The Transfer Of Endoplasmic Reticulum Stress Signals From Hepatocellular Carcinoma Cell To Macrophage And The Effects And Mechanisms Of Exosome Derived From Endoplasmic Reticulum-stressed Macrophage On Apoptosis Of Hepatocellular Carcinoma Cell

BackgroundHepatocellular carcinoma(HCC)is the common malignant tumor,of which incidence is on the rise in the worldwide.The life of liver cancer patients is very short and the recurrence rate of operation is high.Chemotherapy is a main therapeutic strategy for patients with advanced disease,but the existing chemotherapeutics do not achieve significant survival benefits and do cause the phenomenon of the resistance to thermotherapy to a great extent.Endoplasmic reticulum(ER)stress is a state of the pathology of organelles caused by the inside and outside factors of cell pressure,characterized by endoplasmic reticulum cells physiological dysfunction,calcium balance,unfolded or misfolded proteins in the endoplasmic reticulum lumen.Hepatocellular carcinoma,especially the liver cancer in China,is often developed from viral or alcoholic hepatitis.It is infected by viral infection,inflammation stimulation for a long time and combined with ischemic anoxia microenvironment caused by the rapid proliferation of the cancer liver tissue;more easily lead to the ER stress.Macrophages are a type of immune cells with high plasticity and functional diversity.They are the most immune cell in tumor microenvironment and widely involved in the shaping of local immune microenvironment,as well as,play an important role in the tumor growth,invasion and metastasis.Recent studies have found that tumor cells could pass ERS signals to macrophages in the microenvironment,lead to the increase of the ERS gene transcription coding and the secretion of proinflammatory cytokines at the same time.Exosomes are vesicular vesicles which secrete outside cells between 40 and 100nm in diameter,and widely involved in cellular communication.They are closely related to the occurrence,development and treatment of tumor.Exosomes have been widely recognized as carriers of proteins and nucleic acids.However,can exosomes transmit the ER stress signal of tumor cells to the surrounding macrophages?What is the effect of macrophages on the biological behavior of tumor cells after receiving ER stress signal?The information is rarely reported.In the current study,we aim to discuss the way that hepatocellular carcinoma transmit ER stress signals to macrophages,further studies on the effects of ER stress macrophages on liver cancer and possible molecular mechanisms and provide new ideas for the clinical treatment.Objective(1)To discuss the relationship between endoplasmic reticulum stress and clinicopathological characteristics as well as prognosis of hepatocellular carcinoma patients.(2)To clarify that hepatocellular carcinoma transmit the stress signal of endoplasmic reticulum to macrophages in microenvironment through exosomes.(3)To explore the effect and mechanisms of the endoplasmic reticulum stressed macrophages deprived exosomes on the apoptosis of hepatocellular carcinoma.Methods(1)142 HCC cases confirmed by pathology were collected to produce tissue chips,immunohistochemical method was used to detect the expression of GRP78 in the tissues,and further to analyzed correlation between GRP78 expression and the clinicopathological characteristics as well as prognostic data;expression of CD68 and GRP78 was detected by immunofluorescence to analyze the differences of ER stress levels in HCC tissues and para-carcinoma tissues;TM to induce HepG2 in hepatocellular carcinoma to develop ERS,collect the supernatant fluid and to separate the exosomes by differential centrifugation;PMA induced the transformation of thp-1 into macrophages,culture supernatant,exosomes and exosome-free supernatant with macrophage respectively,detect expression of GRP78 in macrophages by Western Blotting.(2)Supernatant were collected to isolate ordinary cultured exosome deprived exosomes(con-exo)and ER stressed macrophage deprived exosomes(ERS-exo)by ExoQuick-TC;Transmission electron microscopy was used to observe the morphology of exosomes;Western Blotting was performed to observe the expression of marker proteins such as CD63 and Calnexin.Atfer dyeing the exosomes with PKH67 and cultured with HepG2 cells,observing the fluorescence intensity of PKH67 in HepG2 cells by laser confocal microscope.(3)In vivo experiment,HCC mouse subcutaneous xenograft models were built by BALB/c nude mice.Normal saline,con-exo and ERS-exo were injected through the tail vein,respectively.Subcutaneous xenograft were isolated after mice model were dead and made into paraffin samples after measuring and weighing;H&E staining were used to observe classical tumor morphological features,TUNEL method were used to detect tumor apoptosis.In vitro experiment,con-exo and ERS-exo were co-cultured with HepG2 and SMMC-7721 cell lines before and after ER stress respectively,Flow cytometry and TUNEL were performed to detect the apoptosis of HCC cells.(4)After co-cultured with con-exo and ERS-exo,immunofluorescence was used to detect LC3 expression in HepG2 cells with or without ER stress;Transmission electron microscopy was used to observe the morphological characteristics of autophagy in HepG2 cells,Western Blotting ws performed to detect the expression of LC3,P62 and Beclinl ad well as the XPB1s expression in both ER stressed macrophages and ERS-exo;After inhibiting IRE1 expression with RNAi,the expression of IRE la and XPBls were detected by Western Blotting;After autophagy was inhibited by pretreated with chloroquine(CQ)and 3-methyladenine(3-MA),con-exo and ERS-exo were co-cultured with HepG2 cells,flow cytometry and TUNEL were performed to observed apoptosis in HepG2 cells.Results(1)Immunohistochemical results showed that GRP78 negative patients,GRP78 low expression patients and GRP78 high expression patients are 5,36 and 101 cases respectively,the GRP78 expression is associated with the clinicopathological characteristics such as hepatitis,cirrhosis and tumor size;There was a significant difference of overall survival between with GRP78 high expression patients and GRP78 low expression patients positive patients,the overall survival of GRP78 high expression patients was shorter.(2)Immunofluorescence results showed that in HCC tissues,GRP78 expression of macrophages was significantly higher than that of the para-carcinoma tissues;The results of Western Blotting showed that GRP78 expression was significantly up-regulated in macrophages co-cultured with the HepG2 supernatant and its exosomes,but not with exosomes-released supernatant.(3)The circular vesicle structure with a diameter of 40-100 nm was observed under the transmission electron microscope,which conformed to the typical characteristics of exosomes.The expression of exosome marker protein CD63 was detected by Western Blotting,while the expression of the exclusion protein Calnexin was not been detected the at the same time.The laser confocal microscope showed that PKH67 green fluorescence was found in cytoplasm of recipient HepG2 cells.(4)In vivo experiment,the volume and weight of the subcutaneous transplanted tumor of the con-exo group were significantly smaller than that of the control group,while the ERS-exo group showed no significant difference compared with the control group.The diffuse distribution of tumor cells,obvious cell atypia and visible pathological mitosis,which are typical morphological characteristics of tumor,was observed in xenograft by H&E staining;The results of TUNEL staining showed that the proportion of apoptotic cells in con-exo group was significantly higher than that in the control group,and the ERS-exo group showed no significant difference compared with the control group.In vitro experiments,results of flow cytometry and TUNEL showed that the apoptotic rate was increased in HepG2 and SMMC-7721 cells co-cultured with con-exo but not ERS-exo.(5)Immunofluorescence showed that LC3 fluorescence intensity was significantly higher in TM+ERS-exo group than in other groups.The typical double-layer membranous structure of autophagosome was observed by transmission electron microscope;and in the same size of view,the number of autophagosomes in TM+ERS-exo group was significantly higher than that of other groups.There was no significant difference between TM+con-exo group and TM group.With Western Blotting,a significant increase in LC3Ⅱ/LC3Ⅰ ratio was detected in TM+ERS-exo group,as well as the the decrease of P62 expression and the increase of Beclinl expression,while the expressions of these proteins showed no significant difference in TM+con-exo group compared with TM group.(6)After TM induced ER stress was activated,the up-regulation of XBP1s was detected in both ER stressed macrophages and ERS-exo by Western Blotting;After IRE1 pathway was inhibited by si-IRE1,the expression of IREla and XBP1s were significantly decreased in ER stressed macrophages.(7)After pre-treated with CQ and 3-MA,the apoptosis was enhanced in HepG2 cells co-cultured with ERS-exo by flow cytometry and TUNEL;Immunofluorescence further confirmed that the autophagy level in HepG2 cells was significantly lower after CQ and 3-MA pretreatment.Conclusions(1)GRP78 expression is generally high in HCC tissues,which is associated with clinicopathological characteristics such as hepatitis,cirrhosis and tumor size;GRP78 high expression is a poor prognostic factor for HCC patients.(2)Exosome is the mediator for HCC cells to transmit ER stress signals to macrophages in tumor microenvironment.(3)Ordinary cultured macrophage deprived exosomes may increase apoptosis in ER stressed HCC cells,while ER stressed macrophage derived exosomes may induce apoptosis resistance.(4)Macrophages can activate autophagy in HCC cells by transmitting exosomal XBPls,which might be a possible mechanism for the apoptosis resistance of ER stressed hepatocellular carcinoma cells.