High-Throughput RNAi Screening Identifies a Role for the Osteopontin Pathway in Proliferation and Migration of Human Aortic Smooth Muscle Cells.
OBJECTIVE
Understanding of the mechanisms of vascular smooth muscle cells (VSMCs) phenotypic regulation is critically important to identify novel candidates for future therapeutic intervention. While HTS approaches have recently been used to identify novel regulators in many cell lines, such as cancer cells and hematopoietic stem cells, no studies have so far systematically investigated the effect of gene inactivation on VSMCs with respect to cell survival and growth response.


RESULTS
257 out of 2000 genes tested resulted in an inhibition of cell proliferation in HaoSMCs. After pathway analysis, 38 significant genes were selected for further study. 23 genes were confirmed to inhibit proliferation, and 13 genes found to induce apoptosis in the synthetic phenotype. 11 genes led to an aberrant nuclear phenotype indicating a central role in cell mitosis. 4 genes affected the cell migration in synthetic HaoSMCs. Using computational biological network analysis, 11 genes were identified to have an indirect or direct interaction with the Osteopontin pathway. For 10 of those genes, levels of proteins downstream of the Osteopontin pathway were found to be down-regulated, using RNAi methodology.


CONCLUSIONS
A phenotypic high-throughput siRNA screen could be applied to identify genes relevant for the cell biology of HaoSMCs. Novel genes were identified which play a role in proliferation, apoptosis, mitosis and migration of HaoSMCs. These may represent potential drug candidates in the future.
BioQuant, Heidelberg University, INF 267, 69120, Heidelberg, Germany.|Clinic for Vascular and Endovascular Surgery, University Hospital, Frankfurt, Germany.
Lei
L
Zhang
BioQuant, Heidelberg University, INF 267, 69120, Heidelberg, Germany.
Holger
H
Erfle
BioQuant and IPMB, University of Heidelberg and DKFZ, Biomedical Computer Vision Group, Heidelberg, Germany.
Nathalie
N
Harder
BioQuant, Heidelberg University, INF 267, 69120, Heidelberg, Germany.
Jürgen
J
Beneke
BioQuant, Heidelberg University, INF 267, 69120, Heidelberg, Germany.
Nina
N
Beil
BioQuant, Heidelberg University, INF 267, 69120, Heidelberg, Germany.
Ruben
R
Bulkescher
BioQuant and IPMB, University of Heidelberg and DKFZ, Biomedical Computer Vision Group, Heidelberg, Germany.
Karl
K
Rohr
Clinic for Vascular and Endovascular Surgery, University Hospital, Frankfurt, Germany. michael.keese@kgu.de.|Clinic for Vascular and Endovascular Surgery, Johann Wolfgang Goethe University Hospital, Theodor Stern Kai 7, 60590, Frankfurt am Main, Germany. michael.keese@kgu.de.
Michael
M
Keese
D001011:Aorta / Q000166:cytology*; D017209:Apoptosis; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D006801:Humans; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction
D034741:RNA, Small Interfering; C506895:SPP1 protein, human; D053495:Osteopontin
HaoSMCs; High-throughput RNAi screening; Nuclear phenotype; Osteopontin pathway; siRNA