Genome Wide Expression Profiling of Cancer Cell Lines Cultured in Microgravity Reveals Significant Dysregulation of Cell Cycle and MicroRNA Gene Networks

<div><p>Zero gravity causes several changes in metabolic and functional aspects of the human body and experiments in space flight have demonstrated alterations in cancer growth and progression. This study reports the genome wide expression profiling of a colorectal cancer cell line-DLD-1, and a lymphoblast leukemic cell line-MOLT-4, under simulated microgravity in an effort to understand central processes and cellular functions that are dysregulated among both cell lines. Altered cell morphology, reduced cell viability and an aberrant cell cycle profile in comparison to their static controls were observed in both cell lines under microgravity. The process of cell cycle in DLD-1 cells was markedly affected with reduced viability, reduced colony forming ability, an apoptotic population and dysregulation of cell cycle genes, oncogenes, and cancer progression and prognostic markers. DNA microarray analysis revealed 1801 (upregulated) and 2542 (downregulated) genes (>2 fold) in DLD-1 cultures under microgravity while MOLT-4 cultures differentially expressed 349 (upregulated) and 444 (downregulated) genes (>2 fold) under microgravity. The loss in cell proliferative capacity was corroborated with the downregulation of the cell cycle process as demonstrated by functional clustering of DNA microarray data using gene ontology terms. The genome wide expression profile also showed significant dysregulation of post transcriptional gene silencing machinery and multiple microRNA host genes that are potential tumor suppressors and proto-oncogenes including <i>MIR22HG</i>, <i>MIR17HG</i> and <i>MIR21HG</i>. The <i>MIR22HG</i>, a tumor-suppressor gene was one of the highest upregulated genes in the microarray data showing a 4.4 log fold upregulation under microgravity. Real time PCR validated the dysregulation in the host gene by demonstrating a 4.18 log fold upregulation of the miR-22 microRNA. Microarray data also showed dysregulation of direct targets of miR-22, <i>SP1</i>, <i>CDK6</i> and <i>CCNA2</i>.</p></div

Quantitative PCR analysis for changes in mRNA expression of significant, candidate genes involved in cell proliferation and cancer.

<p><b>A</b><i>CDK1</i>—Cell cycle kinase gene, <i>CD117</i>—proto-oncogene, <i>JUNB</i>—transcription factor and immediate early gene, <i>MYC</i>—proto-oncogene expression in DLD-1 and MOLT-4 <b>B</b> Real time PCR analysis in HL-60 <i>CCNE1</i> and <i>CDK2</i>, <i>CCNB1</i> and <i>CDK1</i>, Oncogenes: <i>CD117</i> and <i>MYC</i>, Cancer prognostic markers <i>CD105</i>, <i>CD90</i> and <i>CD71</i>.</p

Effect of microgravity on cell morphology and cell viability of DLD-1 cell cultures.

<p><b>A</b> DLD-1 cell cultures; Static culture (control) <b>B</b> DLD-1 Microgravity culture at 16 RPM <b>C</b> DLD-1 Microgravity culture at 27RPM <b>Differential staining to detect apoptotic population D</b> DLD-1Static monolayer cultures <b>E</b> Microgravity cultures of DLD1 at 16 RPM <b>F</b> Microgravity cultures of DLD1 at 27 RPM <b>G Cell adhesion and proliferation assay</b> Top panel—static cultures, bottom panel—microgravity cultures shifted to static TCP <b>H</b> Morphological changes in DLD-1; Crystal violet staining of DLD-1 cells in static monolayer culture <b>I</b> Crystal violet staining of DLD-1 cells after transfer of cell aggregates from microgravity to TCP <b>J</b> Colony forming ability assay; Static cultures <b>K</b> Colony forming ability assay; DLD-1 cells after transfer of cell aggregates from microgravity to TCP</p

Dysregulation of stem cell marker CD44 and tumor suppressor microRNA under microgravity.

<p><b>A</b> Validation of microarray data for <i>CD44</i> by Western blotting for CD44 protein and beta-actin in static and Microgravity (μG) cultures of DLD1 and MOLT-4 and Densitometric analysis of western blots. <b>B</b> MIR22HG expression under microgravity; Overexpressed MIR22HG, host gene of miR-22 microRNA in DLD-1 cells under microgravity, MOLT-4 cells show no differential expression; Levels of dysregulation of direct targets of miR-22 microRNA <i>CDK6</i>, <i>CCNA2</i>, <i>SP1</i> and <i>CDKN1A</i> in microarray data; RT-PCR validation of microRNA miR-22 levels and target genes in DLD-1 shows over expressed microRNA miR-22 in DLD-1 cells under microgravity confirming upregulation in microarray data. No significant dysregulation of direct targets <i>CDKN1A</i> (similar to expression levels in microarray data) and <i>CCND1A</i><b>C</b> GO analysis (by DAVID) of microarray data to depict other dysregulated microRNA host genes in DLD-1 and MOLT-4 cells under microgravity.</p

Functional annotation of microarray data using DAVID.

<p><b>A</b> Functional annotation of upregulated and downregulated genes of DLD-1 cells under microgravity <b>B</b> Functional annotation of upregulated and downregulated genes of MOLT-4 cells under microgravity.</p

Effect of microgravity on gene expression levels in DLD-1 and MOLT-4 cell cultures; Validation of microarray analysis by real time PCR and western blotting.

<p><b>A</b> Log fold change of <i>CCNB1</i>, <i>ROMO1</i> and <i>HES1</i> deregulation in MOLT-4 cells under microgravity as observed by microarray analysis validated by real time PCR <b>B</b> Log fold change of <i>CDK2</i>, <i>HEY1</i> and <i>STAT3</i> deregulation in DLD-1 cells under microgravity as observed by microarray analysis validated by real time PCR <b>C</b> Log fold change of commonly up and downregulated genes <i>CCNE1</i>, <i>TFRC (CD71)</i> and <i>CD44</i> as observed by microarray analysis validated by real time PCR for<i>CCNE1 and TFRC (CD71)</i>.</p

Effect of microgravity on cell viability and cell cycle of DLD-1 and MOLT-4 cell lines.

<p><b>A</b> Cell viability assay for DLD-1 cells; Viability measured for microgravity cultures (16 RPM and 27 RPM) and static cultures using MTT <b>B</b> Cell cycle analysis for DLD-1 cells; Static <b>C</b> Cell cycle analysis; Microgravity <b>D</b> Cell cycle analysis; Static suspensions on agar underlays <b>E</b> The average sub G0 population in replicates of cell cycle analysis for microgravity, static and static suspension cultures of DLD-1 cells <b>F MOLT-4 cell culture</b> Static and Microgravity cultures of MOLT-4 <b>G Cell viability assay</b> Viability measured for microgravity cultures (16 RPM and 27 RPM) and static cultures using MTT <b>H</b> Cell cycle analysis; Static <b>I</b> Cell cycle analysis; Microgravity cultures of MOLT-4.</p

Analysis of microarray data using Gene Functional Classification and Functional Annotation; Dysregulation of genes involved in the Notch signaling system and microRNA processing and regulation.

<p><b>A</b> Regulation of transcription <b>B</b> RNA mediated gene silencing (PTGS) <b>C</b> Notch signaling pathway <b>D</b> Dysregulated microRNA processors and regulators.</p