK snRNA level (G) in control, shLARP7-2 cells and rescue cells (expressing an shRNA-resistant WT LARP7 cDNA) were examined by Western blotting and qRT-PCR, respectively. (H) Phase contrast pictures of control, shLARP7-2, and rescue cells. (I) The wound healing assay. (J) The Transwell migration assay. For C, E, G, and J panels: data are presented as mean ± SD. *: the p values (p<0.05) were determined by the Student's t test.DOI:http://dx.doi.org/10.7554/eLife.02907.006
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fig2: Silencing LARP7 induces EMT in MCF10A cells.(A) Upper panel, the levels of 7SK snRNA and LARP7 protein in MCF10A parental cells and cells stably expressing scramble control, shLARP7-1, or shLARP7-2 were examined by Northern blotting (NB) and Western blotting (WB), respectively. Tubulin was used as a loading control. Lower panel, phase-contrast images of control and two shLARP7 pools. (B) Wound healing assay. Confluent cell monolayers were wounded, and wound closure was monitored at 0 hr and 16 hr. (C) Migration assay. MCF10A control or shLARP7 cells were subjected to a Transwell migration assay. The migrated cells were stained and counted. Data were collected from five fields in three independent experiments. (D) Immunofluorescence staining of E-cadherin (green) and actin stress fibers (red). (E) qRT-PCR analysis of N-cadherin expression in control and two shLARP7 pools. (F and G) The levels of LARP7 protein (F) and mRNA (G) as well as 7SK snRNA level (G) in control, shLARP7-2 cells and rescue cells (expressing an shRNA-resistant WT LARP7 cDNA) were examined by Western blotting and qRT-PCR, respectively. (H) Phase contrast pictures of control, shLARP7-2, and rescue cells. (I) The wound healing assay. (J) The Transwell migration assay. For C, E, G, and J panels: data are presented as mean ± SD. *: the p values (p<0.05) were determined by the Student's t test.DOI:http://dx.doi.org/10.7554/eLife.02907.006
Mentions: To determine the precise role of LARP7 in breast cancer development, we first knocked down the expression of LARP7 in the untransformed MCF10A cells using short hairpin RNAs (shRNAs). Two independent LARP7-targeting sequences (shLARP7-1 and shLARP7-2), when introduced separately into MCF10A cells, markedly decreased the levels of LARP7 protein and 7SK snRNA (Figure 2A, upper panel). We have previously reported that these KD cells are partially transformed as evidenced by the disruption of epithelial polarity and morphological differentiation in the 3D IrECM (He et al., 2008). During the culture of these cells, we noticed a change in cell morphology from the cobble stone-like shape typical of epithelial cells to a more spindle-like and scattered appearance (Figure 2A, lower panel), indicating these cells may be undergoing EMT.10.7554/eLife.02907.006Figure 2.Silencing LARP7 induces EMT in MCF10A cells.

Bottom Line: The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating Pol II and antagonizing negative elongation factors.A reservoir of P-TEFb is sequestered in the inactive 7SK snRNP where 7SK snRNA and the La-related protein LARP7 are required for the integrity of this complex.Decreased levels of LARP7 and 7SK snRNA redistribute P-TEFb to the transcriptionally active super elongation complex, resulting in P-TEFb activation and increased transcription of EMT transcription factors, including Slug, FOXC2, ZEB2, and Twist1, to promote breast cancer EMT, invasion, and metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

Show MeSH
Related in: MedlinePlus