Investigation Into The Functional Significance Of Precusor Messenger RNA Processing Factor19(PRP9) Gene Upregulation In Human Lung Adenocarcinoma

Introduction:Cancer is the uncontrolled or abnormal division of cells and is named according to the cells, tissue or organ concerned. Cancer cells have the ability to migrate from their site of origin via blood and lymph to invade distant tissues, with potentially lethal repercussions if the spread is not controlled. Of all human cancers, lung cancer is the most frequently diagnosed and is responsible for the greatest proportion of annual global cancer-related mortalities. Adenocarcinoma is the most common lung cancer histological subtype and accounts for approximately40%of all lung cancer cases. Early diagnosis and efficient treatment regimens for the disease have historically not been very successful necessitating intensified research into new areas, including biomarker identification, to ameliorate the situation.Precursor messenger RNA Processing Factor19(PRP19) encodes a55kDa protein, PRP19, with ubiquitous nuclear and cytoplasmic subcellular distribution. PRP19is present in prokaryotic and eukaryotic cells as part of the NineTeen Complex (NTC) and the PRP19/Cdc5Complex, respectively and interacts with other proteins such as Cell division cycle5-like protein (Cdc5L), Werner Syndrome RecQ Helicase-like (WRN) and Spliceosome-associated protein factor27(Spf27). PRP19functions in;(1) mRNA splicing,(2) ubiquitination,(3) DNA damage repair,(4) transcription elongation and (5) structurally supporting cellular nuclear framework. PRP19has been implicated in many diseases including cancers. For instance, development of certain cancers has been linked to compromised or defective ubiquitin-proteasome activity and to aberrant splicing stemming from mutations that alter the gene’s E3-ligase activity. This leads to dysregulation of important signaling pathways and/or the altered degradation of critical cell cycle protein regulators culminating in imprudent potentiation of oncoproteins and other cell growth regulators such us NF-κB, C-Myc, N-Myc and Src-like proteins. It may also result in unwarranted degradation or suppression of the functions of tumor suppressor proteins such as p53and p27. PRP19upregulation has been demonstrated in vitro and also in whole organism models to have beneficial effects, including anti-aging and life span extension properties. These pro-survival activities hinge on PRP19’s DNA damage repair ability. Upregulated PRP19expression in vitro led to increased resistance to apoptosis and increased p53phosphorylation, ultimately resulting in an extended cellular life span, whereas cells with depleted PRP19showed significantly diminished resistance to apoptosis.Methods:Employing immunohistological, western blotting (using anti-PRP19antibody) and real-time PCR approaches, the presence, subcellular localization and abundance of PRP19in tumor and non-tumor lung tissues were assessed. In vitro experimental models typifying in situ PRP19overexpression were generated among A549adenocarcinoma cells via plasmid transfection. The transfected cells were then subjected to several in vitro and in vivo cell viability and tumorigenicity assays to assess the impact of PRP19overexpression in cells on proliferation, migration, invasion, cytotoxicity resistance and apoptosis. Control groups were A594cells transfected with the empty vector and an untransfected A549cell group.Results and Discussion:PRP19had a ubiquitous intracellular distribution regardless of disease state with higher expression in tumor tissues than non-tumor tissues. Tissue microarray results from a total of172lung tissue samples (66primary tumors,53metastatic tumors and53normal tissues) showed elevated PRP19expression in tumor tissues (78%) compared with their non-tumor counterparts (43%) with P-value<0.001. There was a20-fold increase (P-value<0.001) in PRP19mRNA in lung tumor tissues compared with non-tumor tissues. Cells overexpressing PRP19showed downregulated p53expression and upregulated p21expression compared with controls with the former suggesting lowered genomic damage and the latter promoting cell growth retardation. In Transwell(?) migration, CCK-8proliferation and clonogenic assays, PRP19-overexpressing cells showed considerably reduced rates of migration and proliferation compared to control groups. Similarly in in vivo tumorigenicity assay, mice that received A549-pEX-PRP19(test group) cells were last to develop tumors, at25±1(SE) days post-inoculation, whereas the control groups developed tumors at18±1.4(SE) days post-inoculation (P-value=0.021). At the end of6weeks post-inoculation, mice in the test group had smaller xenograft tumors [164±79(SE) mm3] than those of the control groups [562±173(SE) mm3].PRP19overexpression also delayed the onset of apoptosis and significantly retarded its progress. At24hours post-cisplatin treatment in a DAPI experiment, control groups had larger populations of cells in both Stage1and Stage2of apoptosis than test group. By48hours, an average of76.6±2.8%of cells of the control groups was in various stages of apoptosis with the predominant being Stage2and Stage3of apoptosis against59.3±2.1%of test group cell being apoptotic with a balanced distribution among Stage1and Stage2of apoptosis and a minimal number in Stage3of apoptosis. Similarly in TUNEL assay, test group had reduced numbers of TUNEL positive (apoptotic) cells compared with control groups at various experimental time points. At the end of48hours of cisplatin treatment, control groups had an average of84±11(SD) TUNEL positive cells per microscope field compared to that of45±8cell per field for the test groups taken at the same magnification (P-value±0.001).Further test by western blotting on key proteins involved in apoptosis regulation revealed unaltered Bax expression in test and control groups but elevated Bcl-2expression and diminished Caspase3expression in test group against control groups. This can explain the lowered apoptotic rate in test group since Bcl-2antagonizes apoptotic. Also, caspase3is an apoptosis executioner protein and its reduced abundance in test groups cells compared with cells of control groups further underpins the observed trends in apoptosis among experimental groups.Conclusion:PRP19is overexpressed in human lung tumor tissues compared with their non-tumor counterparts. Upregulated PRP19expression enhances cell viability and reduces the tumorigenic potential in lung adenocarcinomas. This occurs by suppressing cell proliferation and tumor growth through p21-mediated cell cycle arrest and impeding apoptosis to allow more time for DNA damage repair.