| Object:Chemotherapy resistance in gastric cancer is one of the most common and difficult problems to overcome in patients with gastric cancer(especially advanced gastric cancer).It is also a major cause of tumor recurrence and metastasis.Our previous study found that the expression level of CLIC1 is closely related to the proliferation,apoptosis,cell cycle and invasion and migration of gastric cancer cells.CLIC1 is a Potential Target for Gastric Cancer Gene Therapy.Pre-experimental studies have shown that CLIC1 levels are highly expressed in vincristine-resistant gastric cancer cell lines(SGC-7901/VCR)and their secreted exosomes.We speculate that CLCI1 may be related to tumor resistance.Therefore,this subject was applied to normal SGC7901 cells by extracting the exosomes of SGC7901/VCR.Detection of resistance to vincristine and changes in CLIC1 levels.Simultaneous Silencing of SGC7901/VCR CLCI1 Gene to Detect Resistance to Vincristine.To investigate the role of CLIC1 in gastric cancer resistance in exosomes.Method:CCK-8 was used to determine the drug resistance of sensitive cell SGC-7901 and resistant cell SGC-7901/VCR to vincristine.qRT-PCR was used to determine the expression of CLIC1 gene in SGC-7901 and SGC-7901/VCR.Western Blot and immunofluorescence was used to determine the expression of CLIC1 protein in SGC-7901 and SGC-7901/VCR.Extraction of exosome of gastric cancer-resistant vincristine-resistant SGC-7901/VCR and gastric cancer cell line SGC-7901,Identifying the morphology of exosomes under an electron microscope;Western Blot Identification of Exosomes;Their protein concentrations were determined using BCA;The expression of CLIC1 gene in exosomes secreted by both cells was determined by qRT-PCR.SGC-7901/VCR-CLIC1-kd SGC-7901/VCR CLIC1 Gene Silenced by Lentivirus,qRT-PCR and Western Blot were used to determine the expression of CLIC1 gene and protein in two cells to determine the efficiency of silencing.CCK-8 was used to determine the resistance of both cells to vincristine resistance.The SGC-7901/VCR secreted exosomes were co-cultured in SGC-7901 cells.Get SGC-7901/exo.Observe the changes in the morphology of SGC-7901,SGC-7901/VCR,and SGC-7901/exo cells.Determination of Resistance to Vincristine in SGC-7901 and SGC-7901/exo by CCK-8;RT-PCR analysis of CLIC1 gene expression in SGC-7901 and SGC-7901/exo cells;Western Blot and Immunofluorescence Detection of CLIC1 Protein Expression in SGC-7901 and SGC-7901/VCR/exo Cells.Result:CLCI1 was highly expressed in the resistant cell line SGC-7901/VCR both at the gene and protein levels.The resistance of drug-resistant cells was significantly stronger than that of sensitive cells.Under the electron microscope,the exosome showed obvious typical oval cup-shaped structure,diameter 40-100 nm.;Exosomal marker proteins HSP-70 expressed;In the same volume of cell supernatant,the resistant strain SGC-7901/VCR secreted more exosome(V-exo)than the secreted exosome(7901-exo)of the sensitive cell line SGC-7901;The expression of CLIC1 in the exosomes secreted by both cells was detected by qRT-PCR and the expression of drug resistance was high.After Silencing,Validate SGC-7901/VCR/kd Low Expression CLIC1 by qRT-PCR,CCK-8 assay showed reduced resistance to vincristine in both cells.The morphology of SGC-7901/exo cells co-cultured with exosomes of drug-resistant cell lines was significantly changed compared to that of parental cells SGC-7901.Morphology changes toward drug-resistant cells;q RT-PCR results showed that the SGC-7901 CLIC1 gene was highly expressed in the co-cultured cell line SGC-7901/exo compared to the sensitive cell line SGC-7901;The same results were obtained with Western Blot and immunofluorescence assays for CLIC1 protein levels.In conclusion:(1).Human gastric cancer resistant SGC-7901/VCR is highly expressed at the level of gene and protein relative to human gastric cancer cell line SGC-7901.(2).Resistant cells secrete more exosomes than susceptible cells(3).CLIC1 can mediate the resistance of new drug-resistant strains of Changchun.Silencing CLIC1 can reduce the drug resistance of gastric cancer cell line SGC-7901 to vincristine.(4).The secreted exosomes of SGC-7901/VCR carry the CLIC1 gene and can be passed on to sensitive cell lines to increase its resistance to vincristine. |
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