Mechanisms Of Pristimerin Synergistic PAMAM/miR-144 Inhibiting The Migration And Invasion Of Gastric Cancer

Gastric Cancer(GC)is one of the most common cancers of the digestive system,with a high incidence and mortality rate,ranking in the top five of all cancers respectively.Men are approximately twice as likely to be sick as women compared to the female rate.Most patients are already at an advanced stage when diagnosed,resulting in a very high mortality rate for patients.Previous studies by our group have shown that microRNA-144(miR-144)has an abnormally low expression in gastric cancer cells and has a tumor suppressor-like effect,which has potential clinical application.However,the free miRNAs can be digested by nucleases in serum and are easily degraded by lysosomes even when they reach the target tissue.Therefore,in this study,PAMAM/miR-144 nanocomplexes were constructed to protect miR-144 from nuclease degradation,deliver miR-144 to the tumor microenvironment,increase the content of miR-144 in gastric cancer tissues and inhibit the growth of gastric cancer.Pristimerin is a naturally occurring quinone triterpene monomeric compound that can be extracted from plants of the genera Weeping and Sea Buckthorn.Recently,it was found that Pristimerin significantly prevented the growth and proliferation of tumor cells and induced their apoptosis.This thesis focuses on the effects and molecular mechanisms of Pristimerin in combination with PAMAM/miR-144 to inhibit the migration and invasion of AGS cells.The study is divided into four main parts.Part Ⅰ Correlation analysis of miR-144 expression and gastric cancerObjective:To investigate relationship between the expression level of miR-144 and the occurrence of different types of gastrointestinal malignancies.Methods:Using bioinformatics techniques,data on six gastrointestinal malignancies,namely oesophageal,gastric,liver,bile duct,pancreatic,and colorectal cancers,were mined from the database and one-way analysis of variance was used to compare the differences in miR-144 expression levels between tumor tissue samples and normal tissues samples.Results:The expression of miR-144 was significantly lower in human pancreatic,liver,bile duct and gastric cancer tissues and higher in colorectal cancer than in normal tissues(P<0.05).Conclusion:The expression of miR-144 was significantly reduced in Gastric Cancer.Part Ⅱ Effect of Pristimerin combined with PAMAM/miR-144 on AGS cellsObjective:To investigate the effect and molecular mechanism of Pristimerin in combination with PAMAM/miR-144 to inhibit the invasion and metastasis of AGS cells.Methods:Construction of PAMAM/miR-144 nanoparticles.AGS cells were treated with PAMAM/miR-144 for 24 h.Different concentrations of Pristimerin(0.2,0.4,0.8,1.6 and 3.2μM)were added to the combined treatment for 24 h.The effect of the combination of Pristimerin with PAMAM/miR-144 on the proliferation ability of AGS cells was examined by MTT.Transwell assay and Scratch assay were performed to detect changes in the invasion and migration ability of AGS cells.qRT-PCR was performed to detect the expression level of mTOR mRNA.Western blot was performed to detect the expression of PI3K/AKT/mTOR signaling pathway related proteins.Results:PAMAM/miR-144 nanoparticles were successfully constructed with stable physicochemical properties and biological activity of reducing mTOR mRNA.Compared with the group treated with PAMAM/miR-144 alone,the combination of Pristimerin and PAMAM/miR-144 significantly inhibited the proliferation of AGS cells(P<0.05);reduced the scratch healing of AGS cells(P<0.05);significantly inhibited the migration and invasion of AGS cells;and significantly reduced the expression of mTOR mRNA expression in AGS cells(P<0.05).The expression of mTOR,PI3K,AKT,4EBP1 and other proteins in AGS cells were reduced to different degrees(P<0.05).Conclusion:Pristimerin combined with PAMAM/miR-144 had a killing effect on AGS cells and may inhibit the invasion and migration of AGS cells by mediating the PI3K/AKT/mTOR signaling pathway.Part Ⅲ Safety testing of PAMAM/miR-144 nanoparticleObjective:To investigate whether PAMAM/miR-144 nanoparticles,have biosafety for in vivo therapeutic use.Methods:PAMAM solution(2.5 μg/μL),miR-144-CY5 solution(10 pmol/mL)and PAMAM/miR-144-CY5 solution(PAMAM:miR-144 volume ratio=1:2)were prepared and injected into mice through the tail vein at 3-5 times the normal therapeutic dose,to observe the mental status of mice and to record body weight,diet and other general conditions.The fluorescence intensity of the tail vein of the mice was measured by small animal live imaging at 1 h,16 h and 96 h after injection.On day 7 and day 14,half of the mice in each group were executed and serum was collected for biochemical tests including liver and kidney function.The organs of the heart,liver,spleen,lungs and kidneys were washed and drained,weighed and recorded,and pathological sections were made to observe whether there were pathological changes in the organs of the mice under the microscope.Results:There was no significant change in body weight of the mice within 14 d,indicating that PAMAM/miR-144 had no significant effect on the growth of the mice.The ALP levels and AST/ALT ratios of mice in all groups remained normal,indicating normal liver function.Compared with the normal control group,the blood urea nitrogen(BUN),cholesterol(CHOL),triglyceride(TG)and phosphorus(P)levels did not change significantly in all groups of mice,indicating normal renal function in all mice.No abnormal changes were observed in the histopathological images of the heart,liver,spleen,lung,kidney and stomach of each mouse.No significant differences were observed when comparing the organ index(i.e.organ weight to body weight ratio,mg/g).Small animal live imaging showed no fluorescence in the tail vein of mice in the control and PAMAM groups.miR-144-CY5 group,only one of the eight mice showed fluorescence in the tail vein at 1 h and 16 h after injection,and the fluorescence disappeared at 96 h.In the PAMAM/miR-144-CY5 group,fluorescence was visible in the tail vein of each mouse at 1 h,16 h and 96 h.Conclusion:PAMAM/miR-144 is biocompatible and has a strong protective effect against degradation of miR-144 by nucleases in the blood.The biosafety is relatively good and can be used for in vivo treatment.Part Ⅳ In vivo study of Pristimerin combined with PAMAM/miR-144 in the treatment of gastric cancerObjective:To investigate the distribution of PAMAM/miR-144-CY5 in human gastric cancer tumor-bearing nude mice,and the therapeutic effect of Pristimerin in combination with PAMAM/miR-144 on gastric cancer transplanted tumors in mice.Methods:The distribution of PAMAM/miR-144-CY5 in tumor-bearing mice was first investigated.12 BALB/c nude mice were divided into 4 groups of 3 mice each.After 10 d,when the transplanted tumors were clearly visible to the naked eye,the tail vein was injected with saline,PAMAM(2.5 μg/μL),miR-144-CY5 solution(10 pmol/mL)and PAMAM/miR144-CY5 solution(PAMAM:miR-144 volume ratio=1:2)each at 20 μL,at 6 h,24 h and 48 h after injection,respectively,to observe the targeting transport effect of PAMAM/miR-144CY5 by using small animal live imaging system.After the shooting,the mice in each group were executed and the heart,liver,spleen,lung,kidney,stomach and transplantation tumor were removed for in small animal live imaging system to observe the retention of PAMAM/miR-144-CY5 in different organs and tissues.Twenty-four BALB/c nude mice were randomly divided into 4 groups of 6 mice each.They were the negative control group,the PAMAM/miR-144 group,the Pristimerin combined with PAMAM/miR-144 group and the DDP group,respectively.In the negative control group,20 μL of saline was injected into the tail vein;in the PAMAM/miR-144 group,20 μL was injected into the tail vein;Pristimerin(0.8 mg/kg)was injected intraperitoneally;and DDP(2μg/mL)was injected intraperitoneally with 200 μL.After the administration of each group,the nude mice were observed for their mental and dietary conditions,and the length and diameter of the tumors were measured every 3 days using vernier calipers,and the tumor volumes were calculated.Every 7 days,Small animal live imaging was performed to observe and record the tumor growth in real time.At the end of treatment on day 21,the nude mice were executed,the tumors were peeled off and the volume and weight of the transplanted tumors were compared between the groups.Results:The distribution of PAMAM/miR-144-CY5 in tumor-bearing mice.In four groups of mice,the stripped heart,liver,spleen,lung,kidney,stomach and tumor were imaged in small animal live system,and red fluorescence was found only in the transplanted tumor of the PAMAM/miR-144 group.This indicates that PAMAM/miR-144 is capable of targeted transport into the tumor tissue and exerts a therapeutic effect.Red fluorescence was seen in both the miR-144-CY5 and PAMAM/miR-144-CY5 groups at 6 h.At 24 and 48 h,only the PAMAM/miR-144-CY5 group showed red fluorescence,indicating that PAMAM protected miR-144 from nuclease degradation in the tumor.The therapeutic effect of Pristimerin in combination with PAMAM/miR-144 on gastric cancer transplantation tumors in mice was investigated.Microscopic observation of the transplantation tumor tissues revealed that the tumor cells in each group were disorganized and of different sizes,with deeply stained nuclei and pathological nuclear division,indicating that the transplantation tumor model of gastric cancer in nude mice was successfully constructed.Compared with the control group,treatment with PAMAM/miR-144 alone resulted in shrinkage and weight reduction of the transplanted tumors;compared with the group treated with PAMAM/miR-144 alone,the oncogenic effect of Pristimerin combined with PAMAM/miR-144 was more significant(P<0.05).Conclusion:PAMAM/miR-144 is capable of targeted transport into tumor tissue for therapeutic action.Pristimerin,in combination with PAMAM/miR-144,significantly inhibited the growth of gastric cancer transplanted tumors in nude mice.