The DNA microarray technology has contributed widely in biological studies. Recently, a method to detect the effects of extracellular matrix (ECM) proteins on cells using microarray was developed. In this dissertation, the microarray technology was applied to study the effects of ECM proteins on human mesenchymal stem cell (hMSC) differentiation, and to identify the best growth- supporting ECM conditions for glioblastoma, and to create a method for efficient small molecule drug screening. The importance of ECM on hMSCs is well known. Numerous studies have shown that the ECM deposed by specific types of cells could induce differentiation in hMSCs. However, clinical application of cell-deposited ECM could carry risks such as contamination, immune rejection and batch-to-batch variation. An artificial ECM could avoid these problems. In this dissertation, the effects of ECM proteins were screened on the hMSCs differentiation using the microarray technology. The myogenic and neurogenic differentiation profiles are reported. This information can contribute to the selection of optimal differentiation-inducing coating on the muscle or nerve tissue engineering scaffolds. Cell culture has always been a fundamental tool in cancer research. However, most primary cancer cells are very difficult to maintain. In this dissertation, ECM protein combinations were tested for their efficacy in supporting the growth of glioblastoma cells. Twelve best combinations were identified. The result may lead to successful maintenance of the primary cells of glioblastoma and other types of cancerous tumors. The results may yield larger quantities of the primary cancer cells, which are heretofore limited in supply, and thus facilitate the advancement of cancer research. Genetic marker-based personalized medicine has attracted much attention. However, our understanding of the genes is still limited. A direct drug efficacy screening on the diseased cells of an individual patient may complement the gene-based personalized medicine. In this study, a small-molecule drug microarray method is reported. This method is able to screen a large number of drugs and their combinations by using a very small mount of patients' diseased cells, making it possible to design personalized drug cocktails

Zoledronic acid, a potent nitrogen-containing bisphosphonate (NBP), has been extensively used to limit bone turnover in a various diseases including tumors. Recent clinical studies have demonstrated direct anti-cancer effects of zoledronic acid, in addition to its clinical benefits for skeletal-related events. Here we investigated the effects of 4 clinically available NBPs on human tumor cell proliferation. Our data demonstrate a potent anti-proliferative effect of zoledronic acid against glioblastoma (GBM) cell lines, breast cancer cells and GBM patient-derived lines. Zoledronic acid also effectively inhibited GBM tumor growth in xenograft mouse models. Zoledronic acid strongly stimulated autophagy but not apoptotic signals in all tested cells. Only one intermediate product of cholesterols synthesis pathway, geranylgeranyl diphosphate (GGPP) rescued cells from the cytotoxic effects of zoledronic acid. To further investigate the effect of GGPP, we knocked down RABGGTA, which encodes a subunit of the Rabgeranylgeranyltransferase protein. This knockdown induced an effect similar to zoledronic acid in cancer cell lines. These data are promising and suggested a potential for zoledronic acid as an anti-cancer agent, through its ablation of the function of Rab proteins.

Objective

The site-specificity of endothelial phenotype is attributable to the local hemodynamic forces. The flow regulation of microRNAs in endothelial cells (ECs) plays a significant role in vascular homeostasis and diseases. The objective of this study was to elucidate the molecular mechanism by which the pulsatile shear flow-induced microRNA-23b (miR-23b) exerts antiproliferative effects on ECs.

Approach and results

We used a combination of a cell perfusion system and experimental animals to examine the flow regulation of miR-23b in modulating EC proliferation. Our results demonstrated that pulsatile shear flow induces the transcription factor Krüppel-like factor 2 to promote miR-23b biosynthesis; the increase in miR-23b then represses cyclin H to impair the activity and integrity of cyclin-dependent kinase-activating kinase (CAK) complex. The inhibitory effect of miR-23b on CAK exerts dual actions to suppress cell cycle progression, and reduce basal transcription by deactivating RNA polymerase II. Whereas pulsatile shear flow regulates the miR-23b/CAK pathway to exert antiproliferative effects on ECs, oscillatory shear flow has little effect on the miR-23b/CAK pathway and hence does not cause EC growth arrest. Such flow pattern-dependent phenomena are validated with an in vivo model on rat carotid artery: the flow disturbance induced by partial carotid ligation led to a lower expression of miR-23b and a higher EC proliferation in comparison with the pulsatile flow regions of the unligated vessels. Local delivery of miR-23b mitigated the proliferative EC phenotype in partially ligated vessels.

Conclusions

Our findings unveil a novel mechanism by which hemodynamic forces modulate EC proliferative phenotype through the miR-23b/CAK pathway.