The Mechanism Of Gastric Cancer Cell-Derived Exosomal Let-7b-5p Target MAPK6/ERK3 To Increase The Vascular Endothelial Permeability Via MAPK Signaling Pathway

Objective: gastric cancer(GC)is a high morbidity and high mortality disease,with metastasis being an important cause of the GC-associated mortality.It is difficult to early diagnosis and treatment,and the diagnosis is often in the late stage with poor prognosis.Gastric cancer has obvious heterogeneity,high degree of malignancy,prone to hematogenous metastasis and abdominal planting metastasis.Although gastric cancer radical surgery can clean the lymph nodes,the effect to other distant metastasis such as blood metastasis is limited.Metastasis is an important factor that influences the patients survival.The etiology of metastasis is multifactorial and complex.Thus,detailed understanding of the mechanism underlying metastasis will not only help in understating biology of cancers but also provide guidance for devising a targeted cancer treatment strategy.The infiltration of metastatic tumor cells into the vascular wall is one of the key steps to achieve hematogenous metastasis,and the mechanism is still unknown.The present study aimed to investigate a possible mechanism of hematogenous metastasis in gastric cancer and to investigate the key molecules and signaling pathways involved in this process.Methods: 1.Gastric cancer cell lines with different invasive ability were selected with transwell.2.Vesicles in the supernatant of the cultured fluid were extracted by differential overspeed centrifugation.3.Transmission electron microscopy was used to identify the membrane structure and diameter of the vesicles.4.Exosomal specific biomarkers were detected by Western blot.5.PKH67 was used to mark the exosomes which were co-cultured with Human Umbilical Vein Endothelial Cell(HUVEC).6.transwell was used to assess the effect on the permeability of gastric cancer cell-derived exosomes with different invasive ability after acting on HUVEC.7.CCK-8 assay was used to determine the proliferation of HUVEC after added exosomes.8.P120-catenin and VE-cadherin were detected via western blot after the gastric cancer cell-derived exosomes acting on HUVEC.9.Screening our aim miRNA by exosomal deep sequencing,combined with bioinformatics analysis and literature.10.The sequencing results were verified by quantitative real-time PCR.11.Validate the HUVEC transfection efficiency and the transfection conditions.12.Manufactured let-7b-5p mimics and inhibitor to verify whether let-7b-5p increases vascular endothelial permeability.13.CCK-8 assay was used to determine the proliferation of HUVEC after let-7b-5p mimics and inhibitor transfection.14.To verified the effect of let-7b-5p inhibitor on reversing gastric cancer cell-derived exosomes on increasing vascular endothelial permeability.15.CCK-8 assay was used to determine the proliferation of HUVEC after let-7b-5p inhibitor transfection and added exosomes.16.Flow cytometry assay was used to detect the effect of let-7b-5p inhibitor and exosomes on HUVEC apoptosis.17.Screened target genes of let-7b-5p and double luciferase reporter gene experiment was used to prove the result.18.P120-catenin,VE-cadherin and MAPK6/ERK3 were detected via western blot.19.Overexpressed MAPK6 in HUVEC by lentivirus and verified by Western blot.20.Transwell was used to determine that let-7b-5p can increase the HUVEC permeability via MAPK6.21.The effect of gastric cancer cell derived exosomes in increasing vascular endothelial permeability and promoting metastasis were verified in vivo.Results: 1.The invasion abilities of AGS and MGC80-3 were higher than those of MKN45 and NCL-N87 under the same conditions.2.Exosomes from AGS,MGC80-3and NCL-N87 cells were extracted.The diameter is between 30 and 150 nm and has a typical bilayer structure.3.CD9、CD63 and TSG101 were positive that proved the vesicles derived from gastric cancer cells were exosomes.4.PKH67 labeled exosomes were proved that they can absorbed by DAPI labeled HUVEC through Laser confocal microscopy.5.The permeability of HUVEC added exosomes was significantly increased than normal HUVEC,P<0.0001.This effect was increased with the ability of gastric cell invasion,P<0.0001.6.The proliferation of HUVEC-AGS-Exo was significance down regulation compared to HUVEC,P<0.0001,while HUVEC-NCIN87-Exo and HUVEC-MGC80-3-Exo were significance up regulation,P<0.0001,P=0.0002.7.Western blot showed there was no different in relative expression levels of P120-catenin between HUVEC,HUVEC-AGS,HUVEC-NCI-N87 and HUVECMGC80-3,P>0.05.The relative expression level of VE-cadherin in HUVECMGC80-3 was up regulated but others were no significant change.8.Through deep exosome miRNA sequencing,let-7b-5p was locked as aim gene according to the bio-informatics screening,combined with literature review and analyzed its role in various fields.9.Real-time PCR showed amplification of target genes in all three groups of exosomes and the relative expression levels of let-7b-5p was AGS exosome group> MGC80-3 exosome group> NCL-N87 exosome group.10.The preliminary experiment show that 4-6 hours was the best time for transfection,the transfection efficiency reached 60% with FAM NC(2.5 μl):lipo2000(2 μl).11.The permeability of let-7b-5p mimics transfected group was significantly increased compared with HUVEC-mimics NC,P<0.0001.Meanwhile,the permeability of let-7b-5p inhibitor transfected group was significantly decreased compared with HUVEC-inhibitor NC,P<0.0001.12.The proliferation in HUVEC-let-7b-5p group was decreased and HUVEC-let-7b-5p inhibitor group was decreased,too.13.Compared with HUVEC inhibitor-NC,the permeability of HUVEC-let-7b-5p-inhibitor was significance decreased,P<0,0001.Compared with HUVEC-let-7b-5p-inhibitor group,the permeability was not significant increase in HUVEC-inhibitor +AGS-Exo,HUVEC-inhibitor+MGC80-3-Exo and HUVEC inhibitor+ NCL-N87-Exo,P>0.05.14.Compared with HUVEC-let-7b-5p-inhibitor group,the proliferation of HUVEC inhibitor-NC was decreased,P<0.001.HUVEC-inhibitor+NCL-N87-Exo20/50 group and HUVEC-inhibitor +MGC80-3-Exo 20/50 group were also decreased,P<0.001,while there was no difference in HUVEC inhibitor + AGS-Exo 20/50 group.15.Compared with HUVEC-let-7b-5p-inhibitor group,the apoptosis of HUVEC inhibitor-NC was decreased,P<0.001.HUVEC inhibitor + AGS-Exo 20/50 group and HUVEC-inhibitor +MGC80-3-Exo 20/50 group were increased,P<0.001.The apoptosis of HUVEC-inhibitor+NCL-N87-Exo20 group was increased but there was no difference in HUVEC-inhibitor+NCL-N87-Exo50 group.16.let-7b-5p could regulated the expression of luciferase with the MAPK6 3’UTR(P=0.0008),with a29.21% decreased,and this regulatory relationship disappeared after the binding site mutation(CUACCUCA→ GAUGGAGU).17.There was no difference of the expression of VE-cadherin between HUVEC-mimics NC group and HUVEC-let-7b-5p mimics group,P>0.05.The expression of VE-cadherin in HUVEC-inhibitor group was less than HUVEC-inhibitor NC group,P<0.0001.There was no difference of the expression of P120-catenin between HUVEC-mimics NC group and HUVEClet-7b-5p mimics group,P>0.05.The expression of P120-catenin in HUVEC-inhibitor group was less than HUVEC-inhibitor NC group,P<0.0001.There was no difference of the expression of MAPK6/ERK3 between HUVEC-mimics NC group and HUVEC-let-7b-5p mimics group,P>0.05.The expression of MAPK6/ERK3 in HUVEC-inhibitor group was more than HUVEC-inhibitor NC group significantly,P<0.0001.18.Western blot results showed that MAPK6/ERK3 was overexpression in HUVEC after lentiviral transfection.19.There was no difference between the permeability of natural control group(HUVEC-NC + Exo)and the negative control group(HUVEC-E + Exo),P>0.05,while the permeability of experimental group was significantly less than the two control groups,P<0.0001.20.In vivo experiments showed that gastric cancer cell-derived exosomes could promote the formation of abdominal metastases both in the number of metastases nodes and in terms of metastasis size.Conclusion: 1.Gastric cancer cell-derived exosomes can increase the permeability of vascular endothelium.2.The effect of the exosomes correlates with the invasive ability of their origin gastric cancer cells.Namely,the more invasive gastric cancer cells secreted exosomes to increase the vascular endothelial cells permeability increases accordingly.3.Hsa-let-7b-5p is a key molecule in gastric cancer cell-derived exosomes for increasing vascular endothelial permeability,and MAPK6 / ERK3 is its target gene.4.Exosomal hsa-let-7b-5p increases vascular endothelial permeability via MAPK6 / ERK3.The MAPK signaling pathway was activated and it may be related to hematogenous metastasis of gastric cancer.5.The mechanism of MAPK6 / ERK3 increase the permeability of vascular endothelium is independent on the proliferation of vascular endothelial cells or apoptosis,the potential mechanism is still need further investigation.