Translational Medicine and Oncologists Meet

Hypothesis: The high exposure causes up-regulation of JUN, FOS & AP1; genes in adapted cell lines and over expression of Pseudogenes which has metastatic potential.

Objective: When subjected to high concentration of NO, H&N tumour cell express more aggressive phenotype compared to nonexposed cell. Upon exposure these cells exhibit adaptation causing greater metastatic potential. Five H&N cell lines were adapted to high concentrations of NO. It appears that AP1, which is a transcription factor protein, composed of JUN and FOS family proteins.These proteins are responsible for various cellular process including cell differentiation, proliferation and apoptosis which up regulate these cells. MicroRNA isolation due to CeRNA add to Pseudogenes and their influence on the growth of cancer. One well studied gene is the BRAF pseudogene and its functional BRAF gene. Studies have shown that high levels of the BRAF pseudogene are directly proportional to the formation of aggressive malignancies.

Methods: This study used five human H&N cells lines (SSC-016, SSC-040, SSC-056, SSC-114, and SSC-116). Known pseudogenes were identified in each line, as well as their coding counterparts.Slow exposure of high NO was used on the cell lines to increase quantities of DETA-NONOate (NO donor). Both the parent and NO cell lines were tagged with red/green fluorescent markers and mRNA was isolated. A gene chip analysis was used to assess genome wide gene expression. Via scratch assays cell migration rates were assessed. Within these five cell lines JUN, FOS, and AP1 genes were up-regulated when exposed to high NO. Increased migration velocities was demonstrated among all three genes.

Results: The adenocarcinoma cell lines RP6-159A1.2, RP11-255N24.3, AC004490.1, LDHBP, RP11-572H4.2 were down regulated pseudogenes, and there was no up regulated pseudogenes. The squamous cell carcinomas (SCCs) had the following up regulated pseudogenes: RPL37AP1, AC138972.1, RP11-641D5.1, AC005534.6, AC022431.1, RPL26P12, and they had these down regulated pseudogenes: RP6-159A1.2, RP11-255N24.3, RBMXP1, RP11-20O23.1, RP11-551G24.2. All cell lines showing an increase in a pseudogene expression indicating an increase in the corresponding gene (with the exception of the adenocarcinoma cell lines). JUN,FOS and AP1 genes showed increased migration velocities with up- regulation compared to the parent cell lines.

Conclusions: The high concentration of CeRNA may reduce expressions of microRNA, which would then lead to high concentrations of pseudogenes (likely due to high levels of HNO). Pseudogenes, along with BRAF, in turn reduce the expression of microRNA. Therefore, the pseudogenes and BRAF take the same role as the CeRNA. This results in a feedback loop of over expression of the coding gene.Within these cell lines JUN, FOS, and AP1 genes had an increased migration velocities which demonstrated an increased tumour aggressiveness.