NFIX as a Master Regulator for Lung Cancer Progression

Abstract

About 40% of lung cancer cases globally are diagnosed at the advanced stage. Lung cancer has a high mortality and overall survival in stage I disease is only 70%. This study was aimed at finding a candidate of transcription regulator that initiates the mechanism for metastasis by integrating computational and functional studies. The genes involved in lung cancer were retrieved using <i>in silico</i> software. 10 kb promoter sequences upstream were scanned for the master regulator. Transient transfection of shRNA NFIXs were conducted against A549 and NCI-H1299 cell lines. qRT-PCR and functional assays for cell proliferation, migration and invasion were carried out to validate the involvement of <i>NFIX</i> in metastasis. Genome-wide gene expression microarray using a HumanHT-12v4.0 Expression BeadChip Kit was performed to identify differentially expressed genes and construct a new regulatory network. The <i>in silico</i> analysis identified <i>NFIX</i> as a master regulator and is strongly associated with 17 genes involved in the migration and invasion pathways including <i>IL6ST</i>, <i>TIMP1</i> and <i>ITGB1</i>. Silencing of <i>NFIX</i> showed reduced expression of <i>IL6ST</i>, <i>TIMP1</i> and <i>ITGB1</i> as well as the cellular proliferation, migration and invasion processes. The data was integrated with the <i>in silico</i> analyses to find the differentially expressed genes. Microarray analysis showed that 18 genes were expressed differentially in both cell lines after statistical analyses integration between <i>t</i>-test, LIMMA and ANOVA with Benjamini-Hochberg adjustment at <i>p</i>-value < 0.05. A transcriptional regulatory network was created using all 18 genes, the existing regulated genes including the new genes <i>PTCH1</i>, <i>NFAT5</i> and <i>GGCX</i> that were found highly associated with <i>NFIX</i>, the master regulator of metastasis. This study suggests that <i>NFIX</i> is a promising target for therapeutic intervention that is expected to inhibit metastatic recurrence and improve survival rate.