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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

Isolation of subpopulations of NSCLC cells that have a high metastatic potential(A) Experimental outline for the generation of highly aggressive NSCLC cell lines. H522 or H1975 cells were intravenously injected into the tail vein of nude mice. After 6 weeks, lung nodules were removed and were expanded in vitro and subsequently intravenously re-injected. The high metastatic potential sub-cell lines (after 3 rounds of selection) were compared with the parental cell lines. (B) Cell migration assays. Cells were placed into the upper chamber for 24 h. Migrated cells were stained with crystal violet, and counted for quantitative analysis. (C) Cell invasion assays. Cells in serum-free medium were seeded into the Matrigel pre-coated upper chamber, and the invading cells were stained and counted for quantitative analysis. (D) Wound healing assays. Cell culture plates attached with cells were wounded and cultured for 48 h. The wound healing was determined. Data are presented as mean ± s.e.m.. (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001 for comparing with parental cells.
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Figure 1: Isolation of subpopulations of NSCLC cells that have a high metastatic potential(A) Experimental outline for the generation of highly aggressive NSCLC cell lines. H522 or H1975 cells were intravenously injected into the tail vein of nude mice. After 6 weeks, lung nodules were removed and were expanded in vitro and subsequently intravenously re-injected. The high metastatic potential sub-cell lines (after 3 rounds of selection) were compared with the parental cell lines. (B) Cell migration assays. Cells were placed into the upper chamber for 24 h. Migrated cells were stained with crystal violet, and counted for quantitative analysis. (C) Cell invasion assays. Cells in serum-free medium were seeded into the Matrigel pre-coated upper chamber, and the invading cells were stained and counted for quantitative analysis. (D) Wound healing assays. Cell culture plates attached with cells were wounded and cultured for 48 h. The wound healing was determined. Data are presented as mean ± s.e.m.. (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001 for comparing with parental cells.

Mentions: To identify potential mechanisms of metastasis, we firstly generated a series of highly aggressive NSCLC sub-cell lines using a in vivo selection systems. Two NSCLC cell lines, H522 and H1975, were used to form multiple small nodules in athymic nude mice by tail vein injection. The cells were further isolated from tumor nodules followed by a reinjection. After 3 rounds of in vivo selection, cells with increased metastatic capacity were obtained as shown in Figure 1A. To evaluate the biological aggressiveness, cell migration and invasion assays were performed. As predicted, we found a progressive increase in the migration ability of cell lines obtained from each rounds (Figure 1B). And the collagen invasion assays showed a similar tendency in cellular invasion (Figure 1C). Wound healing assays demonstrated the established cell lines exhibited improved ability to migrate into the wound (Figure 1D). Among these cells, H522M3 and H1975M3 cells exhibited highly metastatic potential compared with the parental cells, and could be used for further study.


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)

Isolation of subpopulations of NSCLC cells that have a high metastatic potential(A) Experimental outline for the generation of highly aggressive NSCLC cell lines. H522 or H1975 cells were intravenously injected into the tail vein of nude mice. After 6 weeks, lung nodules were removed and were expanded in vitro and subsequently intravenously re-injected. The high metastatic potential sub-cell lines (after 3 rounds of selection) were compared with the parental cell lines. (B) Cell migration assays. Cells were placed into the upper chamber for 24 h. Migrated cells were stained with crystal violet, and counted for quantitative analysis. (C) Cell invasion assays. Cells in serum-free medium were seeded into the Matrigel pre-coated upper chamber, and the invading cells were stained and counted for quantitative analysis. (D) Wound healing assays. Cell culture plates attached with cells were wounded and cultured for 48 h. The wound healing was determined. Data are presented as mean ± s.e.m.. (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001 for comparing with parental cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480748&req=5

Figure 1: Isolation of subpopulations of NSCLC cells that have a high metastatic potential(A) Experimental outline for the generation of highly aggressive NSCLC cell lines. H522 or H1975 cells were intravenously injected into the tail vein of nude mice. After 6 weeks, lung nodules were removed and were expanded in vitro and subsequently intravenously re-injected. The high metastatic potential sub-cell lines (after 3 rounds of selection) were compared with the parental cell lines. (B) Cell migration assays. Cells were placed into the upper chamber for 24 h. Migrated cells were stained with crystal violet, and counted for quantitative analysis. (C) Cell invasion assays. Cells in serum-free medium were seeded into the Matrigel pre-coated upper chamber, and the invading cells were stained and counted for quantitative analysis. (D) Wound healing assays. Cell culture plates attached with cells were wounded and cultured for 48 h. The wound healing was determined. Data are presented as mean ± s.e.m.. (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001 for comparing with parental cells.
Mentions: To identify potential mechanisms of metastasis, we firstly generated a series of highly aggressive NSCLC sub-cell lines using a in vivo selection systems. Two NSCLC cell lines, H522 and H1975, were used to form multiple small nodules in athymic nude mice by tail vein injection. The cells were further isolated from tumor nodules followed by a reinjection. After 3 rounds of in vivo selection, cells with increased metastatic capacity were obtained as shown in Figure 1A. To evaluate the biological aggressiveness, cell migration and invasion assays were performed. As predicted, we found a progressive increase in the migration ability of cell lines obtained from each rounds (Figure 1B). And the collagen invasion assays showed a similar tendency in cellular invasion (Figure 1C). Wound healing assays demonstrated the established cell lines exhibited improved ability to migrate into the wound (Figure 1D). Among these cells, H522M3 and H1975M3 cells exhibited highly metastatic potential compared with the parental cells, and could be used for further study.

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