1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the active form of vitamin D, is a key regulator of bone metabolism and osteocyte function. However, the comprehensive transcriptional programs initiated by 1,25(OH)₂D₃ in osteocytes—particularly in a time- and dose-dependent manner—remain insufficiently characterized. This gap in knowledge limits our understanding of how vitamin D signaling dynamically orchestrates osteogenic differentiation and skeletal homeostasis at the molecular level.

This project seeks to delineate the transcriptomic landscape of MLO-A5 cells following treatment with varying concentrations and durations of 1,25(OH)₂D₃. By capturing the temporal and dosage-dependent gene expression changes, this work aims to provide novel insights into the regulatory networks downstream of vitamin D and their potential implications in bone physiology and related pathologies.

MLO-A5 is a murine osteocyte-like cell line that represents a late stage of osteoblast differentiation. These cells exhibit many characteristics of mature osteocytes, including the expression of osteocyte markers, making them a valuable in vitro model for studying osteocyte biology and bone mineralization.

1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the hormonally active form of vitamin D, exerts its biological effects primarily through the vitamin D receptor (VDR), a nuclear receptor that regulates the transcription of target genes. In bone tissue, 1,25(OH)₂D₃ plays essential roles in calcium and phosphate homeostasis, osteoblast/osteocyte differentiation, and bone remodeling. However, the temporal dynamics and dose-specific transcriptional responses of osteocyte-like cells to 1,25(OH)₂D₃ are still not fully understood.

In this study, MLO-A5 cells were treated with varying concentrations of 1,25(OH)₂D₃ across multiple time points to explore how vitamin D signaling affects the global transcriptomic landscape during osteocyte differentiation.

MLO-A5 cells were treated with 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) at three concentrations (0 nM, 10 nM, and 100 nM) for two time points (6 h and 24 h), under two differentiation states: day 0 (undifferentiated) and day 6 (differentiated). This factorial design generated a total of 12 distinct treatment conditions, enabling the investigation of dose- and time-dependent transcriptional responses using Tag-Seq Data across differentiation stages, shown in the following figure. 

For the current phase of the study, transcriptomic profiling was conducted on a selected subset of samples (highlighted in orange in the schematic):

1. Day 0 – 24 h – 0 nM  

2. Day 0 – 24 h – 100 nM

This comparison allows for the focused analysis of vitamin D-induced gene expression changes in undifferentiated osteocyte-like cells following prolonged exposure to a high pharmacological dose.

In this project, I performed the following tasks:      

1. FastQC + MultiQC: Conducted quality control of sequencing data.      

2. STAR: Aligned the sequencing reads to the reference genome and generated BAM files.      

3. FeatureCounts: Created a counting matrix for gene expression analysis.      

4. DESeq2: Analyzed differential gene expression.      

5. Visualization: Visualized results to provide clear insights into the data.