| Objective: Gastric cancer is one of the most common cancers in the world.The incidence rate of gastric cancer is about 11.1% of all types of cancers in 2020 and accout for a population of 8 million.It is the main reason of cancer death among Asian men.Peritoneal metastasis is one of the most common metastasis of gastric cancer.The poor quality of patients with peritoneal metastasis is often due to complications such as ascites,peritoneal adhesion,intestinal obstruction,and the lack of effective treatment.Therefore,exploring the molecular mechanism is of great significance for preventing the occurrence of peritoneal metastasis and making precise treatment strategies.The most classic theory of peritoneal metastasis is the "seed and soil" theory.After the gastric cancer cells migrate as "seed" and infiltrate through the serous membrane,they leave the primary focus and get free in the abdominal cavity,and finally adhere to the "peritoneum" soil.Any factor that enhances the migration,invasion and adhesion ability of gastric cancer cells may promote peritoneal metastasis of gastric cancer.As a common feature of solid tumors,hypoxia plays an important role in tumor proliferation,metastasis and drug resistance.In fact,the hypoxia status of tumor tissue is different,which is also one of the reasons for the heterogeneity of tumor cells in situ.The surrounding tumor tissue is in normoxic state and as for the rapid growth but insufficient blood supply,the middle site of tumor tissue is often in a hypoxic state.The tumor cells under hypoxic state can induce the hypoxia inducible factor HIF-1 α expression and induce blood vessels to restore partial oxygen supply;However,the tumor continues to grow and aggravates hypoxia,which repeatedly forms a chronic hypoxia area.The necrotic cells that could not tolerate the hypoxia environment formed necrotic areas.Our previous studies confirmed that gastric cancer cells tolerant to hypoxia can promote its metastasis by up-regulating UCA1,circ HIPK3 and other non-coding RNA;Hypoxia can also promote the peritoneal metastasis of gastric cancer by up-regulating the expression of VEGFA in peritoneal mesothelial cells.However,it is not clear whether the primary hypoxic microenvironment,especially the hypoxia tolerant gastric cancer cells located in the tumor can promote peritoneal metastasis,and how to promote peritoneal metastasis.Extracellular vesicles are lipid bilayer membrane structures secreted by cells that contain a large number of RNA,protein,lipids and other substances.Because of its stability and non-degradability,it has become an important tool for information transmission between cells.Our previous studies have confirmed that the extracellular vesicles derived from gastric cancer cells can promote the occurrence of peritoneal metastasis by inducing the apoptosis of peritoneal mesothelial cells and the change of mesothelium-mesenchyme;The extracellular vesicles derived from resistant to cetuximab cancer cells can promote the resistance of sensitive cells to cetuximab by inhibiting PTEN.These results suggest that extracellular vesicles can be ingested by heterologous receptor cells and alter receptor cells to promote tumor progression.However,whether the gastric cancer cells in the hypoxic microenvironment can transform the normal gastric cancer cells through the extracellular vesicles,and promote the "seeds" of the normal gastric cancer to be free and planted in the peritoneum,and further promote the occurrence of peritoneal metastasis of gastric cancer,remains to be further explored.This study focuses on exploring whether and how the extracellular vesicles derived from gastric cancer cells can promote the peritoneal metastasis of normoxic gastric cancer cells in hypoxic microenvironment,and further exploring the mechanism of extracellular vesicle protein sorting in hypoxic microenvironment,providing a theoretical basis for the mechanism of extracellular vesicles in the peritoneal metastasis in hypoxic microenvironment.Methods: 1.The human hypoxia tolerant gastric cancer cell line was constructed.Transwell assay,the matrix adhesion test were used to verify the migration,invasion and adhere ability of the hypoxia tolerant cell line compared with the normoxic cell line.2.Extracellular vesicles were extracted by ultracentrifugation and characterized by electron microscopy,particle size analysis and Western blot.PKH26 labeling and uptake assay confirmed the uptake of extracellular vesicles by target cells.3.Extracellular vesicles of hypoxia-tolerant gastric cancer cell lines and gastric cancer cell lines were extracted by ultracentrifugation.Transwell assay,the matrigel adhesion assay and the human peritoneal mesothelial cells(Hmr-SV5)adhesion assay were used to verify the effect of PBS,gastric cancer cell line extracellular vesicles and hypoxia-tolerant cell line extracellular vesicles on the migration,invasion and adhere ability of normoxic cell lines.4.Proteomic analysis of the differential protein expression between hypoxia-tolerant gastric cancer cell line(HRGC)MGC803/Hypo and normoxic gastric cancer cell line MGC803.Data analysis suggests that CAV1 may be the key factor in HRGC extracellular vesicles.5.Transwell assay,the matrigel adhesion assay,the human peritoneal mesothelial cells(Hmr-SV5)adhesion assay were used to verify the effect of EVs-CAV1 by using si RNA knockdown on the migration,invasion and adhere ability of normoxic cell lines.6.Transwell assay,the matrigel adhesion assay,the human peritoneal mesothelial cells(Hmr-SV5)adhesion assay were used to verify the effect of EVs-CAV1 by using the overexpression plasmid CAV1(OECAV1)on the migration,invasion and adhere ability of normoxic cell lines.7.Using lentivirus knockdown plasmid to establish CAV1 knockdown stable transfection cell line(NC,Sh CAV1),the peritoneal metastasis model in mice was used to verify the effect of EVs-CAV1 on the peritoneal metastasis ability of normoxic cells.8.Particle size analysis verified the effect of CAV1 on the secretion of extracellular vesicles.9.Knockdown si RNACAV1,OECAV1,Sh CAV1 cell was used to extract extracellular vesicles,and Western blot verified the effect of CAV1 on the sorting of extracellular vesicles LAMB2.10.Transwell assay,the matrigel adhesion assay and the human peritoneal mesothelial cells(Hmr-SV5)adhesion assay were used to verify the effect of EVs-LAMB2 by using si RNA knockdown on the migration,invasion and adhere ability of normoxic cell lines.11.Knockdown si RNACAV1,si RNALAMB2,OE CAV1,Sh CAV1 was used to extract extracellular vesicles and EV was uptaked by normoxic gastric cells for 24 hours.Western blot verified the expression level of CAV1,LAMB2 and AKT,ERK,STAT3 pathway in normoxic gastric cells.12.The interaction between protein CAV1 and ROCK1 was investigated by co-immunoprecipitation,mass spectrometry,immunofluorescence and Duolink assay.13.Knockdown si RNA ROCK1,ROCK1 inhibitor Y27632 was used to verifies the effect of ROCK1 on the phosphorylation of Y14CAV1 and the sorting of extracellular vesicles LAMB2 in cells by Western blot.14.Construct the continuous phosphorylation plasmid Y14CAV1D(CAV114D)mutation and non-phosphorylation plasmid Y14CAV1F(CAV114F)mutation.Western blot verifies the effect of CAV114 D,CAV114F on Y14CAV1 phosphorylation and the selection of extracellular vesicle LAMB2 in cells.15.The interaction between Rab11 and LAMB2 protein was verified by co-immunoprecipitation in NC,Sh CAV1 stably transfected hypoxia tolerance cell line,CAV114 D,CAV114F mutation plasmid transfected hypoxia tolerance cell line.16.Knockdown si RNA Rab11 was used to verify the effect of Rab11 on the sorting of extracellular vesicles LAMB2.17.The interaction between Rab11 and CD63 protein was verified by co-immunoprecipitation in NC,Sh CAV1 stably transfected hypoxia tolerance cell line,CAV114 D,CAV114F mutation plasmid transfected hypoxia tolerance cell line.18.Immunoprecipitation,immunofluorescence and GTP pulldown assay were used to investigate the interaction between protein CAV1 and Rab11.19.Extracellular vesicles were extracted by ultracentrifugation,and the expression of EVs-CAV1 and EVs-LAMB2 in the patients’ ascites was verified by Western blot.Extracellular vesicles were extracted by the method of extracellular vesicle plasma kit and the expression of EVs-CAV1 and EVs-LAMB2 in the patients’ plasma was clinically verified by Elisa.Results: 1.Hypoxia promoted gastric cancer cells metastasis.The human hypoxia tolerant gastric cancer cell line was constructed.Transwell,the matrigel adhesion assay was used to verify that the migration,invasion and adhension ability of the hypoxia tolerant cell line was stronger than that of the normoxic cell line.2.Hypoxia promoted extracellular vesicles secretion.After the extracellular vesicles were extracted by ultracentrifugation,the structure of the extracellular vesicles was verified by electron microscopy.The particle size analysis showed that the number of extracellular vesicles secreted by the hypoxia tolerant gastric cancer cell line increased compared to nomoxic gastric cancer cell line.Western blot showed that CD63,CD9,and TSG101 were highly expressed in the extracellular vesicles of the hypoxia tolerant gastric cancer cell line than the nomoxic gastric cancer cell line.3.Extracellular vesicles secreted by hypoxia tolerance gastric cancer cell line promote the migration,invasion and adhesion of normoxic gastric cancer cells.PKH26 labeling and uptake assay was used to confirm the uptake of extracellular vesicles of hypoxia tolerance gastric cancer cell line by normoxic gastric cancer cells.Transwell assay,the matrigel adhesion assay and the human peritoneal mesothelial cells(Hmr-SV5)adhesion assay were used to verify HRGC-EVs enhanced the ability of migration,invasion and adhesion of GC compared to PBS and GC-EVs.4.HRGC EVs-CAV1 promoted peritoneal metastasis of gastric cancer cells in vivo and vitro.Data analysis suggests that CAV1 may be the key factor that plays a role in extracellular vesicles.Western blot confirmed that the expression of CAV1 was increased in the HRGC and HRGC-EVs.Si RNA knockdown CAV1,plasmid overexpression CAV1,Transwell assay,the matrigel adhesion assay and the human peritoneal mesothelial cells(Hmr-SV5)adhesion assay were used to verify the increased ability of EVs-CAV1 on migration,invasion and adhesion of GC.Using lentivirus knockdown plasmid to establish CAV1 knockdown stable transfection cell line,the peritoneal metastasis model of mice in vivo verifies that the EVs-CAV1 promotes the gastric peritoneal metastasis of normoxic cells.5.CAV1 sorting Evs-LAMB2 under hypoxic environment.Particle size analysis showed that CAV1 had no significant effect on the secretion of extracellular vesicles.Knockdown si RNACAV1,OE CAV1,Sh CAV1 were used.Western blot verified that CAV1 increased the expression of EVs-LAMB2.6.EVs-LAMB2 promoted the migration,invasion and adhesion of normoxic cells.EVs-LAMB2 promoted the migration,invasion and adhere ability of normoxic cells was verified by using si RNA knockdown LAMB2 on transwell assay,matrigel adhesion assay and human peritoneal mesothelial cells(Hmr-SV5)adhesion assay.7.EVs-LAMB2 activated AKT pathway.Knockdown si RNACAV1,OE CAV1,Sh CAV1 cell lines,knockdown si RNALAMB2 were extracted extracellular vesicles and uptaked by normoxic cells for 24 hours.Western blot showed that HRGC Evs increased the expression of CAV1,LAMB2,p AKT in normoxic gastric cells.8.ROCK1 promotes Y14CAV1 phosphorylation and promotes extracellular vesicle LAMB2 sorting under hypoxic microenvironment Immunoprecipitation mass spectrometry,immunofluorescence and Duolink assay confirmed that CAV1 was bound to ROCK1.Extracellular vesicles were extracted by si RNAROCK1 knockdown and ROCK1 inhibitor Y27632 HRGC.Western blot verified that si RNA ROCK decrease the expression level of Y14CAV1 phosphorylation decreased,the expression level of LAMB2 did not change significantly,and the expression level of LAMB2 in extracellular vesicles decreased.And the same was obseved by ROCK1 inhibiter.The plasmids of continuous phosphorylation after Y14CAV1D(CAV114D)mutation and non-phosphorylation after Y14CAV1F(CAV114F)mutation were constructed.Western blot verified that the expression level of Y14CAV1 phosphorylation decreased in CAV114 F group,the expression level of LAMB2 did not change significantly,and the expression level of LAMB2 in extracellular vesicles decreased in CAV114 F group.9.Y14CAV1 phosphorylation increases Rab11 activation and promotes extracellular vesicle LAMB2 sorting under hypoxic microenvironment.In Sh CAV1,CAV114 D,CAV114F HRGC,immunoprecipitation verified that Y14CAV1 phosphorylation promoted the binding of Rab11 and LAMB2.After si RNARab11 was used to knocked down rab11,the expression level of LAMB2 did not change significantly in cells,but the expression level of LAMB2 in the extracellular vesicles decreased.In Sh CAV1,CAV114 D,CAV114F HRGC immunofluorescence verified that Y14CAV1 phosphorylation increased Rab11 and CD63 co-localization.Immunoprecipitation and immunofluorescence assay confirmed that CAV1 combined with Rab11.GTP pulldown assay verified that Y14CAV1 phosphorylation promoted the GTP activation of Rab11.10.EVs-CAV1 and EVsLAMB2 are biomarkers for predicting peritoneal metastasis of gastric cancer.Extracellular vesicles in ascites of patients were extracted by ultracentrifugation.Western blot confirmed that the expression of EVs-CAV1 and EVs-LAMB2 in ascites of patients with gastric cancer was higher than that of patients with cirrhosis.Extracellular vesicles in plasma were extracted by the method of extracellular vesicle plasma kit,and the expression of EVs-CAV1 and EVs-LAMB2 in the plasma of patients was verified by Elisa.Nonpaired T test showed that the expression of EVs-CAV1 and EVs-LAMB2 in the plasma of patients with peritoneal metastasis was higher than that of patients without peritoneal metastasis.Logistic analysis verified that EVs-CAV1 and EVs-LAMB2 in plasma were independent prognostic factors for gastric cancer peritoneal metastasis,and established risk score.The patients were divided into two groups by using the EVs-CAV1 and EVsLAMB2 risk score.The survival analysis showed that the OS of patients in the high-risk group decreased.Conclusion: ROCK1 promoted the phosphorylation of Y14CAV1,and Y14CAV1 activated Rab11,promoted the sorting of extracellular vesicle LAMB2,and promoted the peritoneal metastasis of gastric cancer under hypoxic microenvironment. |
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