Inhibition of nucleoporin member Nup214 expression by miR-133b perturbs mitotic timing and leads to cell death

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• 作者：Sumana Bhattacharjya (1)
  Kumar Singha Roy (1)
  Abira Ganguly (2)
  Shreya Sarkar (3)
  Chinmay K Panda (3)
  Dibyendu Bhattacharyya (2)
  Nitai P Bhattacharyya (4) (5)
  Susanta Roychoudhury (1)
  
  1. 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
  2. Tata Memorial Centre ; ACTREC Sector 22 ; Navi Mumbai ; Kharghar ; 410210 ; India
  3. Department of Oncogene Regulation ; Chittaranjan National Cancer Institute ; 37 ; S.P. Mukherjee Road ; Kolkata ; 700 026 ; India
  4. Crystallography and Molecular Biology Division ; Saha Institute of Nuclear Physics ; 1/AF ; Bidhannagar ; Kolkata ; 700 064 ; India
  5. Present address ; Biomedical Genomics Centre ; PG Polyclinic Building (3rd floor) ; 5 ; Suburban Hospital Road ; Kolkata ; 700 020 ; India
  
• 关键词：Nucleoporin ; Nup214 ; miR ; 133b ; Mitosis ; Apoptosis ; Cell cycle ; MicroRNA ; Cancer ; Head and neck cancer ; Chromosomal abnormality
• 刊名：Molecular Cancer
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：14
• 期：1
• 全文大小：1,911 KB
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• 刊物主题：Cancer Research; Oncology;
• 出版者：BioMed Central
• ISSN：1476-4598

文摘

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.