| Background Hepatocellular carcinoma(HCC)is one of the common malignant tumors.The prognosis of hepatocellular carcinoma is poor,and the 5-year survival rate is less than10%.In recent years,the incidence has been increasing year by year.More importantly,there has been no breakthrough in the treatment of liver cancer in the past few decades.The liver is considered to be a unique immune organ in the body.Macrophage is an important part of the immune system,as well as an important cellular component involved in liver inflammation and damage-repair,which plays a key role in maintaining the stability of the liver environment.However,more and more studies have found that macrophages infiltrated in the tumor microenvironment cannot perform normal anti-tumor immune functions,and even promote tumor escaping from immunological surveillance.More importantly,the mechanisms that promote the conversion of macrophages to M2-macrophages in the tumor microenvironment are still unclear,which brings great difficulties to the development of targeted treatment strategies.Therefore,it is of great significance to deeply explore the mechanisms of the transformation of macrophages into M2-macrophages in the tumor microenvironment.Nutrient deficiency,hypoxia and calcium dyshomeostasis can lead to the accumulation of unfolded or misfolded proteins in the cavity of endoplasmic reticulum,leading to endoplasmic reticulum stress(ERS).Then the three transmembrane sensors of the unfolded protein response(UPR)were activated,including requires Inositol requires 1α(IRE1α),protein kinase RNA-like ER kinase(PERK)and activated transcription factor-6(ATF6).The activated UPR pathway could maintain endoplasmic reticulum homeostasis,thereby promoting cell survival.However,continuous and severe ERS can initiate cell apoptosis through a variety of mechanisms.Studies have shown that abnormal activation of ERS exists in a variety of tumors,and continuous activation of ERS is closely related to tumor occurrence,development,drug resistance and immune escape.However,the effects and mechanisms of activated endoplasmic reticulum stress on the M1/M2 polarization of macrophages in the liver cancer microenvironment still need to be further clarified.Macrophages account for about 50% of the inflammatory cells in the microenvironment,and play key role in the intercellular communication with tumor cells.It was initially thought to be derived from bone marrow-derived monocytes.Later,it was discovered that macrophage could also originate embryonic yolk sacs.According to the macrophage balance hypothesis,macrophages are divided into classical activated macrophages(M1 type)and alternative activated macrophages(M2type).Numerous studies have shown that macrophages resident in the hepatocellular carcinoma tissues were mainly M2-macrophages.These macrophages could secrete anti-inflammatory factors and immunosuppressive molecules to create an immunosuppressive tumor microenvironment,thereby promoting tumor progression.However,the precise mechanism by which endoplasmic reticulum stressed hepatocellular carcinoma cells promote macrophages M2 polarization is still unclear.Exosomes are a type of double-layered cystic vesicles with a diameter of 30 nm to 150 nm.At first,exosomes were considered as metabolic waste excreted by various cells.Recently,researchers have discovered that exosomes can loaded numerous biologically active substances,such as genetic material,lipids,and proteins,and these bioactivators can be transmitted to the recipient cells in the microenvironment to directly or indirectly modulating the biological behaviors of the recipient cells.Tumor cell-derived exosomes can cross-talk with immune cells,and cause tumor immune escaping.However,little is known about the effect and mechanism of exosomes secreted by ER stressed hepatocellular carcinoma cells on macrophages polarization.Our topic will explore the effects and the possible molecular mechanisms of endoplasmic reticulum-stressed hepatocellular carcinoma cells on the phenotypic transformation of macrophages.Purpose(1)To clarify the effect of exosomes derived from ER stressed hepatocellular carcinoma cells on macrophages M2 polarization.(2)To explore the precise mechanisms by which endoplasmic reticulum stress-related exosomes affect the M2 polarization of macrophage.Methods(1)Tunicamycin(TM)stimulated hepatocellular carcinoma cells.Western-blot analysis was used to detect the changes of endoplasmic reticulum stress marker proteins,and determined the concentration and time conditions for constructing the endoplasmic reticulum stress model of hepatocellular carcinoma cells.(2)After the endoplasmic reticulum stress model was established,the exosomes released from the unstressed and stressed-hepatocellular carcinoma cells were isolated by the Exo Quick-TC kit;Structural characteristics of exosomes was observed under transmission electron microscope;Western-blot was used to detect the marker proteins of exosomes;The immunofluorescence was used to detect that exosomes are taken up by macrophages.(3)Flow cytometry(FCM)experiments was used to detect the effect of ERS-related exosomes on the expression of CD206 in macrophages;The expression of arginase-1(Arg-1),Transforming growth factor β1(TGF-β1)were detected by Western-blot analysis;CBA Mouse Inflammation kit were used to evaluate the expression of inflammatory factors secreted by macrophages.(4)Affymetrix gene chip and bioinformatics analysis was employed to analyse the impact of endoplasmic reticulum stress-related exosomes on the expression of macrophage-related immune regulation genes,and the expression of TLR4 on the surface of macrophages was detected by flow cytometry.Results(1)Western-blot analysis showed that tunicamycin can up-regulate the expression of GRP78 protein in hepatocellular carcinoma cells in a dose-and timedependent manner.After stimulating the hepatocellular carcinoma cells with2.5μmol/L of tunicamycin for 24 hours,the expression level of GRP78 reached to a plateau.We then using this condition to construct endoplasmic reticulum stress in the following experiment.(2)The pellet harvested from the supernatants of ER stressed HCC cells or normal control demonstrated round or class round vesicles with membrane like structures diameter in 40 to 100 nm under transmission electron microscope.In addition,compared with control group,endoplasmic reticulum stress-related vesicles had a larger diameter.CD63,TSG101 and HSP70 proteins,but not the ER molecular chaperones Calnexin were observed in the harvested pellets in the Western-blot analysis,which further confirmed that the pellets were true exosome without cellular components.Laser confocal microscope showed that macrophages effectively engulf PKH-67 labeled exosomes.(3)FCM analysis revealed that endoplasmic reticulum stress-related exosomes promoted the expression of CD206 in macrophages.And Western-blot analysis showed that endoplasmic reticulum-related exosomes can up-regulate the expression of Arg-1 and TGF-β1.Moreover,we also found that endoplasmic reticulum stress-related exosomes could increase the expression of IL-6 and IL-10 in macrophages via using a CBA mouse inflammation kit.(4)Affymetrix gene chip screens showed that 129 genes were up-regulated and123 genes down-regulated in Exo-TM stimulated macrophages,compared with the Exo-con group.Bioinformatics analysis suggested that these differentially expressed genes were closely related to the Toll-like receptor family.FCM verified that Exo-TM significantly up-regulates the expression of TLR4 receptor on the surface of macrophages.Conclusion(1)Endoplasmic reticulum stress-related exosomes of hepatocellular carcinoma cells can up-regulate the expression of CD206,Arg-1,TGF-β1 and IL-10 in macrophages,and promote macrophages polarized to M2 phenotype.(2)Endoplasmic reticulum-stressed hepatocellular carcinoma cells may promote macrophages polarization via HSP70 enriched exosomes and activation of TLR4. |
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