Preliminary Study Of Endoplasmic Reticulum Stress And Epithelial-mesenchymal Transition Induced By Hypoxia In Human Hepatocellular Carcinoma

Endoplasmic reticulum(ER) is one of the most important organelle of eukaryotic cells. It is a net-like structure which consists of a closed membrane system and the membrane chamber. It is involved in the synthesis and assembling, secretion and translocation of proteins, etc., and plays a very important role in the cell activities. The unfolded protein response(UPR) is activated upon the accumulation of misfolded or unfolded proteins in the ER. The most sensitive sensor of ER stress glucose-regulated protein 78(GRP78), also known as binding immunoglobulin protein(BiP), dissociates from the three ER-transmembrane transducers: IRE1(Inositol-requiring transmembrane kinase/endonuclease1), PERK(PKR-like Endoplasmic Reticulum Kinase), and ATF6(Activating Transcription Factor 6)leading to their activation. PERK phosphorylates eukaryotic initiation factor 2ɑ(eIF2ɑ) resulting in global mRNA translation attenuation, and concurrently selectively increases the translation of several mRNAs, including the transcription factor ATF4, and its downstream target CHOP. IRE1ɑ autophosphory- lation activates the RNase activity to splice XBP1 mRNA to produce the active transcription factor known as sXBP1, it also recruits and activates the stress kinase JNK because of its kinase activity. ATF6ɑ transits to the Golgi compartment and is cleaved by intramembrane proteolysis to generate a new form of active transcription factor. These UPR pathways act in concert to expand the ER protein folding capacity, degrade misfolded or unfolded proteins, they also reduce the new synthetic proteins at the same time. The UPR could promote cell survival in the face of stimulations such as hypoxia, high or low glucose, calcium concentration change and nutrition deficiency etc. However, theses adaptations start to fail and apoptosis occurs with persistent and a long time ER stress.The epithelial-mesenchymal transition(EMT) is a biological process, which elongates an epithelial shape cell in order to convert into a spindle-shaped. The main features of EMT is the reduction of cadherin proteins such as E-cadherin, cytoskeletal proteins are converted to vimentin resulting in weak connection between the cells with the basement membrane, a higher invasion and migration, elevated resistance to apoptosis and the production of extra cellular matrix components.As an adaptive reaction, ER stress is considered to be related with the development and of many diseases such as nerve degeneration, infectious disease caused by virus and malignant tumors etc. And also, a growing research evidence shows that EMT makes malignant epithelial cells obtaining more invasion and migration capacities. It has been considered to play a important role in tumor development process. Often there is insufficient oxygen supply in the cancer tissues because of high metabolism and excessive proliferation in malignant solid tumors. Therefore, the main purpose of this paper is to explore the role of the hypoxia-induced ER stress and epithelial-mesenchymal transition in hepatocellular carcinoma(HCC). The preliminary experimental results have been obtained as follows:(1) Constructing the ER stress sentitive senor protein BiP plasmid and verifying its expression in different hepatoma cell lines;(2) Finding the up-regulation expression of ER related proteins and EMT in HCC tissue by immunohistochemical staining;(3)Verifying the results from the mRNA and protein levels, respectively, that the ER stress and EMT related genes increased expression in the different hepatoma cell lines;(4) It was unexpectedly found that the ectopic expression of BiP in the nucleus of HCC, and the existence of co-localization of BiP and the hypoxia-inducible factor(HIF1ɑ) in HCC tissue;(5) Hypoxia promoted the nucleus location of BiP, which was associated with the expression of HIF1ɑ;(6) It was observed within a certain time hypoxia promoted cell survival using MTT assay.These results suggest that there indeed exsit ER stress and EMT, and they were both up-regulated in HCC. Hypoxia simulated BiP nuclear translocation.