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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

MYO10 is identified as a functional downstream target of miR-124(A) Western blot analysis of the expression of MYO10 in aggressive M3 and parental cells. (B) Relative MYO10 expression in human normal lung or NSCLC tumors with lymph nodes positive or negative was determined by qRT-PCR. *p < 0.05. (C) The correlation between the expression of MYO10 and miR-124 was analyzed. (D) After transfection of P-miR-124, the expression of MYO10 in H522M3, H1975M3 and H1299 cells was examined by Western blot. (E) Schematic representation of the human MYO10 3′UTR showing the highly conserved miR-124 binding site, and the pGL3 reporter vectors carrying the wide type or mutated MYO10 3′UTR are indicated. (F) The human MYO10 3′UTR vector containing a putative binding site for miR-124 was co-transfected into H522 and Calu-3 cells along with P-miR-124, and the luciferase activity was messured 24 h post-transfection. **p < 0.01.
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Figure 4: MYO10 is identified as a functional downstream target of miR-124(A) Western blot analysis of the expression of MYO10 in aggressive M3 and parental cells. (B) Relative MYO10 expression in human normal lung or NSCLC tumors with lymph nodes positive or negative was determined by qRT-PCR. *p < 0.05. (C) The correlation between the expression of MYO10 and miR-124 was analyzed. (D) After transfection of P-miR-124, the expression of MYO10 in H522M3, H1975M3 and H1299 cells was examined by Western blot. (E) Schematic representation of the human MYO10 3′UTR showing the highly conserved miR-124 binding site, and the pGL3 reporter vectors carrying the wide type or mutated MYO10 3′UTR are indicated. (F) The human MYO10 3′UTR vector containing a putative binding site for miR-124 was co-transfected into H522 and Calu-3 cells along with P-miR-124, and the luciferase activity was messured 24 h post-transfection. **p < 0.01.

Mentions: In order to demonstrate the molecular mechanisms by which miR-124 impairs tumor metastasis in NSCLC, several computational algorithms were used to identify the potential functional targets for miR-124. Using miRecords, an integrated resource for microRNA-target interactions, a panel of molecules was predicted to be potential targets of the miR-124 with eight miRNA target prediction programs (Supplementary Table 2). Expression difference of the predicted potential targets between parental and aggressive cells was further examined. It was identified that MYO10 was commonly up-regulated in the two aggressive cells compared to parental cells (Figure 4A). The increased expression of MYO10 was also observed in NSCLC, particularly in lymph node-positive ones (Figure 4B), and was negatively correlated with miR-124 level in NSCLC tissue biopsies (Figure 4C). This prompted us to study the expression of MYO10 in ectopic miR-124 expressed cells revealing a significant down-regulation (Figure 4D). Additionally, inhibition of miR-124 with anti-miR-124 markedly increased the expression of MYO10, together with the potential of cell migration (Supplementary Figure 1A–1D). Using the miRNA prediction tool, miRanda (v1.9), we identified the phylogenetically conserved miR-124 seed-matching sequences in human MYO10 3′UTR (Figure 4E). Ectopic miR-124 repressed the activity of MYO10 wild-type 3′UTR reporter constructs in dual luciferase reporter assays (Figure 4F), while mutation in miR-124-binding site completely abrogated this repression. Therefore, it was demonstrated that the MYO10 expression could be directly regulated by miR-124 via conserved seed-matching sequences and correlates with the node metastasis of NSCLC disease.


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)

MYO10 is identified as a functional downstream target of miR-124(A) Western blot analysis of the expression of MYO10 in aggressive M3 and parental cells. (B) Relative MYO10 expression in human normal lung or NSCLC tumors with lymph nodes positive or negative was determined by qRT-PCR. *p < 0.05. (C) The correlation between the expression of MYO10 and miR-124 was analyzed. (D) After transfection of P-miR-124, the expression of MYO10 in H522M3, H1975M3 and H1299 cells was examined by Western blot. (E) Schematic representation of the human MYO10 3′UTR showing the highly conserved miR-124 binding site, and the pGL3 reporter vectors carrying the wide type or mutated MYO10 3′UTR are indicated. (F) The human MYO10 3′UTR vector containing a putative binding site for miR-124 was co-transfected into H522 and Calu-3 cells along with P-miR-124, and the luciferase activity was messured 24 h post-transfection. **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: MYO10 is identified as a functional downstream target of miR-124(A) Western blot analysis of the expression of MYO10 in aggressive M3 and parental cells. (B) Relative MYO10 expression in human normal lung or NSCLC tumors with lymph nodes positive or negative was determined by qRT-PCR. *p < 0.05. (C) The correlation between the expression of MYO10 and miR-124 was analyzed. (D) After transfection of P-miR-124, the expression of MYO10 in H522M3, H1975M3 and H1299 cells was examined by Western blot. (E) Schematic representation of the human MYO10 3′UTR showing the highly conserved miR-124 binding site, and the pGL3 reporter vectors carrying the wide type or mutated MYO10 3′UTR are indicated. (F) The human MYO10 3′UTR vector containing a putative binding site for miR-124 was co-transfected into H522 and Calu-3 cells along with P-miR-124, and the luciferase activity was messured 24 h post-transfection. **p < 0.01.
Mentions: In order to demonstrate the molecular mechanisms by which miR-124 impairs tumor metastasis in NSCLC, several computational algorithms were used to identify the potential functional targets for miR-124. Using miRecords, an integrated resource for microRNA-target interactions, a panel of molecules was predicted to be potential targets of the miR-124 with eight miRNA target prediction programs (Supplementary Table 2). Expression difference of the predicted potential targets between parental and aggressive cells was further examined. It was identified that MYO10 was commonly up-regulated in the two aggressive cells compared to parental cells (Figure 4A). The increased expression of MYO10 was also observed in NSCLC, particularly in lymph node-positive ones (Figure 4B), and was negatively correlated with miR-124 level in NSCLC tissue biopsies (Figure 4C). This prompted us to study the expression of MYO10 in ectopic miR-124 expressed cells revealing a significant down-regulation (Figure 4D). Additionally, inhibition of miR-124 with anti-miR-124 markedly increased the expression of MYO10, together with the potential of cell migration (Supplementary Figure 1A–1D). Using the miRNA prediction tool, miRanda (v1.9), we identified the phylogenetically conserved miR-124 seed-matching sequences in human MYO10 3′UTR (Figure 4E). Ectopic miR-124 repressed the activity of MYO10 wild-type 3′UTR reporter constructs in dual luciferase reporter assays (Figure 4F), while mutation in miR-124-binding site completely abrogated this repression. Therefore, it was demonstrated that the MYO10 expression could be directly regulated by miR-124 via conserved seed-matching sequences and correlates with the node metastasis of NSCLC disease.

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