The Function Study Of A New PGRN Binding Protein-FAM76B Protein

Progranulin(PGRN)is a kind of secretory glycoprotein and consists of 593 aminoacids.The molecular weight of mature glycosylated protein is 88 kD while the protein without glycosylation is 68 kD.PGRN contains a secretory signal peptide consisting of 17 aa in N terminal followed by seven and one half granulin-like domains.From N terminal to C terminal,these domains were sequentially named as paragranulin,Granulin G,F,B,A,C,D and E.Different domains are connected by polypeptide linkers.PGRN is involved in many kinds of physiological processes,including embryonic development,wound healing,inflammation inhibition,immunoregulation,cartilage development and nutrition of the neurons.Besides,PGRN plays important roles in many pathological processes,including obesity,insulin resistance and neurodegeneration etc..But mechanisms mediating biological functions of PGRN are still not clear at present.In previous study,FAM76B,a new protein interacting with PGRN,was found by our lab through screening of yeast two hybrid system.And the interaction between PGRN and FAM76B has been confirmed by several experiments,including Co-IP,GST-Pull down and cellular co-localization staining in vitro.At present,the function of FAM76B is still unknown,but FAM76B as a PGRN interacting protein,may have the similar biological functions with PGRN theoretically.Therefore,based on the previous studies in this laboratory,this project on one hand explored the function of FAM76B in tumor drug resistance,cell survival,cell senescence and proliferation in vitro.On the other hand the functions of FAM76B and PGRN in the inflammation mediated by macrophage were investigated.These studies would provide ceratin help for explicating the molecular mechanism of functional diversity of PGRN.At same time,the processing,distribution and relationship with FAM76B co-localization of PGRN in cell under different conditions were explored and the possible mechanism of change of PGRN distribution in cell was analysed.Based on the above studies,not only the biological functions of FAM76B could be further understood,the foundation for the research to elucidate molecular mechanism mediating PGRN biological functions was laid.The specific contents of this research include the following 5 aspects.(1)Establishment of HEK293 cell line with FAM76B gene knockout.The sgRNA against FAM76B was designed to knockout FAM76B gene in HEK293 cell using the CRISPR/Cas 9 system.After cell cloning,the HEK293 cell line with FAM76B knockout was detected by PCR and confirmed by DNA sequencing and WB.(2)Study of the function of FAM76B in breast cancer resistance to Tamoxifen.Based on MCF-7 cell lines stably overexpressing FAM76B or PGRN established by lentiviral vector expressing FAM76B or PGRN,using the promoter activity reporter vector of luciferase expression controlled by estrogen response element,the synergistic enhancing effect of FAM76B or PGRN on estrogen was studied on one hand,on the other hand the effect of FAM76B or PGRN on antagonistic action of Tamoxifen on estrogen was also examined.At the same time,the effect of FAM76B or PGRN on the expression of Bcl-2,an apoptosis inhibitor gene,was analysed in the process of apoptosis induced by Tamoxifen.(3)Study of the function of FAM76B in cell senescence and proliferation.Using the MEF cell and HEK293 cell line with FAM76B gene knockout,the function of FAM76B in MEF senescence was determined by staining for senescence associated beta galactosidase activity,and the effect of FAM76B knockout on the proliferation of MEF and HEK293 cell was examined by MTT method.(4)Study of the function of FAM76B in cell survival.The U87 and SHSY5Y cells with high endogenous expression of PGRN,the U87 and SHSY5Y cells stably overexpressing exogenous FAM76B and HEK293 cells with FAM76B knockout were chosen respectively and then subjected to different concentration of H2O2 to induce cell death.The effect of FAM76B on cell death induced by H202 in U87,SHSY5Y and HEK293 cells was explored by morphology observation and MTT method.(5)Study of the function of FAM76B and PGRN in inflammation mediated by macrophages.The U937 cells stably overexpressing exogenous FAM76B or PGRN and co-overexpressing exogenous FAM76B and PGRN were chosen and treated with PMA only or combined with LPS/IFNy or IL4/IL13.And then the effect of FAM76B or PGRN overexpression on the expression of IL6,TNFa,PTGS2 and IL10 was examined by real time PCR.Meanwhile the cellular localization of FAM76B and PGRN in the process of differentiation induced by treatment with PMA only or combined with LPS/IFNy was examined by immunofluorescence staining and the mechanism was also explored,which would lay the foundation for the study on the mechanism of FAM76B and PGRN function in inflammation mediated by macrophages.Through the above studies,the results of this subject were obtained as follows.(1)In the study of FAM76B gene knockout in HEK293,the results of PCR and DNA sequencing indicated that two alleles of FAM76B were inserted with exogenous DNA fragment with different lengths,respectively.And the result of WB also showed that the expression of endogenous FAM76B was undetected in cells.The results suggested that the HEK293 cell lines with both of two alleles of FAM76B knockout was obtained successfully.(2)In breast cancer cell line MCF-7,overexpression of PGRN could enhance the estrogen effect synergistically,inhibit the antagonistic effect of Tamoxifen on estrogen and increase the expression of apoptosis inhibitor gene Bcl-2.But overexpression of FAM76B had no effects on estrogen effect and function of Tamoxifen.(3)In MEF cells,complete deficiency of FAM76B could accelerate cell senescence,but also inhibit the MEF cell proliferation.But there was no significant difference between the chimeric MEF cell with FAM76B knockout and the wildtype MEF cell in mediating cell senescence and proliferation.The FAM76B gene knockout in HEK293 cell could also inhibit cell proliferation.(4)In U87 and SHSY5Y cells,overexpression of FAM76B could enhance the tolerance to cell death induced by H2O2 and increase cell survival.But in HEK293 cell,the knockout of FAM76B had no effects on cell survival while being treated with H2O2-(5)In U937 cells,overexpression of FAM76B or PGRN could inhibit the expression of proinflammatory cytokines IL6,TNFa and PTGS2 induced by PMA combined with LPS/IFNy.And overexpression of PGRN could also increase the expression of anti-inflammatory cytokine IL10 while FAM76B had no this function.Meanwhile overexpression of FAM76B or PGRN could not change the expression of proinflammatory cytokines IL6,TNFa and PTGS2 when being treated with PMA combined with IL4/IL13 but could inhibit the expression of anti-inflammatory cytokine IL10 to some extent.These results suggested that FAM76B and PGRN could inhibit inflammation by suppressing the expression of proinflammatory cytokines and PGRN could also increase the anti-inflammatory cytokine to inhibit inflammation.These two genes could regulate the dynamic balance between the proinflammatory and anti-inflammatory cytokines expression.Based on the above results,further validation experiments in HEK293 cells showed that knockout of FAM76B could increase the hIL6 and hPTGS2 promoter activity.Besides,overexpression of PGRN or FAM76B could inhibit apoptosis of U937 cells induced by treatment with PMA combined with LPS/IFN? by inhibiting activation of Caspase 3 and increasing expression of Bcl-2.(6)After being treated with PMA,endogenous FAM76B was mostly localized in cytoplasm in addition to nucleus of U937 cell while endogenous PGRN was distributed into nucleus in inclusion-like body in addition to cytoplasmic distribution.And after U937 cells expressing FAM76B or PGRN only and coexpressing FAM76B and PGRN being treated with PMA combined with LPS/IFNy,FAM76B was localized in nucleus while PGRN was distributed into nucleus in inclusion-like body in addition to cytoplasmic distribution,which indicated that the change of PGRN cellular localization might mediate its function of anti-inflammation.The expression of SORT1 could be increased by treatment with PMA in U937 cell.Besides,overexpression of FAM76B or PGRN could increase the expression of SORT1 when being treated with PMA combined with LPS/IFNy in U937 cell.But in HEK293 cells,overexpression of SORT1 could decrease the expression of PGRN in cel1 culture supernant and cytoplasm while it did not change the cytoplasmic distribution of PGRN.These results suggested that SORT1 might mediate the cellular distribution change of PGRN,but other proteins might be necessary for SORT1 mediating the cellular distribution change of PGRN.In summary,FAM76B had the functions of inhibiting cell senescence,inhibiting cell apoptosis,promoting cell proliferation and inhibiting inflammation,which were very similar to PGRN functions.The above results could provide important help to clarify the mechanism of PGRN function further.