Western Blot validation of microarray results for key genes.

<p>Validation at the protein level by Western Blot was made at different time terms (basal, 24 hr, 3, and 7 days). Western blot demonstrated protein expression was higher in CEMP1 expressing cells than in controls.</p

CEMP1 Induces Transformation in Human Gingival Fibroblasts

<div><p>Cementum Protein 1 (CEMP1) is a key regulator of cementogenesis. CEMP1 promotes cell attachment, differentiation, deposition rate, composition, and morphology of hydroxyapatite crystals formed by human cementoblastic cells. Its expression is restricted to cementoblasts and progenitor cell subpopulations present in the periodontal ligament. CEMP1 transfection into non-osteogenic cells such as adult human gingival fibroblasts results in differentiation of these cells into a “mineralizing” cell phenotype. Other studies have shown evidence that CEMP1 could have a therapeutic potential for the treatment of bone defects and regeneration of other mineralized tissues. To better understand CEMP1’s biological effects in vitro we investigated the consequences of its expression in human gingival fibroblasts (HGF) growing in non-mineralizing media by comparing gene expression profiles. We identified several mRNAs whose expression is modified by CEMP1 induction in HGF cells. Enrichment analysis showed that several of these newly expressed genes are involved in oncogenesis. Our results suggest that CEMP1 causes the transformation of HGF and NIH3T3 cells. CEMP1 is overexpressed in cancer cell lines. We also determined that the region spanning the CEMP1 locus is commonly amplified in a variety of cancers, and finally we found significant overexpression of CEMP1 in leukemia, cervix, breast, prostate and lung cancer. Our findings suggest that CEMP1 exerts modulation of a number of cellular genes, cellular development, cellular growth, cell death, and cell cycle, and molecules associated with cancer.</p></div

Time series expression pattern of key genes.

<p><b>Expression of CDH1 before and after RNAi of CEMP1.</b> Expression patterns of the key genes were obtained in an independent time series experiment at 3, 6, 12, 24, 48, hours and 3, 7 and 14 days. The major expression changes as a result of CEMP1 overexpression initiated 24hrs (A). The relationship between CEMP1 and CDH1 was evaluated by RT-qPCR before and after knockdown of CEMP1 expression by RNAi. The RNAi directed against CEMP1 resulted in a significant decrease of CDH1 expression (B).</p

CEMP1 is expressed on cancer cells.

<p>CEMP1 expression screening on a variety of human cancer cell lines by qRT-PCR. the mean ± standard deviations are presented (A). Western Blot was developed and Fold of CEMP1 expression compared against cementoblastic-like cell line (CEM) is presented (B).</p

Genome-wide expression profiles HGF-CEMP1 vs. HGF.

<p>Total RNA was extracted from HGF and HGF/CEMP1 cell lines. Results from 3 independent experiments were subjected to a microarray analysis. (A). Volcano plot of 1039 differentially expressed genes between HGF-CEMP1 vs. HGF controls. Genes were selected on the basis of the significance of the differential gene expression (vertical red line; <i>P</i> < 0.001) and the level of induction or repression (horizontal red line; fold-change ≥ 2). (B). Heat-map of genes differentially expressed in microarray analysis.</p

Expression of CEMP1 in HGF/CEMP1.

<p>The expression of CEMP1 after transfection in HGF and HGF/CEMP1 was evaluated using quantitative real time PCR at 3, 7 and 14 days. Fold increase of CEMP1 expression are represented, the graph shows that expression was higher in HGF/CEMP1 in all time points. * p = 0.0001 (A). CEMP1 protein expression levels were corroborated by western blot assays (B).</p

RT-qPCR validation set.

<p>RT-qPCR validation set.</p

CEMP1 induces anchorage-independent growth in NIH3T3.

<p>CEMP1 induces anchorage-independent growth in NIH3T3.</p

CEMP1 induces anchorage-independent growth in HGF cells.

<p>Cells were plated in a soft agar assay and the number of colonies was evaluated 21 days after the seeding. Representative plates of HGF, HGF/CEMP1 and HGF/CEMP/RNAi are presented. RT-qPCR showed that RNAi against CEMP1 decreased its expression in more than 50% at transcriptional level (A). All soft agar assays were performed in triplicates, and the mean ± standard deviations are presented (B and C).</p

Functional analysis of differentially expressed HGF-CEMP1 genes.

<p>Ingenuity pathway analysis showed Diseases and Bio-Functions significantly modulated by CEMP1 expression in HGF/CEMP1 cells (p < 0.05). A total of 1,390 genes were differentially expressed: 260 were up-regulated and 779 were down-regulated. The most significant diseases (A) and bio-functions (B) are shown. Genes that met the p-values <0.05 threshold were associated with biological functions or diseases in the Ingenuity Pathway knowledge. Fischer's exact test was used to calculate the p-value to determine the probability that each biological function/disease assigned to the data set is not due to chance alone.</p