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NF-κB-mediated miR-124 suppresses metastasis of non-small-cell lung cancer by targeting MYO10.

Sun Y, Ai X, Shen S, Lu S - Oncotarget (2015)

Bottom Line: Over-expression of miR-124 robustly attenuated migration and metastatic ability of the aggressive cells.Knockdown of MYO10 inhibited cell migration, whereas forced MYO10 expression markedly rescued miR-124-mediated suppression of cell metastasis.Additionally, we found an activated NF-κB-centered inflammatory loop in the highly aggressive cells leading to down-regulation of miR-124.

View Article: PubMed Central - PubMed

Affiliation: Lung Tumor Clinical Medical Center, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030, China.

ABSTRACT
Recently, dysregulation of microRNAs plays a critical role in cancer metastasis. Here, an in vivo selection approach was used to generate highly aggressive NSCLC sub-cell lines followed by comparing the microRNAs expression using microarrays. miR-124 was notably deregulated in both highly invasive sub-cell lines and node-positive NSCLC specimens. Over-expression of miR-124 robustly attenuated migration and metastatic ability of the aggressive cells. MYO10 was subsequently identified as a novel functional downstream target of miR-124, and was up-regulated in node-positive NSCLC tissues. Knockdown of MYO10 inhibited cell migration, whereas forced MYO10 expression markedly rescued miR-124-mediated suppression of cell metastasis. Additionally, we found an activated NF-κB-centered inflammatory loop in the highly aggressive cells leading to down-regulation of miR-124. These results suggest that NF-κB-regulated miR-124 targets MYO10, inhibits cell invasion and metastasis, and is down-regulated in node-positive NSCLC.

No MeSH data available.


Related in: MedlinePlus

Re-expression of MYO10 rescues miR-124-mediated suppression of cell aggressivenessH522M3 and H1975M3 cells were tansfected with control siRNA (si-control) or MYO10 specific siRNA (si-MYO10), (A) the expression of MYO10 was determined by Western blot. (B) After transfection, the migratory properties were examined by transwell migration assays. The migrated cells were stained with crystal violet, and (C) counted for quantitative analysis. *p < 0.05 and **p < 0.01. (D) The expression of MYO10 in the stable transfected cells following re-expression of MYO10 in pRTR-miR-124 cells were analyzed by Western blot. (E) After re-expression of MYO10, cell invasion was analyzed by matrigel invasion assays. The invaded cells were summarized in the bar graph. *p < 0.05 and **p < 0.01. (F) Representative EGFP imaging revealing metastatic foci on the surface of lungs excised from mice after re-expression of MYO10. Bar, 5 mm. Quantification of EGFP-positive metastatic foci on the surface of the lung. **p < 0.01.
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Figure 5: Re-expression of MYO10 rescues miR-124-mediated suppression of cell aggressivenessH522M3 and H1975M3 cells were tansfected with control siRNA (si-control) or MYO10 specific siRNA (si-MYO10), (A) the expression of MYO10 was determined by Western blot. (B) After transfection, the migratory properties were examined by transwell migration assays. The migrated cells were stained with crystal violet, and (C) counted for quantitative analysis. *p < 0.05 and **p < 0.01. (D) The expression of MYO10 in the stable transfected cells following re-expression of MYO10 in pRTR-miR-124 cells were analyzed by Western blot. (E) After re-expression of MYO10, cell invasion was analyzed by matrigel invasion assays. The invaded cells were summarized in the bar graph. *p < 0.05 and **p < 0.01. (F) Representative EGFP imaging revealing metastatic foci on the surface of lungs excised from mice after re-expression of MYO10. Bar, 5 mm. Quantification of EGFP-positive metastatic foci on the surface of the lung. **p < 0.01.

Mentions: Our findings that elevated MYO10 expression in NSCLC patients was associated with lymph node metastasis led us to hypothesise that MYO10 might be involved with cell motility. MYO10-specific (si-MYO10) and scrambled (si-control) small interfering RNA were used, and the efficacy was examined by Western blot (Figure 5A). As expected, MYO10 depletion with small interfering RNA markedly abrogated cell migration capabilities in aggressive NSCLC cells (Figure 5B and 5C). To further study the roles of MYO10 in miR-124-mediated suppression of aggressiveness in NSCLC cells, we rescued the expression of MYO10 in cells stably expressing miR124 (H522M3/pRTR-miR-124 and H1975M3/pRTR-miR-124 cells) by transfecting the plasmids carrying MYO10 (pcDNA3-MYO10). While cells expressed stably high levels of miR-124 exhibited a decrease in invasion (Figure 3C), re-expression of MYO10 in H522M3/pRTR-miR-124 and H1975M3/pRTR-miR-124 cells, as confirmed by Western blot analysis (Figure 5D), restored the capacity of invasion (Figure 5E). Furthermore, H522M3/pRTR-miR-124 cell containing pcDNA3-control or pcDNA3-MYO10 were injected into the tail vein to investigate MYO10-dependent distant metastasis. The number of metastatic foci on the lung surface was significantly increased after re-expression of MYO10 (Figure 5F).


NF-κB-mediated miR-124 suppresses metastasis of non-small-cell lung cancer by targeting MYO10.

Sun Y, Ai X, Shen S, Lu S - Oncotarget (2015)

Re-expression of MYO10 rescues miR-124-mediated suppression of cell aggressivenessH522M3 and H1975M3 cells were tansfected with control siRNA (si-control) or MYO10 specific siRNA (si-MYO10), (A) the expression of MYO10 was determined by Western blot. (B) After transfection, the migratory properties were examined by transwell migration assays. The migrated cells were stained with crystal violet, and (C) counted for quantitative analysis. *p < 0.05 and **p < 0.01. (D) The expression of MYO10 in the stable transfected cells following re-expression of MYO10 in pRTR-miR-124 cells were analyzed by Western blot. (E) After re-expression of MYO10, cell invasion was analyzed by matrigel invasion assays. The invaded cells were summarized in the bar graph. *p < 0.05 and **p < 0.01. (F) Representative EGFP imaging revealing metastatic foci on the surface of lungs excised from mice after re-expression of MYO10. Bar, 5 mm. Quantification of EGFP-positive metastatic foci on the surface of the lung. **p < 0.01.
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Related In: Results  -  Collection

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Figure 5: Re-expression of MYO10 rescues miR-124-mediated suppression of cell aggressivenessH522M3 and H1975M3 cells were tansfected with control siRNA (si-control) or MYO10 specific siRNA (si-MYO10), (A) the expression of MYO10 was determined by Western blot. (B) After transfection, the migratory properties were examined by transwell migration assays. The migrated cells were stained with crystal violet, and (C) counted for quantitative analysis. *p < 0.05 and **p < 0.01. (D) The expression of MYO10 in the stable transfected cells following re-expression of MYO10 in pRTR-miR-124 cells were analyzed by Western blot. (E) After re-expression of MYO10, cell invasion was analyzed by matrigel invasion assays. The invaded cells were summarized in the bar graph. *p < 0.05 and **p < 0.01. (F) Representative EGFP imaging revealing metastatic foci on the surface of lungs excised from mice after re-expression of MYO10. Bar, 5 mm. Quantification of EGFP-positive metastatic foci on the surface of the lung. **p < 0.01.
Mentions: Our findings that elevated MYO10 expression in NSCLC patients was associated with lymph node metastasis led us to hypothesise that MYO10 might be involved with cell motility. MYO10-specific (si-MYO10) and scrambled (si-control) small interfering RNA were used, and the efficacy was examined by Western blot (Figure 5A). As expected, MYO10 depletion with small interfering RNA markedly abrogated cell migration capabilities in aggressive NSCLC cells (Figure 5B and 5C). To further study the roles of MYO10 in miR-124-mediated suppression of aggressiveness in NSCLC cells, we rescued the expression of MYO10 in cells stably expressing miR124 (H522M3/pRTR-miR-124 and H1975M3/pRTR-miR-124 cells) by transfecting the plasmids carrying MYO10 (pcDNA3-MYO10). While cells expressed stably high levels of miR-124 exhibited a decrease in invasion (Figure 3C), re-expression of MYO10 in H522M3/pRTR-miR-124 and H1975M3/pRTR-miR-124 cells, as confirmed by Western blot analysis (Figure 5D), restored the capacity of invasion (Figure 5E). Furthermore, H522M3/pRTR-miR-124 cell containing pcDNA3-control or pcDNA3-MYO10 were injected into the tail vein to investigate MYO10-dependent distant metastasis. The number of metastatic foci on the lung surface was significantly increased after re-expression of MYO10 (Figure 5F).

Bottom Line: Over-expression of miR-124 robustly attenuated migration and metastatic ability of the aggressive cells.Knockdown of MYO10 inhibited cell migration, whereas forced MYO10 expression markedly rescued miR-124-mediated suppression of cell metastasis.Additionally, we found an activated NF-κB-centered inflammatory loop in the highly aggressive cells leading to down-regulation of miR-124.

View Article: PubMed Central - PubMed

Affiliation: Lung Tumor Clinical Medical Center, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030, China.

ABSTRACT
Recently, dysregulation of microRNAs plays a critical role in cancer metastasis. Here, an in vivo selection approach was used to generate highly aggressive NSCLC sub-cell lines followed by comparing the microRNAs expression using microarrays. miR-124 was notably deregulated in both highly invasive sub-cell lines and node-positive NSCLC specimens. Over-expression of miR-124 robustly attenuated migration and metastatic ability of the aggressive cells. MYO10 was subsequently identified as a novel functional downstream target of miR-124, and was up-regulated in node-positive NSCLC tissues. Knockdown of MYO10 inhibited cell migration, whereas forced MYO10 expression markedly rescued miR-124-mediated suppression of cell metastasis. Additionally, we found an activated NF-κB-centered inflammatory loop in the highly aggressive cells leading to down-regulation of miR-124. These results suggest that NF-κB-regulated miR-124 targets MYO10, inhibits cell invasion and metastasis, and is down-regulated in node-positive NSCLC.

No MeSH data available.


Related in: MedlinePlus