| Background:Gastric cancer(GC)is the fifth most common cancer in the world,with a very high mortality rate.Since most patients are diagnosed in the middle and advanced stages,most patients miss the chance of surgery,cauce chemotherapy to be the main treatment,and platinum is an important part of chemotherapy.Clinically,however,patients usually develop drug resistance after several cycles of chemotherapy,which limits the overall efficacy.Therefore,it is necessary to find new ways to improve the sensitivity of chemotherapy.c-Met is overexpressed in a variety of tumors,including gastric cancer,and acts as a "driver" for carcinogenesis in the proliferation,invasion,and metastasis of gastric cancer cells,leading to poor prognosis of gastric cancer.Therefore,it is regarded as a key target for the treatment of gastric cancer.As a new method,small interfering RNA(siRNA)has great potential in the treatment of diseases caused by pathogenic genes,and exosomes serve as a new carrier for the delivery of various biologically active substances for treatment.Therefore,reducing c-Met expression level in gastric cancer cells by exosomes carrying siRNA may be a promising way to increase the sensitivity of cancer cells to chemotherapy and reverse the resistance of refractory gastric cancer to cisplatin.Objective:The purpose of this study was to determine whether exosomes coated with si-c-Met can reduce the expression level of c-Met in gastric cancer,and then used in combination with traditional chemotherapy drug cisplatin in drug-resistant gastric cancer to reverse the resistance of gastric cancer to cisplatin to improve the treatment effect of gastric cancer patients.Methods:1.Clinical level:(1)We used Kaplan-Meier plotter analysis to demonstrate that the correlation between the difference in c-Met protein expression and the survival of gastric cancer patients;(2)Gastric cancer tissues and adjacent noncancerous tissues were collected,and detect the expression of c-Met at the protein level and mRNA level;2.In vitro:(1)SGC7901 and SGC7901/DDP were transfected with c-Met siRNA,Western blot and RT-qPCR were used to detect the expression levels of c-Met respectively.(2)HEK293T cells were transfected with siRNA of c-Met and exosomes with si-c-Met were obtained by sequential differential centrifugation,exosomes were co-cultured with SGC7901 and SGC7901/DDP for subsequent cell functional experiments;3.In vivo:(1)Transplantation tumor model in nude mice:the SGC7901/DDP cells were selected to set up tumor models in nude mice subcutaneously in the left groin of nude mice,when tumors grew to about 8mm in diameter,20μg of different exosomes(suspended in 40μl PBS)or PBS were injected through the caudal vein every other day,and intraperitoneal injection of 5 mg/kg DDP every other 3 days.(2)The length and short diameter of tumors were recorded every 2 days and calculate the volume,the growth rate of tumor injected with si-c-Met exosomes was slower than that of the other two control groups;(3)The expression level of c-Met in tumors of different groups of mice were verified by Western blot and RT-qPCR.Results:1.Clinical level:Kaplan-Meier plotter database analysis shows,during the follow-up,the survival rate of c-Met high expression patients were consistently higher than the patients with c-Met low expression.It can be seen from the graph that the overall survival of c-Met high expression patients group was lower than that with low expression group during the follow-up period.Then we selected 8 pairs of cancer tissues and adjacent normal tissues from GC patients.Level of c-Met was detected by immunohistochemistry,and it was found that the expression of c-Met was significantly upregulated in cancer tissues compared with that in noncancerous tissues,followed by western blot and got consistent results.2.In vitro:To explore the inhibitory effect of small interfering RNA on c-Met,si-c-Met and si-NC were transfected into SGC7901 and SGC7901/DDP cells,the level of c-Met was detected by qRT-PCR,and c-Met protein expression was verified by western blot,and results indicate that si-c-Met could significantly reduce the expression level of c-Met.We added gradient concentrations of cisplatin to SGC7901 and SGC7901/DDP cells,based on preliminary results,the concentration of cisplatin in SGC7901 was 0.09375-6μg/ml,while ranged from 0.1875-12μg/ml in SGC7901/DDP.We found that it has the most significance difference of inhibition rate at the concentration of 3μg/ml,so 3μg/ml is the most suitable concentration for subsequent experiments.We tested cell viability by CCK-8 reagent and then calculate the inhibition rate.Si-c-Met could increase the sensitivity of two cell lines to cisplatin and significantly reversed the resistance of SGC7901/DDP cells to cisplatin.Extracting exosomes from HEK293-T medium,and co-cultured with SGC7901 and SGC7901/DDP cells,it was confirmed by qRT-PCR that exosomes loaded si-c-Met could significantly down-regulate the expression of c-Met at the gene level compared with exosomes carrying si-NC and obtained consistent results by western blot that exo-si-c-Met could down-regulate the expression of c-Met consistent with transfecting si-c-Met directly.A series of subsequent cell function experiments confirmed that exosomes with si-c-Met can significantly increase the sensitivity of gastric cancer cells to cisplatin,significantly inhibit the migration and proliferation of gastric cancer cells and promote apoptosis level of gastric cancer cells,and this effect is more obvious in drug-resistant cells SGC7901/DDP,which proving that exo-si-c-Met could reverse the resistance of gastric cancer cells to cisplatin.3.In vivo:SGC7901/DDP was selected to set up tumor models in nude mice subcutaneously.When tumors grew to about 8mm in diameter,when tumors grew to about 8mm in diameter,20μg of different exosomes(suspended in 40μl PBS)or PBS were injected through the caudal vein every other day,and intraperitoneal injection of 5 mg/kg DDP every other 3 days.The length and short diameter of tumors were recorded every 2 days and calculate the volume.Compared with the other two control groups,tumor volumes did not increase significantly in the treatment group.After 2 weeks,tumors were removed and weighed.Extracted RNA from each tumor and qRT-PCR was performed to detect the level of mRNA of c-Met in each group of tumors.The results showed that the level of c-Met mRNA in the exo-si-c-Met group was significantly lower than that in control groups,then verified by western blot,the expression level of c-Met was also significantly decreased in exo-si-c-Met group.The above results demonstrate that exo-si-c-Met could reverse the resistance of cisplatin in vivo.Conclusion:1.c-Met is overexpressed in gastric cancer tissues,and high expression of c-Met is associated with poor prognosis in gastric cancer patients;2.si-c-Met can significantly reduce the expression level of c-Met in SGC7901 and SGC7901/DDP cells and reverse the resistance of gastric cancer cells to cisplatin;3.Exosome with si-c-Met can inhibit the expression level of c-Met in gastric cancer cells as transfection of si-c-Met directly;4.Under the treatment of cisplatin,exosomes with si-c-Met can significantly inhibit the migration and proliferation ability of gastric cancer cells and promote the apoptosis of gastric cancer cells,which can reverse the resistance to cisplatin in gastric cancer cells;5.It has been confirmed that under the treatment of cisplatin,exosomes with si-c-Met can reverse the resistance of gastric cancer to cisplatin in vivo and significantly inhibit the growth of mouse tumors. |
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