Differential Expression of microRNAs in Osteoblasts and Their Roles in Regulation of Cell Survival and Metabolism

Bone structure is maintained by bone remodeling. Osteoblasts play crucial roles in mediating bone formation. A complicated network of local or systemic mediators can modulate osteoblast activities. Cell survival and metabolism are two typical factors to determine osteoblast functions. Our previous studies (NSC90-2314-B-002-196, NSC91-2314-B-038-031, NSC92-2314-B-038-010, NSC94-2314-B-038-013) provided several lines of evidence to show that oxidative stress can induce osteoblast death via an apoptotic mechanism. At the same time, the possible signal-transducing mechanisms of osteoblast death had also been evaluated in our lab (Chen et al, 2002, Chen et al., 2005a, 2005b, and Ho et al., 2005). In the study of NSC95-2314-B-038-029-MY3, we further showed that pretreatment with low nitric oxide (NO) can protect osteoblast from oxidative stress-induced apoptotic insults possibly through regulation of survival gene Bcl-2 and Bcl-XL expressions and their protein translocations from the cytoplasm to mitochondria (Chang et al., 2006; Tai et al., 2007). Recently, our transcriptional analyses showed that transcription factors Runx2 and GATA-3 were involved in gene regulation of Bcl-2 and Bcl-XL (Chang et al., 2008; Ho et al., 2008). In addition to transcriptional regulation, MicroRNAs (miRNAs), which are 22 nucleotides long, have been reported to inhibit translation or promote mRNA degradation by annealing to complementary sequences in the 3′-untranslated regions (3’-UTR) of specific target mRNAs. In response to stimulation of oxidative stress, previous studies have shown that miRNAs can be differentially expressed and contribute to the control of cell survival or death. Our preliminary results showed that oxidative stress can induce miR-210 in osteoblasts. A variety of bone marker genes are involved in regulation of osteoblast metabolism. miRNA may target these bone marker gene expression and regulate bone metabolism. Meanwhile, roles of miRNAs in regulation of osteoblast survival and metabolism and their possible molecular mechanisms are still unknown. This is a 3-year-period project which is specified to evaluate the roles of miRNAs in regulation of osteoblast survival and metabolism using primary neonatal rat calvaria osteoblasts and human osteosarcoma MG63 cells as the experimental models. We hypothesize that miRNAs can contribute to the regulation of osteoblast survival and metabolism through repressing mRNA translation following binding to the complementary elements in the 3’-UTR of apoptosis-related and marker gene mRNAs. To verify our hypothesis, the sub-hypothesis and its specific aims for each year’s sub-project are designed and described as below: Sub-hypothesis-1: During osteoblast survival and metabolism, miRNAs can regulateapoptosis-related and bone marker gene expression. Specific Aims: 1. To determine the differential expressions of miRNAs in osteoblasts exposed to oxidative stress (death agent), low NO pretreatment (survival agent), or dexamethasone (differentiated agent) 2 using miRNAmicroarrays. 2. To evaluate the differential expressions of apoptosis-related and the bone marker genes in osteoblasts exposed to oxidative stress (death agent), low NO pretreatment (survival agent), or dexamethasone (differentiated agent) using cDNA microarrays. 3. To confirm the expressions of these miRNAs, and apoptosis-related and the bone marker genes in osteoblasts using real-time PCR analyses. 4. To validate the correlations among the expressions of miRNAs and apoptosis-related and the bone marker genes in osteoblasts using bioinfomatic approaches. Sub-hypothesis-2: The molecular mechanisms of miRNA-involved regulation of apoptosis-related and the bone marker genes occur through the binding to the complementary elements in the 3’-UTR of mRNAs, and consequently leading to the repression of mRNA translation. Specific Aims: 1. To determine the specificity of certain miRNAs to regulation of the target mRNAs by: a) designing the wild-type and mutant antisense oligonucleotides of specific miRNAs. b) transfecting these antisense oligonucleotides into osteoblasts to inhibit the expression of specific miRNAs. c) after knocking-down the specific miRNA expression, measuring the rates of that mRNA translation to determine if a miRNA specifically targets that mRNA expression. 2. To determine the transactivation activity (binding affinity) of a specific miRNA to its target mRNA by: a) constructing the wild-type and mutant miRNA-binding elements in the 3’-UTR of the target mRNAs. b) cloning these wild-type and mutant elements into reporter plasmids. c) transfecting these reporter plasmids into osteoblasts and detecting the luciferase activity. Sub-hypothesis-3: miRNAs participate in the regulation of osteoblast survival and metabolism through targeting mRNA translation of apoptosis-related and marker genes. Specific Aims: 1. To determine if miRNAs can regulate osteoblast survival via downregulation of mRNA translation of apoptosis-related genes using the miRNA inhibition strategy. 2. To evaluated the effects of differentiated agents or survival agents on osteoblast metabolism using the mineralization assays (von Kossa and Alizarin red staining). 3. To determine if miRNA can regulate osteoblast metabolism through suppression of mRNA translation of the bone marker genes. 4. To validate the roles of miRNA-targeting the bone marker genes in regulation of osteoblasts metabolism using RNA interference technique.