| Objective: In this study,we examined the biological characteristics of micro RNA-744 in colorectal cancer and its relationship with gastrin-stimulated proliferation of colorectal cancer cell line SW480.The purpose of this study was to further clarify the possible regulatory role and molecular mechanism of gastrin and micro RNA-744 in the synergistic participation in the occurrence and development of colorectal cancer,not only for the early molecular diagnosis and individual of patients with colorectal cancer.Sexual therapy provides a reliable theoretical basis,and also provides a hidden target and experimental basis for the clinical diagnosis,treatment and prevention of colorectal cancer.Methods: 1.Biological characteristics of micro RNA-744 in colorectal cancer(1)Detection of the expression of micro RNA-744 in colorectal cancer tissues and cells In this study,real-time RT-PCR was used to detect the expression of micro RNA-744 in colorectal cancer tissues,non-cancer tissues and cells.(2)The effect of micro RNA-744 on the biological characteristics of colorectal cancer cells in vitro To detect the effects of micro RNA-744 on proliferation,apoptosis,migration and invasion of colorectal cancer cells in vitro.(3)Prediction and validation of downstream target genes of micro RNA-744 Target Scan,micro RNA anda and micro RNA.org were used to preliminarily predict and screen the target genes of micro RNA-744.Combined with the double luciferase reports in related literature,real-time fluorescent quantitative PCR and WB(Western Blot)were used to verify the results.2.The role of Micro RNA-744/Notch1 signaling pathway in gastrin-promoting cell proliferation(1)Real-time RT-PCR was used to detect the expression of micro RNA-744 in colorectal cancer cell line SW480 treated with pentapeptide gastrin(6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L)at different concentration gradients for 24 hours,and Western Blot technique was used to detect the changes of the target gene protein level of micro RNA-744.(2)Group I: blank control group,adding 10% fetal bovine serum medium RPMI-1640.Group II: In negative transfection group,micro RNA mimic Negative Control was transfected into cells instantaneously.After 24 hours,10% fetal bovine serum medium RPMI-1640 was replaced for further culture.Group III: Gastrin group,adding 10% fetal bovine serum medium RPMI-1640 containing pentapeptide gastrin(the optimum concentration).Group IV: Mi-744 mimic interference group was transfected into cells instantaneously.After 24 hours,10% fetal bovine serum medium RPMI-1640 was cultured.The expression abundance of micro RNA-744 in each group could be determined by real-time RT-PCR.CCK-8 method was used to detect the changes of cell proliferation in each group.Then Western Blot method was used to verify whether the protein levels of target genes in each group changed accordingly.In this study,statistical software SSPS22.0 was used for statistics and analysis.Measurement data were expressed in the form of mean(+standard deviation).Variance analysis and SNK-q test were used to compare the differences between groups.P<0.05 was considered to have statistical differences.Results: 1.The expression of micro RNA-744 was decreased in colorectal cancer tissues and cell lines.After overexpression of micro RNA-744 in SW480 cells,the cell proliferation,migration and invasion ability were significantly lower than those in the negative control group(P<0.05),and the apoptotic rate was higher than that in the negative control group(P<0.05).There was no significant difference between negative transfection group and blank control group(P>0.05).2.Using bioinformatics technology,combined with Target Scan(www.targetscan.org),Mianda(www.micro RNA.org)and micro RNA.org,the potential targets of micro RNA-744 were predicted,and Notch1 was predicted to be the target gene of micro RNA-744.Combining with the results of double luciferase reporter gene detection in related literatures,we preliminarily found that the 3'UTR region of Notch1 binds to the 5'end of micro RNA-744.The expression of Notch1 may be directly regulated by micro RNA-744.Notch1 expression and protein levels in SW480 cells overexpressed by micro RNA-744 were significantly decreased.3.Gastrin concentration in the range of 6.25-100 mg/L can down-regulate the expression of micro RNA-744 in colorectal cancer SW480 cells,and 25 mg/L has the strongest effect.Western_Blot results showed that the expression of Notch1 increased,and the highest expression was found at 25 mg/L gastrin concentration.4.Cell proliferation ability of mimic interference group(Group IV)transfected with micro RNA-744 was significantly lower than that of negative transfection group(Group II)and blank control group(Group I)(P<0.05);cell proliferation ability of gastrin group(Group III)was higher than that of negative transfection group(II group)and blank control group(Group I)(P<0.05);cell proliferation ability of gastrin group(Group III)was significantly higher than that of mimic interference group transfected with micro RNA-744(Group IV)(P<0.05).There was no significant difference in proliferation between the blank control group(Group I)and the negative transfectiongroup(Group II)(P>0.05).The results of PCR showed that the expression level of micro RNA-744 in Group IV was significantly higher than that in other three groups(P<0.05),while the expression level of micro RNA-744 in Group III was significantly lower than that in other three groups(P<0.05),while the expression level of Notch1 in WB was opposite to that of PCR,and the difference was statistically significant(P<0.05).However,there was no significant difference in the results of PCR and WB between negative transfection group(Group II)and blank control group(Group I)(P > 0.05).Conclusion: 1.The expression of micro RNA-744 in colorectal cancer decreased.2.Micro RNA-744 acts as a tumor suppressor in colorectal cancer and may inhibit the proliferation,migration and invasion of tumor cells by inhibiting the expression of Notch1 oncogene.3.The micro RNA-744/Notch1 signaling pathway play a regulatory role in the process of gastrin-induced proliferation of colorectal cancer cells... |
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