| Background&Purpose:the incidence rate of gastric cancer ranks fifth all over the world,of which,more than 70%gastric cancer cases exsits in the Asian population,gastric cancer ranks second in cancer-related deaths.Most gastric cancer patients in China are advanced stage,which results in a very poor prognosis.At the same time,the benefits of comprehensive treatment based on surgery have gradually reached the bottleneck.Targeted therapy,immunotherapy and cellular adoptive therapy have gradually become important complementary therapies.In addition to insufficient secondary prevention,high heterogeneity,early lymphatic metastasis,and chemotherapy resistance also contribute to poor prognosis.Experimental research is the basis of opening up a new treatment situation for gastric cancer.Exosome is one of the current research hotspot and acts as the"messenger"and"vanguard troops"for tumor metastasis,which represents an important mechanism in the material and information exchange between tumor cells and tumor microenvironment.Exosomal mi RNA plays a key role in tumor invasion and progress.Currently,there are few studies on exosomal mi RNAs in gastric cancer.This study aims to find exosomal mi RNAs related to gastric cancer progression and further study their specific molecular mechanisms,so as to provide reference for follow-up precision therapy.Methods:Serum exosomes were extracted from specific gastric cancer patients,and differentially expressed exosomal mi RNAs related to gastric cancer stage were detected by high-throughput sequencing.Differentially expressed exosomal mi RNAs were determined after validation of clinical samples.Interested exosomal mi RNAs were selected based on literature review.Cell lines were divided into four groups according to different transfected substances（mi R-inhibitor and/or Exosome）.MTT assay,Annexin-V assay,Transwell assay,scratch assay and angiogenesis assay were used to fully study the changes of tumor phenotypes between different groups to study the influence of exosomal mi RNA on cell biological behavior.The xenograft tumor model in nude mice was established to verify the function of exosomes in vivo.Confocal microscopy was used to verify mi RNA transport mediated by exosomes.The target genes of mi RNA were found by using bioinformatics database,and the interested target genes were selected based on literature review and gene oncology.The classical dual luciferase reporting assa was used to further verify the exact interaction relationship between interested mi RNA and the screened target genes.Overexpression lentivirus vectors of mi RNA and target gene were constructed.Gastric cancer cells were divided into four groups according to different transfected substances（overexpression lentivirus vectors of mi RNA and/or overexpression lentivirus vectors of target gene）,and functional experiments were conducted to fully study the changes of tumor phenotypes between different groups,and determine the influence of target genes on cell biological behavior.RT-PCR and Western-blot were used to explore the possible downstream signaling pathways of target genes.Results:A total of 36 differentially expressed mi RNAs were found after high-throughput sequencing and PCA grouping between 4 advanced cases and 5 early cases,among which 9 mi RNAs with higher differential multiple were selected for subsequent verification（mi R-193b-5p,mi R-3173-5p,mi R-1228-5p,mi R-548a-3p,mi R-3613-3p,mi R-374b-5p,mi R-509-5p,mi R-6884-5p,and mi R-450b-5p）.Rt-Pcr was conducted in the selected 20 cases of early and 20 cases of late gastric cancer,using2–ΔΔCT method with U6 as an internal reference.The mi RNA expression level showed significant differences between the two groups for all nine selected mi RNA.Combined with the significance of the differences and the results of literature search,we selected mi R-450b-5p for the subsequent study.The expression levels of exosomal mi R-450b-5p in culture medium of different gastric cancer cell lines were measured.According to the different expression level,AGS cells were selected as target cell.Cell supernatant of SGC-7901 was selected as exosome donor.The gastric cancer cell lines were divided into four groups according to 2-level treatment（mi R-inhibitor and/or Exosome）.The functional experiments indicated that the gastric cancer cells cultured with the exosome showed stronger invasion,migration,proliferation and angiogenesis than those in the other three groups（P<0.05）;the xenograft tumors in exosome incubation group grew significantly faster than that in single cell injection group;Confocal microscopic imaging showed that exosomes could transport mi R-450b-5p into AGS cells.After the analysis of the biological information database,20 target genes of mi R-450b-5p met the screening criteria.After sequencing by correlation and combining with literature review,we selected CMTM8 for subsequent verification.The dual luciferase report assay showed that the inhibit effect of mi R-450b-5p was significant in the 3’UTR-WT group,and the inhibition effect disappeared in the 3’UTR-MUT group.The gastric cancer cell lines were divided into four groups according to 2-level treatment（overexpression lentivirus vectors of mi RNA and/or overexpression lentivirus vectors of target gene）.The results indicated that target gene CMTM8 had the ability to inhibit tumor invasion and migration,and this effect could be weakened by mi R-450b-5p.The key proteins in EGFR signal-related pathway were detected by Western-blot method,and the results showed that the high expression of CMTM8 could inhibit p-Akt and p-ERK levels,while mi R-450b-5p could weaken this inhibition effect.CONCLUSIONS:Exosomal mi R-450b-5p can promote invasion,migration,proliferation,anti-apoptosis and angiogenesis of gastric cancer cells.CMTM8 is a target gene of mi R-450b-5p.Overexpression of CMTM8 can inhibit EGFR-related pathways PI3K/Akt and RAF/MEK/ERK,and exosome mi R-450b-5p plays a role by inhibiting CMTM8 expression. |
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