| Objective:Investigate the differential expression of exosomal RNA in plasma and saliva of spleen deficiency syndrome of gastric cancer.Develop a new noninvasive method for early diagnosis of gastric cancer and identification of spleen deficiency syndrome.Based on the changes of exosomal RNA of gastric cancer in the treatment of spleen deficiency syndrome of gastric cancer with Sijunzi decoction to explore the potential molecular mechanism of Sijunzi Decoction intervention of gastric cancer and spleen deficiency syndrome.Methods: The syndrome model of spleen deficiency in nude mice was established by combining the method of relieving bitter cold and abnormal hunger and satiety.Human gastric cancer cell line NCI-N87 was inoculated into the armpit of nude mice to grow gastric cancer tissue blocks.Subcutaneous tissue blocks were extracted and transplanted into spleen deficiency syndrome model of nude mice to establish the orthotopic mouse model of spleen deficiency syndrome of gastric cancer.The model nude mice were randomly divided into model group and traditional Chinese medicine group.The traditional Chinese medicine group was given routine feeding after surgery,and Sijunzi decoction was given once /d by gavage for 2w,while the other two groups were given the same amount of normal saline by gavage.Regular monitoring of body weight,drinking water and food intake in nude mice during modeling period,as well as macro-characterization assignment score of TCM.Postoperative weight changes and tumor metastasis of the nude mice were monitored regularly.Saliva samples were collected on the 22 nd day and the 24 th day of the experiment.At the end of the experiment,blood was collected with orbital venous sinus bleeding and plasma was obtained by centrifugation.The solide tumor was exfoliated for tumor weight measurement.Tumor inhibition rate was calculated.The exosomal RNA from plasma and saliva was extracted.The small RNA libraries were constructed and analyzed with next generation sequencing technology.The exosomal RNA of differential expression was analyzed by bioinformatics and biostatistical analysis.Result: Ⅰ.Experimental results of animal model construction of orthotopic transplanted mice with spleen deficiency syndrome of gastric cancer(1)After the construction of spleen deficiency syndrome model,the nude mice were observed to be listless,with fatigue and hudding,squinting of eyes and bowing of the back,emaciation,soft stool and loose stools.Compared with the normal group,the food intake,water intake and body weight of nude mice were significantly decreased(P< 0.01).The macroscopic evaluation scores of TCM in nude mice were all above 5points,and the scores were significantly higher than those in normal group(P < 0.01).(2)Based on the construction of spleen deficiency syndrome type,gastric cancer was successfully constructed by in situ transplantation in nude mice.Ⅱ.Explore the experimental results of the molecular mechanism of Sijunzi Decoction in the treatment of spleen deficiency syndrome of gastric cancer based on exo RNA.(1)Compared with the normal group,the body weight of the model group and the SJZD group decreased on the 12 th,15th,18 th,21st and 24 th days,which was statisti cally significant;after SJZD treatment,the body weight of the SJZD group increased significantly compared with the model group on the 18 th,21st and 24 th days(P <0.01),and the overall state of the nude mice was more active than before,with less squinting and huddle reactions,and moderate softness and hardness of stool.compared with the model group,the tumor weight in SJZD group was significantly lower than that in the model group(P < 0.01).(2)In this study,based on the standard of P<0.05,|Log2(FC)|>1,bioinformatics and biostatistics were used to analyze the expression of differential miRNA.Compared with the human genome sequence,the expression of miR-328-3p(Log2(FC)=-2.637)and miR-339-5p(Log2(FC)=-2.338)in the model group was up-regulated compared with the control group,and the expression was decreased after Sijunzi Decoction intervention.The expressions of miR-1-3p(Log2(FC)= 1.450),miR-151a-3p(Log2(FC)=3.267),miR-184(Log2(FC)=1.880),miR-202-5p(Log2(FC)= 2.511)and miR-34C-5p(Log2(FC)= 2.960)in the model group were decreased compared with the control group,and the expressions were up-regulated after Sijunzi Decoction intervention.Compared with the mouse genome sequence,the expression of miR-328-3p(Log2(FC)=-2.595)and miR-339-5p(Log2(FC)=-2.274)in the model group was up-regulated compared with the control group,and the expression was decreased after Sijunzi decoction intervention.The expressions of miR-184-3p Log2(FC)=1.929),miR-1a-3p(Log2(FC)=1.507),miR-202-5p(Log2(FC)=2.563),miR-3470a(Log2(FC)= 3.001),miR-3470b(Log2(FC)=3.609),miR-34c-5p(Log2(FC)= 3.015),and miR-466i-5p(Log2(FC)= 4.990)in the model group were decreased compared with the control group,and the expressions were up-regulated after Sijunzi decoction intervention.Sijunzi Decoction has a good effect of invigorating spleen and replenishing qi in the treatment of spleen deficiency syndrome of gastriccancer,which may be related to its effect on target genes of differential miRNA.It participates in the occurrence,development and process of spleen deficiency syndrome of gastric cancer through the regulation of target genes.(3)Analysis of miRNA expression in saliva showed that miR-184 was in the model group compared with the human genome sequence.Compared with the control group,the expression was higher and decreased after medication.Mi R-151a-3p in the model group was lower than that in the control group.The expression was upregulated after treatment.No significant differentially expressed genes were found by comparing with the mouse genomic sequences.(4)The model group was compared with the control group,human differences in interacting saliva miRNA and human differences in plasma miRNA found that miR-145-3p,miR-1-3p,miR-3615,miR-151A-3p,miR-142-3p,miR-184,miR-126-3p,among the 11 plasma samples,miR-28-5p,miR-500a-3p,miR-7-5p,and miR-320a-3p,human-specific miRNAs also existed in salivary exosomes.That is to say,exosomes secreted by gastric cancer cells reach salivary glands through fluid circulation,are absorbed by salivary gland cells through fusion and endocytosis,and are eventually released into saliva.Conclusion:(1)Sijunzi Decoction may play a role in the occurrence and development of spleen deficiency syndrome of gastric cancer by regulating the plasma and saliva human common differential target gene miR-151a-3p,and play a role in strengthening spleen and replenishing qi.(2)miR-145-3p,miR-1-3p,miR-3615,miR-151A-3p,miR-142-3p,miR-184,miR-126-3p,among the 11 plasma samples,miR-28-5p,miR-500a-3p,miR-7-5p,and miR-320a-3p,human-specific miRNAs also existed in salivary exosomes,Saliva diagnosis is expected to be a new non-invasive method for early diagnosis of gastric cancer and syndrome identification of spleen deficiency. |
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