Inhibition of nucleoporin member Nup214 expression by miR-133b perturbs mitotic timing and leads to cell death
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
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| Title |
Inhibition of nucleoporin member Nup214 expression by miR-133b perturbs mitotic timing and leads to cell death
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| Creator |
Bhattacharjya, Sumana
Singha Roy, Kumar Ganguly, Abira Sarkar, Shreya Panda, Chinmay K Bhattacharyya, Dibyendu, Bhattacharyya, Nitai P Roychoudhury, Susanta |
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| Subject |
Cancer Biology and Inflammatory Disorder Division
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| Description |
Background: Nucleoporins mediate nucleocytoplasmic exchange of macromolecules and several have been assigned active mitotic functions. Nucleoporins can participate in various mitotic functions like spindle assembly, kinetochore organisation and chromosome segregation- important for genome integrity. Pathways to genome integrity are frequently deregulated in cancer and many are regulated in part by microRNAs. Indeed, altered levels of numerous microRNAs have frequently been associated with tumorigenesis. Here, we unveil a microRNA-mediated
regulation of the nucleoporin Nup214 and its downstream effect on genome integrity.
Methods: Databases/bioinformatic tools such as miRBase, Oncomine and RNAhybrid predicted Nup214 as a miR-133b
target. To validate this, we used luciferase reporter assays, Real-Time PCR and immuno-blotting. Flow cytometry and immuno-blots of mitotic markers were used to analyse cell cycle pattern upon thymidine synchronization and miR-133b treatment. Mitotic indices and chromosomal abnormalities were assessed by immuno-fluorescence for FITC-tagged phospho-H3 as well as video-microscopy for GFP-tagged histone H4. Annexin V/propidium iodide staining, caspase3/ PARP cleavage and colony formation assays were done to investigate cell death upon either miR-133b transfection or NUP214 knockdown by siRNA. UPCI:SCC084, HCT116, HeLa-H4-pEGFP and HEK293 (human oral squamous cell
carcinoma, colorectal, cervical carcinomas and embryonic kidney cell lines, respectively) were used. miR-133b and
NUP214 expressions were validated in cancer cell lines and tissues by Real-Time PCR.
Results: Examination of head and neck tumour tissues and cancer cell lines revealed that Nup214 and miR-133b
expressions are negatively correlated. In vitro, Nup214 was significantly downregulated by ectopic miR-133b. This
downregulation elevated mitotic indices and delayed degradation of mitotic marker proteins cyclinB1 and cyclinA
and dephosphorylation of H3. Moreover, this mitotic delay enhanced chromosomal abnormalities and apoptosis.
Conclusions: We have identified NUP214, a member of the massive nuclear pore complex, as a novel miR-133b
target. Thus, we have shown a hitherto unknown microRNA regulation of mitosis mediated by a member of the
nucleoporin family. Based on observations, we also raise some hypotheses regarding transport-dependent/independent
functions of Nup214 in this study. Our results hence attempt to explain why miR-133b is generally downregulated in tumours and lay out the potential for Nup214 as a therapeutic target in the treatment of cancer.
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| Publisher |
BioMed Central
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| Date |
2015
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| Type |
Article
PeerReviewed |
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| Format |
application/pdf
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| Identifier |
http://www.eprints.iicb.res.in/2243/1/MOLECULAR_CANCER__V._14_____Article_Number_42.pdf
Bhattacharjya, Sumana and Singha Roy, Kumar and Ganguly, Abira and Sarkar, Shreya and Panda, Chinmay K and Bhattacharyya, Dibyendu, and Bhattacharyya, Nitai P and Roychoudhury, Susanta (2015) Inhibition of nucleoporin member Nup214 expression by miR-133b perturbs mitotic timing and leads to cell death. Molecular Cancer, 14 (A.N.42). |
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| Relation |
http://dx.doi.org/10.1186/s12943-015-0299-z
http://www.eprints.iicb.res.in/2243/ |
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