| BackgroundPancreatic cancer is one of the most malignant tumors, and is called "silence killer" due to its low rate of early diagnosis. Most patients lost the opportunity for surgical treatment when they were diagnosed. About ninety percent patients relapse after surgical treatment, pancreatic cancer have a median survival of only4-5months and a very low5-year survival rate. Unfortunately, pancreatic cancer shows obvious chemoresistance, there were no effective interventions against pancreatic cancer so far. Gemcitabine, as the first-line treatment of pancreatic cancer, only prolonged the median survival period for a few months. Therefore it is imperative to find an effective therapic strategy against pancreatic tumor.Researches indicate that human membrane mucin MUC4participated in the development of pancreatic cancer, MUC4has been reported in various cancers and inflammatory diseases; it has aberrant expression in pancreatic adenocarcinoma and pre-cancerous pancreatic intraepithelial neoplasias but minimal expression in normal pancreatic tissue and in chronic pancreatitis. High molecular weight makes it difficult to study the molecule mechanisms of MUC4associated in pancreatic cancer. One of the characteristic structures of MUC4is that it has24transcription variants, which only occurs in pancreatic cancer. The other one comes from the perspective of evolution progression of MUC4, the ancestral proteins which the structure domain evolve from (NIDO and vWD domain evolved from a common precursor to Susd2protein, AMOP and EGF-like domain evolved from a common progenitor to the nidogen protein) evolved conservative, particularly for tandem repeat domain (TR), it does not crossover with other structure domain in MUC4spatial structure.MUC4/Y, as one natural transcript of MUC4, lacks exon2compared to foil length transcript of MUC4, and the decrease of molecular weight allows us make it up-regulated in cell lines with lentivirus system. MUC4/Y still remains the N-terminal as well as the transmembrane and cytoplasmic domains. In this condition, whether MUC4/Y still has certain biological function remains unknown, investigation on MUC4/Y may help us to better understand the role of MUC4play in pancreatic cancer.ObjectiveIn this study, we will investigate the mRNA expression levels of MUC4/Y in pancreatic cancer cells, including subcellular localisation of MUC4/Y in MIApaca-2cell and the effect of MUC4/Y on subcellular structure and cell morphology, we will focus on the effect of MUC4/Y on cell proliferation and cell apoptosis, associated molecular mechanism also included.Methods1. Real time PCR was used to detect the expression of MUC4/Y in pancreatic cancer cell lines, MUC4/Y up-regulated cell model was established by a lentivirus system2. Immunohistochemistry, western blot, Real time PCR, cell immunofluorescence were used to confirm subcellular localisation and the up-expression of MUC4/Y in MIApaca-2cell lines, cell proliferation was detected by CCK-8and cell apoptosis was investigated by flow cytometry analysis.3. Apoptosis associated signaling pathways were studied by western blot Results1. The expression level of MUC4/Y was high in HPAC, COLO-357and PL-45but very low in MIA-PACA and PANC-1. Stable MUC4/Y up-regulated cell model was constructed successfully.2. Cell immunofluorescence showed that MUC4/Y can anchor on the cytomembrane and cytoplasm. Formatted colony showed that MUC4/Y up-regulation affected cell morphology. Electronic speculum indicated that MUC4/Y increased the number of mitochondria. CCK-8and flow cytometry analysis indicated MUC4/Y potentiates proliferation and suppresses apoptosis3. In vivo, the role of MUC4/Y play on potentiating proliferation and suppressing apoptosis was investigated again4. AKT and JNK cell signaling pathways were activated, and the phosphorylation level of HER2, a Membrane Partner of MUC4,was slightly up-regulated by MUC4/Y.Conclusion1. MUC4/Y can anchor itself on the cytomembrane, suggesting that it can be classified into the membrane-bound forms. Cell morphology and mitochondria was affected by MUC4/Y2. MUC4/Y can potentiate proliferation and suppress apoptosis, the underlying molecular mechanism may be the activation of AKT and JNK associated signaling pathways.3. This study suggests that the presence of the transcript variant of MUC4may have its own significance. Based on this study, we can further investigate the characteristic structure domain of MUC4... |
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