| Background and objectives Esophageal cancer is one of the common malignant tumors around the global with high morbidity and mortality.Due to the atypical clinical symptoms of esophageal cancer,lack of non-invasive and effective screening methods,poor prognosis and low five-year survival rate,it has become one of the major public health problems.Exosomal miRNA is a novel discovered small molecule involved in intercellular communication.It plays an important role in the tumor microenvironment and affects the development of tumor cells.With the high specificity of exosomal miRNA,tumor exosomal miRNA could be used as a biomarker for early diagnosis and prognosis so as to improve early screening rate and identify potential patients.This study explored the effect of esophageal cancer exosomal miRNA on the polarization state of macrophages in the esophageal cancer microenvironment,as well as its role and mechanism involved in esophageal cancer metastasis.It provides research foundation and data support of the exosomal miRNA as a tumor biomarkers in early diagnosis of esophageal cancer.Methods 1.36 cases of esophageal cancer which diagnosed by pathology or gastroscopy in Huai'an area and 36 cases of healthy with no history of digestive system were collected,they are paired by age and sex.The 36 pairs of esophageal cancer and healthy control blood samples were collected.Plasma Exosome Isolation kit was used to extract plasma exosomes.Anti-EpCAM was used to purify tumor cell secretory exosomes and extract small RNA.The expression of miR-21-5p was detected by qRT-PCR.2.The human leukemia mononuclear cell THP-1 was induced into a mononuclear macrophage,M0-type macrophage,by Phorbol myristate acetate(PMA),and then was induced by lipopolysaccharide(LPS)and Human Interleukin-4(IL-4)to establish M1 and M2 type macrophage,respectively.The ELISA and qRT-PCR were used to detect the expression of macrophage markers.3.The esophageal cancer cells exosomes were collected by gradient centrifugation combined with ultracentrifugation.Western blot was used to detect CD63,TSG101 and GM-130 proteins.4.A Transwell co-culture model was established.Cy3-miR-21-5p was transfected into esophageal cancer cell EC109,after 24 h,the transfer characteristics of exosomal miR-21-5p in cells were observed.qR-PCR was used to detect miR-21-5p delivery efficiency in the receptor.Exosomes were collected by esophageal cancer cells transfected with cy3-miR-21-5p mimic,followed by addition of THP-1-derived macrophages.Confocal microscopy was used to confirm the upatake process.5.The biological function changes of recipient cells(M0 macrophages)were observed in the Transwell co-culture model.The effect of exosomal miR-21-5p in recipient cells(macrophages)on polarization was detected by qRT-PCR and ELISA.The effect of exosomal miR-21-5p on the phagocytosis,cycle and proliferation was detected by flow cytometry and EdU staining.6.The macrophage supernatant was collected and used as the culture medium of esophageal cancer cell EC109.Transwell chamber were used to detect migration and invasion of esophageal cancer cells.7.The target gene of exosomal miR-21-5p was verified by qRT-PCR,Western blot and dual luciferase reporter gene experiments(DLR).After treatment by PI3K/AKT inhibitor(LY294002),Western blot was used to detect the expression of related proteins in the PTEN/AKT/STAT6 pathway.TGF-? RI/II inhibitor(LY2109761)was used to treat esophageal cancer cells cultured in macrophage supernatant.The expression of p-Smad2 and EMT-related proteins were detected by Western blot.Results I.Characteristics of exosomal miR-21-5p in esophageal cancer 1.Characteristics of miRNA in esophageal cancer cell and exosomes Western blot analysis showed that exosomes of esophageal cancer cells expressed exosomes-specific protein CD63 and TSG101,but no specific protein GM-130.Meantime,the candidate miRNAs,miR-21-5p,shows the highest expression in EC109 cells and exosomes.These provided a basis for subsequent screening of miRNAs.2.Variance analysis of miR-21-5p in plasma exosomes between esophageal cancer patients and healthy controlsThe expression of miR-21-5p in the tumor cell-specific secretion exosomes of plasma samples from esophageal cancer was significantly higher than that in the control group,about 7.62 times(P<0.05).It confirmed the miR-21-5p was highly expressed in plasma exosomes from esophageal cancer patients.II.Exosomal miR-21-5p promote macrophage M2 polarization1.Characteristics of exosomal miR-21-5p transfer between cellsThe confocal microscopy showed that red fluorescence is observed in the cytoplasm of M0 macrophages,which is Cy3-labeled miR-21-5p,suggesting that miR-21-5p is transmitted by exosomes and taken up by macrophages.2.Establishment of macrophage polarization modelIn the established M1 type macrophage,the M1 type macrophage markers IL-6,TNF-?,IL-1? were highly expressed.The M2 type macrophage markers TGF-?,IL-10,CD206,CD209 were highly expressed in the established M2 type macrophage.The results confirmed that the M1 and M2 macrophage models were successfully established.3.Exosomal miR-21-5p promote macrophage M2 polarizationFour experimental groups were set as follows: the co-culture group of EC109 cells which were transfected with miR-NC mimics and macrophage(Exo-miR-NC group),the co-culture group of EC109 cells which were transfected with miR-21-5p mimics and macrophage(ExomiR-21-5p group),the M2 macrophage group(M2 macrophage group)and the M2 type macrophage+the miR-21-5p inhibitor group(M2+miR-21-5p inhibitor group).After 24 hours of co-culture,exosomal miR-21-5p were found to promote the polarization of macrophages to M2.(1)The mRNA expression levels of M1 and M2 markers were detected by qRT-PCR.The mRNA expression of M2-type macrophage markers TGF-?,IL-10,CD206 and CD209 in Exo-miR-21-5p group were significantly higher than those in Exo-miR-NC group(P<0.05).The M1-type macrophage markers TNF-?,IL-6,IL-1? mRNA levels were significantly inhibited(P<0.05).Compared with those in the M2 macrophage group,the mRNA expression of M2 macrophage markers TGF-?,IL-10,CD206,CD209 and CCL13 were significantly lower than those in the M2+miR-21-5p inhibitor group(P<0.05).(2)The protein levels of of M1 and M2 markers were detected by ELISA.Compared with the Exo-miR-NC group,the Exo-miR-21-5p group showed the inhibition on protein IL-6 significantly,which was decreased by 28.75%(P<0.05),but there is no effect on TNF-?.The protein expression of M2 macrophage markers IL-10 and TGF-? were significantly increased with 1.38 times and 1.49 times respectively in the Exo-miR-NC group.Compared with the M2 macrophage group,the expression of M2 macrophage markers IL-10 and TGF-? were decreased by 43.78%,59.51%(P<0.05).M1 macrophage markers were significantly decreased in the M2+miR-21-5p inhibitor group.The TNF-? was increased significantly(P<0.05)and there is no effect on IL-6.4.Effect of exosomal miR-21-5p on the biological function of macrophagesExosomal miR-21-5p inhibited macrophages phagocytic function and promoted macrophage proliferation,but there is no effect on the cycle distribution of macrophages.(1)Flow cytometry was used to detect changes in macrophage phagocytosis.The phagocytosis rate in Exo-miR-NC group was(75.33±2.13)%,which was lower than that in ExomiR-21-5p group(64.43±7.34)%.(2)The proliferation rate of macrophages was detected by EdU staining.The cell proliferation rate of Exo-miR-NC group was(27.86±0.06)%,which was lower than that of ExomiR-21-5p group was(32.32±0.06)%.(3)Flow cytometry was used to detect the distribution of macrophage cycle.The period distribution of Exo-miR-NC group was G1 phase(49.79±1.17)%,S phase(25.57±1.49)%,G2 phase(24.64±0.97)%,and Exo-miR-21-5p group cycle distribution was G1 phase(48.66±3.74)%,S phase(25.74±1.44)%,G2 phase(25.60±3.64)%.There was no statistical difference between the two groups(P>0.05).III.Exosomal miR-21-5p affects metastasis of esophageal cancer cells through promoting macrophage polarizationFor the above four experimental groups,the macrophage supernatant was collected and used as a culture medium for esophageal cancer cells EC109 to detect the effects of migration and invasion in esophageal cancer cells.(1)Transwell chamber was used to detect the migration ability of esophageal cancer cells.The number of transmembrane cells in Exo-miR-NC and Exo-miR-21-5p groups were 29.20±3.29 and 41.73±7.30,with the increase of 42.91%(P<0.01).The number of transmembrane cells in the M2 macrophage and M2+miR-21-5p inhibitor groups were 46.60±4.56 and 19.80±3.77,with the 57.51% decrease(P<0.01).It is indicated that polarized M2-type macrophages can promote the migration ability of esophageal cancer cells,while inhibiting the expression of miR-21-5p in M2 macrophages,this result is the opposite.(2)Transwell chamber was used to detect the invasion ability of esophageal cancer cells.The number of transmembrane cells in Exo-miR-NC and Exo-miR-21-5p groups were 18.93±3.51 and 24.47±6.33,with the increase of 29.27%(P<0.01).The number of transmembrane cells in the M2 macrophage and M2+miR-21-5p inhibitor groups were 41.60±4.72 and 16.80±4.60,with the decrease of 59.62%(P<0.01).It is indicated that polarized M2-type macrophages can promote the invasion ability of esophageal cancer cells,while inhibiting the expression of miR-21-5p in M2 macrophages,this result is the opposite.(3)The expression levels of EMT-related proteins E-cadherin,N-cadherin,Snail and ?-SMA were detected by Western blot.Compared with the Exo-miR-NC group,the expression of N-cadherin,Snail and ?-SMA were increased significantly in the Exo-miR-21-5p group.The expression of E-cadherin protein was decreased significantly.Compared with the M2 macrophage group,the expression of N-cadherin,Snail and ?-SMA were decreased in the M2+miR-21-5p inhibitor group significantly.The expression of E-cadherin protein was increased significantly.It is indicated that polarized M2-type macrophages can promote the process of EMT,while inhibiting the expression of miR-21-5p in M2 macrophages,this result is the opposite.IV.Mechanism of exosomal miR-21-5p promoting macrophage polarization on esophageal cancer cell metastasis(1)The target gene PTEN of exosomal miR-21-5p was verified by qRT-PCR,Western blot and dual luciferase reporter gene experiments.The PTEN mRNA in Exo-miR-21-5p group was significantly lower than that in Exo-miR-NC group,with the reduction of 35.6%(P<0.05).The PTEN protein in Exo-miR-21-5p group was down-regulated by 59.5% significantly.The fluorescence value of the miR-21-5p+PTEN(WT)experimental group was significantly lower than that of the miR-21-5p+PTEN(Mut)experimental group,with 0.64 times decrease(P<0.05).It confirmed that miR-21-5p specifically binds to the PTEN 3'UTR region so as to regulate the expression of PTEN mRNA and protein.(2)To study the mechanism of M2 type polarization of macrophages induced by exosomal miR-21-5p in esophageal cancer cells.The expression levels of related protein in M0-tybe macrophage the PTEN/AKT/STAT6 pathway were detected by Western blot.Compared with the Exo-miR-NC group,the PTEN protein was decreased in the Exo-miR-21-5p group.After adding PI3K/AKT inhibitor(LY294002,50 ?M),PTEN protein recovered by 38.23%,Akt phosphorylation level decreased by 60.16% significantly.STAT6 phosphorylation level decreased significantly by 48.32%.It is suggested that the expression of p-Akt and p-STAT6 in Exo-miR-21-5p group can be decreased after blocking PI3K/Akt signaling pathway.(3)To investigate the mechanism of the effect of polarized M2-type macrophages on esophageal cancer metastasis.The supernatants of the above four treatment groups were collected,and applied to esophageal cancer cells EC109.The proteins was extracted to detecd the expression of p-Smad2 and EMT-related proteins.Compared with the Exo-miR-NC group,the phosphorylation level of Smad2 protein in Exo-miR-21-5p group was increased significantly as well as N-cadherin,?-SMA,Snail protein levels,but E-cadherin protein were decreased significantly.After adding the TGF-? RI/II inhibitor(LY2109761),the phosphorylation level of Smad2 protein was decreased by 80.16% in Exo-miR-21-5p group(20 ?M).The expression of N-cadherin,?-SMA and Snail were inhibited to 52.98%,69.62%,77.93% respectively,but E-cadherin protein expression level was recovered to 82.39% Conclusion1.The plasma exosomal miR-21-5p from esophageal cancer were found higher than that in the control,showing potential role in the occurrence of esophageal cancer.2.Exosomal miR-21-5p was taken up by macrophages(receptor cells)to promote macrophage M2 polarization.In addition,exosomal miR-21-5p could inhibit macrophage phagocytosis and promote proliferation of macrophages.3.The exosomal miR-21-5p could target the PTEN gene in M0-tybe macrophage,promoting the macrophage M2 polarization through PTEN/AKT/STAT6 pathway.4.The M2 macrophage induced by exosomal miR-21-5p could freed TGF-? to promote migration and invasion of esophageal cancer through activating TGF-?/Smad2/EMT pathway. |
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