Preparation And Immune Activity Study Of Silkworm Peptide By Enzymatic Hydrolysis

Colorectal carcinoma(CRC)is a type of digestive system malignant solid tumor arising from large intestines with increasing incidences and poor prognosis.Recently,with the development of our country's economy,the incidences of colorectal carcinoma was higher than before in China,which affected the quality of the patients' life seriously.micro RNA(mi RNA)is a kind of single stranded RNA contained 18-25 nucleotides,which could not encode protein.Mio RNAs can exert various biological functions in tumors by influencing the expression of oncogenes and / or tumor suppressors.In recent years,precise treatment of colorectal carcinoma had achieved initial efficacy.Therefore,it had become an academic focus to further research the molecular mechanism of colorectal carcinoma development and to seek new and more effective therapeutic targets and methods.In recent years,some reports had been shown that the expression of mi R-552 was increased in the tumor tissues of colorectal carcinoma partents.Moreover,mi R-552 could induce the migration of colon cancer cell.However,the molecular mechanism by which mi R-552 affected colorectal carcinoma has not been well identified.Therefore,the purpose of this study was to investigate the role of mi R-552 in the occurrence and development-colorectal of carcinoma.Our research analyzed the expression of mi R-552 in colorectal carcinoma tissues and normol tissues firstly,and then the effect of mi R-552 on the proliferation and igration of colon cancer cell HT29 was detected,then we explored the molecular mechanism furtherly.This study supplied new idea of the molecular mechanism by which mi R-552 affect cancer occurrence and development.Part one Expression of mi R-552 in colorectal carcinoma and its relationships with clinicopathological features and prognosisObjective: To analyze the changement of mi R-552 expression in cancer tissues via detecting the expression of mi R-552 in colorectal carcinoma tissues and matched tissues;To explore the relationship between mi R-552 expression and clinicopathological features and prognosis of colorectal carcinoma patients.Methods:1.183 colorectal carcinoma patients were chosed randomly,and then the colorectal carcinoma tissues and matched normal tissues were got.The expression of mi R-552 in the colorectal carcinoma tissues and matched normal tissues were detected by q RT-PCR assay.2.All the patients were divided into two group according to the mi R-552 expression level: mi R-552 high expression group and mi R-552 low expression group.The relationships between mi R-552 expression and the clinicopathological features was analyzed by Logistic multivariate regression analysis.The survival time after surgery of all the patients were counted to analyz the relationships between mi R-552 expression and prognosis.Results:1.the results of q RT-PCR showed that the mi R-552 expression level in matched normal tissues was(3.12 ± 1.242),and the expression level of mi R-552 in tumor tissues was(9.87 ± 2.257).Compared with the matched normal tissues,the mi R-552 expression level in tumor tissues was significantly higher(P < 0.05).2.In 183 patients with colorectal carcinoma,mi R-552 expression was significantly associated with tumor differentiation,tumor TNM stage,and lymphatic metastasis,but mi R-552 expression was not significantly associated with the gender and age of patients.Using multiple linear regression analysis,there was a significant positive correlation between tumor differentiation,tumor TNM stage,lymphatic metastasis and the expression of mi R-552.3.The survival time of the patients in mi R-552 high expression group was significantly shorter than that of patients in mi R-552 low expression group(P < 0.05).Conclusions:1.The expression of mi R-552 was significantly higher in colorectal carcinoma tissues than in paracancer tissues,suggesting that mi R-552 may play a role in the development of colorectal carcinoma.2.The expression of mi R-552 was positively correlated with tumor differentiation,tumor TNM stage,lymphatic metastasis,which suggested that mi R-552 may be involved in the proliferation and migration of colorectal carcinoma.3.The expression level of mi R-552 was significantly associated with DFS in patients with colorectal carcinoma.It suggested mi R-552 may be a potential prognostic factor for colorectal carcinoma.Part two The effect of mi R-552 on the growth of mouse model of colon cancer xenograftObjective: To explore the effect of mi R-552 on the growth of mouse colorectal carcinoma by analyzing the effect of mi R-552 inhibitor on the growth of mouse model of colon cancer xenograft.Methods:1.12 male BALB/C mouse(18-20 g)were chosed randomly,and then4×106 SW620 cells were right armpit subcutaneous injection to get the mouse model of colon cancer xenograft.All the mouse were divided into two group:inhibitor group and control group when the cancer in mouse reached to 5-8mm.mi R-155 inhibitor(30 n M/100 ?L)was injected into the cancer in inhibitor group and the PBS was injected into the cancer in control group2.5 days later,the diameter of cancer was detcted by vernier calipers and count the volume of cancer: the volume(mm3)=the lengh(mm)× vertical width(mm)2/2?3.30 days later,the mouse were killed and the solid tumor was departed to detetc the weigth.Results:1.Compared with control group,the volume of solid tumor in inhibitor group was significantly decreased.2.The weight of control group mouse were 359 mg,421 mg,472 mg,489 mg,541 mg,519 mg;the weight of inhibitor group mouse were 258 mg,231 mg,220 mg,205 mg,174 mg,186 mg.compared with control group,the weight of solid tumor in inhibitor group was significantly decreased.Conclusions: mi R-552 expression in mice induced the growth of colon cancer xenografts.Part three mi R-552 induced the proliferation and migration of HT29 csell and to explore the molecular mechanismObjective: To investigate the effect of mi R-552 in the proliferation and migration of HT29,and to explore the regulation mechanism of mi R-552 in the colorectal carcinoma occurrence and development.Methods:1.Mi R-552 inhibitors were transfected into colon cancer cell line HT29 to achieve silence expression of mi R-552.2.WST1 assay was used to detecetd the cell proliferation,the ability of cell proliferation was represented by A540.3.The wound healing assay was used to detecetd the cell migration,and the ability of cell migration was represented by relative migration ratio.The relative migration ratio was defined as the ratio of the migration of mi R-552 silenced group and the migration of control group.4.Transwell assay was used to detecetd the cell migration,and the ability of cell migration was represented by A570.5.Western blot assay was used to detecetd the expression of ?-catenin.6.To get the ?-catenin silence expression HT29 cells.7.WST1 assay was used to detecetd the proliferation of ?-catenin silence expression HT29 cells and normal HT29 cells;8.The wound healing assay was used to detecetd the cell migration of?-catenin silence expression HT29 cells and normal HT29 cells.9.Transwell assay was used to detecetd the cell migration of ?-catenin silence expression HT29 cells and normal HT29 cells;Results:1.The results of WST1 assay showed that the A450 of control grouop cells was1.13.And the A450 of anti-mir-552 grouop cells was 0.521,which was significantly lower than that of control group(P < 0.05).The A450 of negative group was 1.08,which has no significant difference compared with control group.2.The results of wound healing assay showed that the relative migration of control group was(1.0 ± 0.0534);the relative migration of negative group was(1.04 ± 0.0461);the relative migration of anti-mir-552 group was(0.62 ±0.0218),which was significantly lower than that of control group(P < 0.05).3.The results of Transwell assay showed that the relative migration of control group was(1.0 ± 0.0534);the relative migration of negative group was(0.97 ± 0.121);the relative migration of anti-mir-552 group was(0.52 ±0.0415),which was significantly lower than that of control group(P < 0.05).4.The results of western blot showed that the phosphorylation level of?-catenin inanti-mir-552 group was significantly lower than that of control group(P < 0.05).5.The results of WST1 assay showed that the A450 of control grouop cells was 0.865.And the A450 of ?-catenin silence expression HT29 cells cells was0.487,which was significantly lower than that of control group(P < 0.05).6.The results of wound healing assay showed that the relative migration of control group was(0.87 ± 0.0331);the relative migration of ?-catenin silence expression HT29 cells was(0.42 ± 0.0251),which was significantly lower than that of control group(P < 0.05).7.The results of Transwell assay showed that the relative migration of control group was 1.0;the relative migration of ?-catenin silence expression HT29 cells was was 0.53,which was significantly lower than that of control group(P < 0.05).Conclusions: mi R-552 could induce the HT29 cell proliferation and migration via Wnt/?-catenin signal pathway which was activated by upregulating the phosphorylation of ?-catenin.In a word,the result of the study showed that mi R-552 could induce the activation of Wnt / ?-catenin signal pathway contributing to the happence and development of CRC.