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Analysis on Molecular Chaperone BiP Domain and Influence on Proliferation and Apoptosis in Hepatoma Cells
Li Xiangzhu, Liu Yanna, Zhao Wenjun, Zhou Jinghua, Guo Fengjin*
Department of Cell Biology and Genetics, Chongqing Medical University, Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, China
Abstract: Binding immunoglobulin protein (BiP) is a resident member of Hsp70 family and master regulator for Endoplasmic Reticulum Stress (ERS). To study the relationship of Bip domain with the physiological function， BiP DNA sequence and protein structure were analysed， two deletion mutants of BiP and BiP full-length eukaryotic expression vectors with myc tag were successfully constructed with the method of overlapping PCR mutagenesis respectively. BiPa deletion mutant is BiP mutant with ATPase domain deleted， BiPp deletion mutant is BiP mutant with peptide-binding domain deleted. After these eukaryotic expression vectors were transfected into LO2 and SMMC-7721 cells， they were identified the expression by Western blot. Then cell proliferation and apoptosis were analysed by MTT assay， BrdU immunohistochemistry and flow cytometry (FCM) respectively， next the relationship between cell proliferation/apoptosis and BiP/BiPa/BiPp were also analysed. Both the full-length BiP and the deletion mutants BiPa， BiPp can effectively improve SMMC-7721 cells proliferation by MTT assay and BrdU immuno-histochemistry. Besides， every group of cell proliferation rate results showed that ATPase domain might be inhibit cell growth， while peptide-binding domain might be increase cell growth. FCM results showed that both the full-length BiP and the deletion mutants BiPa， BiPp can enhance SMMC-7721 cells apoptosis. The difference of apoptosis rate between group BiPa and group BiP were no significance， however， the apoptosis rate of group BiPp was clearly lower than group BiP (P<0.05). It was showed that ATPase domain might be unconcerned with cell apoptosis， while peptide-binding domain might be increase cell apoptosis. These two domains， ATPase and peptide-binding， can coregulate cell proliferation and cell poptosis when they combined with each other. The molecular mechanism of them need to be studied deeply.
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