High-throughput Functional Genomics Identifies Regulators of Primary Human Beta Cell Proliferation

Karine Robitaille; Jillian L Rourke; Joanne E McBane; Accalia Fu; Stephen Baird; Qiujiang Du; Tatsuya Kin; A M James Shapiro; Robert A Screaton

doi:10.1074/jbc.M115.683912

High-throughput Functional Genomics Identifies Regulators of Primary Human Beta Cell Proliferation

J Biol Chem. 2016 Feb 26;291(9):4614-25. doi: 10.1074/jbc.M115.683912. Epub 2016 Jan 6.

Authors

Karine Robitaille¹, Jillian L Rourke², Joanne E McBane¹, Accalia Fu³, Stephen Baird¹, Qiujiang Du², Tatsuya Kin⁴, A M James Shapiro⁴, Robert A Screaton⁵

Affiliations

¹ From the Children's Hospital of Eastern Ontario Research Institute, Ottawa, Ontario K1H 8L1.
² the Sunnybrook Research Institute, Toronto, Ontario M4N 3M5.
³ From the Children's Hospital of Eastern Ontario Research Institute, Ottawa, Ontario K1H 8L1, the Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5.
⁴ the Clinical Islet Transplant Program, University of Alberta, 8215-112 Street, Edmonton, Alberta T6G 2C8, and.
⁵ the Sunnybrook Research Institute, Toronto, Ontario M4N 3M5, the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada [email protected].

Abstract

The expansion of cells for regenerative therapy will require the genetic dissection of complex regulatory mechanisms governing the proliferation of non-transformed human cells. Here, we report the development of a high-throughput RNAi screening strategy specifically for use in primary cells and demonstrate that silencing the cell cycle-dependent kinase inhibitors CDKN2C/p18 or CDKN1A/p21 facilitates cell cycle entry of quiescent adult human pancreatic beta cells. This work identifies p18 and p21 as novel targets for promoting proliferation of human beta cells and demonstrates the promise of functional genetic screens for dissecting therapeutically relevant state changes in primary human cells.

Keywords: RNA interference (RNAi); beta cell (β-cell); cell cycle; high throughput screening (HTS); pancreatic islet; primary human cells.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adolescent
Adult
Aged
Alberta
Biomarkers / metabolism
Cell Proliferation
Cells, Cultured
Cyclin-Dependent Kinase Inhibitor p18 / antagonists & inhibitors
Cyclin-Dependent Kinase Inhibitor p18 / genetics
Cyclin-Dependent Kinase Inhibitor p18 / metabolism*
Cyclin-Dependent Kinase Inhibitor p21 / antagonists & inhibitors
Cyclin-Dependent Kinase Inhibitor p21 / genetics
Cyclin-Dependent Kinase Inhibitor p21 / metabolism*
Feasibility Studies
Female
Genomics / methods
Green Fluorescent Proteins / genetics
Green Fluorescent Proteins / metabolism
High-Throughput Screening Assays
Humans
Insulin-Secreting Cells / cytology
Insulin-Secreting Cells / metabolism*
Male
Microscopy, Fluorescence
Middle Aged
Pilot Projects
RNA Interference
Recombinant Fusion Proteins / chemistry
Recombinant Fusion Proteins / metabolism
Tissue Donors
Young Adult

Substances

Biomarkers
CDKN1A protein, human
CDKN2C protein, human
Cyclin-Dependent Kinase Inhibitor p18
Cyclin-Dependent Kinase Inhibitor p21
Recombinant Fusion Proteins
enhanced green fluorescent protein
Green Fluorescent Proteins

Grants and funding

MOP 111186/Canadian Institutes of Health Research/Canada