Applied numerical analysis & computational mathematics : ANACM

Annexin 1 (ANXA1) is an endogenous anti-inflammatory protein implicated in cancer. ANXA1 was previously shown to be regulated by hsa-miR-196a. However, whether ANXA1 itself regulates microRNA (miR) expression is unknown. Therefore, we investigated the regulation of miR by ANXA1 in MCF7 breast cancer cells. MCF7-EV (Empty vector) and MCF7-V5 (ANXA1-V5 expressing cells) were subjected to a miR microarray. Microarray analysis revealed a number of miRNAs which were dysregulated in MCF7-V5 cells. 2 novel miRNAs (miR562 and miR26b*) were validated, cloned and functionally characterized. As ANXA1 constitutively activates NF-κB activity to modulate breast cancer metastasis, we found that miR26b* and miR562 directly targeted the canonical NF-κB pathway by targeting the 3' UTR and inhibiting expression of Rel A (p65) and NF-κB1 (p105) respectively. MiR562 inhibited wound healing, which was reversed when ANXA1 was overexpressed. Overexpression of either miR562 or miR26b* in MCF-7 cells enhanced endothelial tube formation when cocultured with human umbilical cord endothelial cells while conversely, treatment of MCF7 cells with either anti-miR562 or anti-miR26b* inhibited endothelial tube formation after co-culture. Further analysis of miR562 revealed that miR562-transfected cell conditioned media enhances endothelial cell tube formation, indicating that miR562 increased angiogenic secreted factors from MCF-7 breast tumor cells. TNFα was increased upon overexpression of miR562, which was reversed when ANXA1 was co-transfected In conclusion, this data suggests that ANXA1-regulated miR26b* and miR562 may play a role in wound healing and tumor-induced endothelial cell tube formation by targeting NF-κB expression and point towards a potential therapeutic target for breast cancer

Primer sequences used for quantitative PCR.

<p>Primer sequences used for quantitative PCR.</p

miR26b* and miR562 overexpression modulates NF-κB activity.

<p>(A,B) qPCR expression of NFKB1 after miR26b* and miR562 overexpression in MCF7 and MCF7-V5 cells (overexpressing ANXA1). (C) MCF7 cells were transfected with empty vector (p-sil), miR26b* or miR562 together with a NF-κB luciferase promoter and NF-κB luciferase activity was measured. * p<0.05 ** p<0.01 vs EV. (D) miR26b* and miR562 expression after transfection with plasmid (E) qPCR analysis of NFKB regulated genes after transfection with miR26b* or miR562. Values are represented as fold change vs EV.</p

Microarray and qPCR validation of microRNA dysregulation in MCF-7 cells overexpressing ANXA1.

<p>(A) Heat map showing pattern of dysregulation observed in miRs during miR microarray analysis when ANXA1 was over-expressed in MCF7 cells. Green bars represent down-regulation of miR expression and red bars represent up-regulation of miR expression. The intensity corresponds to the degree of dysregulation of expression compared to MCF7-EV cells. (B) Correlations between 3 microarray runs and 3 qPCR validations of 12 miRs chosen. p values shown are vs MCF7-EV control cells. (C) Chromosome location of miR26b* and miR562. (E) miR26b* and (F) miR562 expression in breast cancer cell lines. * p<0.05 ** p<0.01 vs MCF10A cells</p

3′UTR cloning and target analysis of miR26b*/RelA and miR562/NF-κB1.

<p>WT and mutant (A) RELA and (B) NF-κB1 3′UTR were subcloned into psiCHECK-2 luciferase vector. The predicted miR binding sites within the respective 3′UTRs is shown. The mutated binding site is represented by asterisks (*). (C,E) 293T cells were transfected with the RELA or NF-κB1 3′UTR plasmid or the mutant RELA or NF-κB1 3′UTR plasmid together with their respective miRs. (D, F) PCR analysis of RELA or NF-κB1 expression after transfection with their respective miRs. (G, H) Western blot analysis of NF-κB protein products p65 and p105/50 after transfection with their respective miRs. A positive control of MCF7-V5 cells overexpressing ANXA1 is shown in lane 3.</p

Expression of miR26b* and miR562 in breast cancer cells.

<p>(A–C) RNA from MCF10A breast epithelial cells, MCF7 and MDA-MB231 breast cancer cells were isolated and expression levels of ANXA1, miR26b* and miR562 were measured. (D–E) MCF7 cells were stably transfected with a ANXA1 overexpression vector and levels of ANXA1, miR26b* and miR562 were assessed. * p<0.05 ** p<0.01 vs MCF-10A cells or EV. Values are represented as fold change vs MCF-10A cells or EV.</p

miR26b* and miR562 overexpression in MCF7 cells enhances endothelial cell angiogenesis while silencing miR26b* and miR562 inhibits angiogenesis.

<p>MCF7 cells were transfected with empty vector (EV), miR26b* or miR562 and a co-culture using transwells was performed with HUVEC in matrigel. (A,C) Average number of tubes formed per field of view and (B,D) average tube length was analyzed. Similar co-culture experiments were performed with MCF7 cells silenced with control, anti-miR26b* and anti-miR562. (D,G) Average number of tubes formed per field of view and (F,H) average tube length was analyzed. * p<0.05 ** p<0.01 vs control cells.</p

MiR562 overexpression in MCF7 cells induces secreted factors which enhance angiogenic activity of endothelial cells.

<p>(A,B) MCF7 cells were transfected with empty vector (EV), miR562, ANXA1 or cotransfected with ANXA1 and miR 562 and conditioned media was used to culture HUVEC in matrigel. Average number of tubes formed per field of view and average tube length was analyzed. (C) Representative images of the four treatments are shown. (D, E) qPCR array of pro/anti angiogenesis genes modulated after miR562 overexpression in MCF7 cells. (F) qPCR array of pro-angiogenesis genes modulated after miR562 and ANXA1 co-overexpression in MCF7 cells. * p<0.05, ** p<0.01 vs EV cells; ξ p<0.05 vs miR562 transfected cells.</p

Postmitotic nuclear pore assembly proceeds by radial dilation of small membrane openings

The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and the structures of assembling NPCs in a correlative 3D EM time course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets whose holes progressively shrink. NPC precursors are found in small membrane holes and dilate radially during assembly of the inner ring complex, forming thousands of transport channels within minutes. This mechanism is fundamentally different from that of interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport competent