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Pancreatic adenocarcinoma (PAC) is one of the most lethal cancers. Only about 5 % of the person with pancreatic adenocarcinoma may survive up to a maximum of 5 years. Therefore, a novel approach to pancreatic adenocarcinoma diagnosis and treatment is required to improve the clinical outcome of the PAC patients.
Isobaric tags for relative and absolute quantitation (iTRAQ) in combination with two-dimensional liquid chromatography-tandem mass spectroscopy (2D LC-MS/MS) is a powerful tool that is frequently used in the quantitative proteomic analysis. During this procedure, iTRAQ reagent is used to label the primary amines of the peptides obtained from the trypsin digesting. There are four to eight different tags of the iTRAQ reagent that provides multiple tagging.
In this study, researchers investigated the overexpression of a membrane-associated protein, myoferlin that is required for muscle development and regeneration. Evidence from the immunohistochemical studies from Human Protein Atlas reveals that myoferlin is overexpressed in most cancers such as colorectal cancer, ovarian, cervical, pancreatic and liver cancers.
Therefore, researchers performed experimental validation to demonstrate the role of myoferlin as a pancreatic adenocarcinoma prognosis and its role in the regulation of the cancer cell invasion revealing that depletion of the myoferlin in the breast cancer promotes the differentiation of the mesenchymal cells to the epithelial shape and stall invasion.
Researchers examined additional 154 patients with pancreatic adenocarcinoma to determine the role of overexpression of the myoferlin in the pancreatic adenocarcinoma prognosis. Furthermore, they performed RNA interference-mediated knockout of the gene encoding myoferlin and they found alleviation of the malignant phenotypes in vitro and in vivo.
For this study, researchers obtained 60 surgically resected tissues fro 40 pancreatic adenocarcinoma patients from the Zhongshan Hospital in between the jun-2010 to Jan-2012 and divided into three categories; low-grade PAC, high-grade PAC, and non-tumor pancreatic tissues with n=20. These samples were further divided into four groups based on the extent of glandular differentiation.
The low-grade PAC was divided into two; tumor grade-1 with well-differentiated and tumor grade-2 with moderately differentiated while high-grade PAC was divided into tumor grade-3 with poorly differentiated and tumor grade-4 with undifferentiated. Tissue samples from these 40 patients were analyzed for quantitative proteomics while an additional 154 patients from the Zhongshan Hospital between Jan-2005 and Dec-2009 with PAC were further analyzed for the myoferlin expression analysis.
Protein was extracted from the tissue sample and quantified by a 2D Quantification Kit. The equal amount of proteins from five different individuals were mixed to generate a common sample for each type the tissue sample and were subjected to trypsin digestion followed by iTRAQ labeling.
The mixture of the peptide was fractioned by HPLC based cation exchange chromatography using a polysulfoethyl column. The fractions obtained were further analyzed by mass spectrometry. Western blotting of all the individual samples was also performed using the polyvinylidene fluoride membrane and anti-myoferlin antibody.
Researchers obtained BXPC-3 and CFPAC-1 of PAC cell lines from the Shanghai Institute of life science to perform the knockout of gene encoding myoferlin (MYOF) using small interference RNA (siRNA). Researchers also performed cell cycle and apoptosis assay followed by cell proliferation assay and colony-forming assay as well as cell migration assay.
Researchers were able to identify the maximum of 2844 different proteins based on the iTRAQ experimentation and when they performed iTRAQ based experiments twice, they found a total of 1623 different proteins.
To analyze the proteins express differentially in the high-grade PAC tissue, researchers compared the protein profiles of the high-grade PAC tissue vs. low-grade PAC tissue. What they found is that 10 different proteins were significantly upregulated while 5 different proteins were downregulated in the high-grade PAC tissues as compared to the low-grade PAC tissues.
Myoferlin was the one among the upregulated proteins. This is why researchers further analyzed the level of myoferlin expression by western blotting to validate the proteomic results. As a result, researchers found that myoferlin-positive and myoferlin-negative tumor tissues were significantly different in their histology.
They further analyzed the correlation between the myoferlin with prognostic data and found that myoferlin positive PAC tissues had a significantly worse prognosis than those with the myoferlin-negative PAC tissues in terms of survival rate.
All these results reveal that myoferlin gene (MYOF) could be a good candidate target to intervene in the development of PAC. To test these further, researchers used primary and metastasis PAC cell lines such as BXPC-3 and CFPAC-1 and found that targeting the MYOF gene expression by siRNA alleviates the malignant phenotypes. Furthermore, it reduces cell proliferation, colony-forming ability, and cell migration as well as causes the cell death-inducing apoptosis.
In conclusion, iTRAQ labeling in combination with 2D LC-MS/MS can be used to identify the differentially expressed proteins in the high-grade pancreatic adenocarcinoma tissues as compared with other tissues. Myoferlin is a membrane-associated protein essential for muscle growth and differentiation. Expression profiling of myoferlin can be used as a biomarker of the pancreatic adenocarcinoma prognosis.
Reference: Journal of Proteomics (iTRAQ-based quantitative proteomics reveals myoferlin as a novel prognostic predictor in pancreatic adenocarcinoma)
Article DOI: 10.1016/j.jprot.2013.06.032