Functional Expression, Purification and Identification of Interaction Partners of PACRG.

PACRG (Parkin co-regulated gene) shares a bi-directional promoter with the Parkinson's disease-associated gene Parkin, but the physiological roles of PACRG have not yet been fully elucidated. Recombinant expression methods are indispensable for protein structural and functional studies. In this study, the coding region of PACRG was cloned to a conventional vector pQE80L, as well as two cold-shock vectors pCold II and pCold-GST, respectively. The constructs were transformed into Escherichia coli (DE3), and the target proteins were overexpressed. The results showed that the cold-shock vectors are more suitable for PACRG expression. The soluble recombinant proteins were purified with Ni2+ chelating column, glutathione S-transferase (GST) affinity chromatography and gel filtration. His6 pull down assay and LC-MS/MS were carried out for identification of PACRG-binding proteins in HEK293T cell lysates, and a total number of 74 proteins were identified as potential interaction partners of PACRG. GO (Gene ontology) enrichment analysis (FunRich) of the 74 proteins revealed multiple molecular functions and biological processes. The highest proportion of the 74 proteins functioned as transcription regulator and transcription factor activity, suggesting that PACRG may play important roles in regulation of gene transcription.

The Development of Three-DNA Methylation Signature as a Novel Prognostic Biomarker in Patients with Colorectal Cancer.

AIMS: The prognosis of colorectal cancer (CRC) remains poor. This study aimed to develop and validate DNA methylation-based signature model to predict overall survival of CRC patients. METHODS: The methylation array data of CRC patients were retrieved from The Cancer Genome Atlas (TCGA) database. These patients were divided into training and validation datasets. A risk score model was established based on Kaplan-Meier and multivariate Cox regression analysis of training cohort and tested in validation cohort. RESULTS: Among total 14,626 DNA methylation candidate markers, we found that a three-DNA methylation signature (NR1H2, SCRIB, and UACA) was significantly associated with overall survival of CRC patients. Subgroup analysis indicated that this signature could predict overall survival of CRC patients regardless of age and gender. CONCLUSIONS: We established a prognostic model consisted of 3-DNA methylation sites, which could be used as potential biomarker to evaluate the prognosis of CRC patients.