Background
Esophageal cancer (EC) is one of the malignant tumors with a poor prognosis. The early stage of EC is asymptomatic, so identification of cancer biomarkers is important for early detection and clinical practice.
Methods
In this study, we compared the protein expression profiles in esophageal squamous cell carcinoma (ESCC) tissues and adjacent normal esophageal tissues from five patients through high-resolution label-free mass spectrometry. Through bioinformatics analysis, we found the differentially expressed proteins of ESCC. To perform the rapid identification of biomarkers, we adopted a high-throughput protein identification technique of Quantitative Dot Blot (QDB). Meanwhile, the QDB results were verified by classical immunohistochemistry.
Results
In total 2297 proteins were identified, out of which 308 proteins were differentially expressed between ESCC tissues and normal tissues. By bioinformatics analysis, the four up-regulated proteins (PTMA, PAK2, PPP1CA, HMGB2) and the five down-regulated proteins (Caveolin, Integrin beta-1, Collagen alpha-2(VI), Leiomodin-1 and Vinculin) were selected and validated in ESCC by Western Blot. Furthermore, we performed the QDB and IHC analysis in 64 patients and 117 patients, respectively. The PTMA expression was up-regulated gradually along the progression of ESCC, and the PTMA expression ratio between tumor and adjacent normal tissue was significantly increased along with the progression. Therefore, we suggest that PTMA might be a potential candidate biomarker for ESCC.
Conclusion
In this study, label-free quantitative proteomics combined with QDB revealed that PTMA expression was up-regulated in ESCC tissues, and PTMA might be a potential candidate for ESCC. Since Western Blot cannot achieve rapid and high-throughput screening of mass spectrometry results, the emergence of QDB meets this demand and provides an effective method for the identification of biomarkers.
Site‐specific recombinase‐mediated genetic technology, such as inducible Cre‐loxP recombination (CreER), is widely used for in vivo genetic manipulation with temporal control. The Cre‐loxP technology improves our understanding on the in vivo function of specific genes in organ development, tissue regeneration, and disease progression. However, inducible CreER often remains inefficient in gene deletion. In order to improve the efficiency of gene manipulation, we generated a self‐cleaved inducible CreER (sCreER) that switches inducible CreER into a constitutively active Cre by itself. We generated endocardial driver Npr3‐sCreER and fibroblast driver Col1a2‐sCreER, and compared them with conventional Npr3‐CreER and Col1a2‐CreER, respectively. For easy‐to‐recombine alleles such as R26‐tdTomato, there was no significant difference in recombination efficiency between sCreER and the conventional CreER. However, for alleles that were relatively inert for recombination such as R26‐Confetti, R26‐LZLT, R26‐GFP, or VEGFR2flox/flox alleles, sCreER showed a significantly higher efficiency in recombination compared with conventional CreER in endocardial cells or fibroblasts. Compared with conventional CreER, sCreER significantly enhances the efficiency of recombination to induce gene expression or gene deletion, allowing temporal yet effective in vivo genomic modification for studying gene function in specific cell lineages.
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