Inhibition of nucleoporin member Nup214 expression by miR-133b perturbs mitotic timing and leads to cell death.
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.
Cancer Biology and Inflammatory Disorder Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4, Raja S.C. Mullick Road, Kolkata, 700 032, India. sumana.rb@gmail.com.
Sumana
S
Bhattacharjya
Cancer Biology and Inflammatory Disorder Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4, Raja S.C. Mullick Road, Kolkata, 700 032, India. kumarsingharoy88@gmail.com.
Kumar Singha
KS
Roy
Tata Memorial Centre, ACTREC Sector 22, Navi Mumbai, Kharghar, 410210, India. asgc.ganguly@gmail.com.
Abira
A
Ganguly
Department of Oncogene Regulation, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata, 700 026, India. shreya.sarkar.momo@gmail.com.
Shreya
S
Sarkar
Department of Oncogene Regulation, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata, 700 026, India. ckpanda.cnci@gmail.com.
Chinmay K
CK
Panda
Tata Memorial Centre, ACTREC Sector 22, Navi Mumbai, Kharghar, 410210, India. dbhattacharyya@actrec.gov.in.
Dibyendu
D
Bhattacharyya
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata, 700 064, India. nitai.bhattacharyya@gmail.com.|Present address: Biomedical Genomics Centre, PG Polyclinic Building (3rd floor), 5, Suburban Hospital Road, Kolkata, 700 020, India. nitai.bhattacharyya@gmail.com.
Nitai P
NP
Bhattacharyya
Cancer Biology and Inflammatory Disorder Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4, Raja S.C. Mullick Road, Kolkata, 700 032, India. susantarc@gmail.com.
Susanta
S
Roychoudhury
D017209:Apoptosis / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics; D016923:Cell Death / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D019925:Cyclin A / Q000235:genetics; D056744:Cyclin B1 / Q000235:genetics; D015536:Down-Regulation / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D045325:HCT116 Cells; D057809:HEK293 Cells; D006258:Head and Neck Neoplasms / Q000235:genetics; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D008938:Mitosis / Q000235:genetics*; D028861:Nuclear Pore Complex Proteins / Q000235:genetics*; D010766:Phosphorylation / Q000235:genetics
C534699:CCNB1 protein, human; D019925:Cyclin A; D056744:Cyclin B1; C548234:MIRN133 microRNA, human; D035683:MicroRNAs; C085914:NUP214 protein, human; D028861:Nuclear Pore Complex Proteins