| Objective:With the development of gene sequencing technology and bioinformatics technology,we have gradually discovered the important role of non-coding RNA.As a member of non-coding genes,mi RNAs participate in various life activities of human cells and play a regulatory role in many human diseases.It also plays an important role in the occurrence and development of tumors.Colorectal cancer is currently a major disease that endangers human health and is one of the most common malignant tumors in the world.Studies have shown that mi RNAs can not be ignored in the occurrence,metastasis,invasion,and prognosis of colorectal cancer.Studies have shown that mi R-30a-5p is abnormally expressed in tumors such as lung cancer,breast cancer,and ovarian cancer,and can regulate various pathophysiological processes of tumor cells at the post-transcriptional level.The purpose of this study was to study the expression and significance of mi R-30a-5p in colorectal cancer,in order to further explain the mechanism of colorectal cancer development and provide new potential targets for the treatment and prognosis of colorectal cancer.Methods:First,we collected mi R-30a-5p levels in 20 groups of colorectal cancer tissue samples and their corresponding adjacent tissue samples using q PT-PCR technology.Second,in normal intestinal epithelial cell line NCM460 and colorectal cancer cell lines DLD1,HCT116,and SW620,the levels of mi R-30a-5p were detected using q PT-PCR technology.The DLD1 cell line with the largest differential expression was selected for further experiments.Third,mi R-30a-5p overexpression model was constructed in DLD1 cells by cell transfection and counting,and cell functional experiments were performed.CCK8 was used to detect cell proliferation ability;plate cloning experiment was used to detect cell clone formation ability;plate scratches and The Transwell invasion assaymeasures the invasion ability of the cells.Then,the downstream targets of mi R-30a-5p were predicted by bioinformatics software,and verify its expression in clinical samples and cells.Finally,in the established mi R-30a-5p overexpression model,the expression of KRAS gene was restored by transfection technology,and the third part of the same cell functional experiment was used to observe whether the biological function of the cell could be reversed,which reversely confirmed mi R Interaction between-30a-5p and KRAS gene.The experimental raw data were analyzed and processed using SPSS 22.0 and Graph Pad 7.0.The data were expressed in the form of MEAN ± SD,and the differences between groups were analyzed by t test.* Represents P <0.05,**represents P <0.01,and P <0.05 is regarded as There is statistical significance.Results:1.Reduced mi R-30a-5p content in colorectal cancer cell lines Compared with the corresponding paracancerous tissue,the mi R-30a-5p content in colorectal cancer tissue was significantly down-regulated.Compared with normal intestinal epithelial cell line NCM460,the expression of mi R-30a-5p in colorectal cancer cell lines DLD1,SW620,and HCT116 decreased significantly,p <0.05,the difference was statistically significant,of which mi R-30a-5p in DLD1 The maximum expression difference was(0.092 ± 0.006),indicating that mi R-30a-5p was abnormally reduced in colorectal cancer tissues and cell lines.2.mi R-30a-5p overexpression can inhibit the proliferation,colony formation and invasion of colorectal cancer cells In DLD1 cells stably transfected with mi R-30a-5p mimic,we successfully constructed a mi R-30a-5p overexpression model.The results of cell functional tests performed in this model suggested that mi R-30a-5p was overexpressed.Can inhibit the proliferation,colony formation and invasion of colorectal cancer cells.3.KRAS is a downstream target gene of mi R-30a-5p Using Target Scan,mi RDB,and mi Randa databases,we predicted that the KRAS gene is a downstream target gene of mi R-30a-5p.The q RT-PCR results showed that the expression trend of KRAS m RNA in colorectal cancer tissues was opposite to that of mi R-30a-5p,indicating that the expression of KRAS gene may be directly regulated by mi R-30a-5p.4.Up-regulating the expression level of KRAS in mi R-30a-5p overexpression model can significantly reverse the inhibitory effect of mi R-30a-5p overexpression on cell function.By transfection of KRAS c DNA in the successfully constructed mi R-30a-5p overexpression model,we successfully increased the KRAS expression content in the mi R-30a-5p overexpression model,and confirmed the upregulation of KRAS expression by cell functional experiments Can significantly reverse the inhibition of mi R-30a-5p overexpression on colorectal cancer cell proliferation,colony formation and invasive ability.So far we have verified at the cellular level in vitro that mi R-30a-5p inhibits the key role of the malignant phenotype of colorectal cancer by negatively regulating the expression of the KRAS gene.Conclusions:1.The low expression of mi R-30a-5p in colorectal cancer tissues and colorectal cancer cell lines,indicating that mi R-30a-5p may be a molecule that has a suppressive effect on colorectal cancer;2.KRAS was the downstream target gene of mi R-30a-5p,and the expression of KRAS gene in colorectal cancer was opposite to that of mi R-30a-5p.3.Up-regulating the expression level of mi R-30a-5p in colorectal cancer cell DLD1 can significantly inhibit the proliferation,colony formation and migration ability of colorectal cancer cells.The above experimental results can be reversed by up-regulating KRAS expression,indicating that mi R-30a-5p is a tumor suppressor molecule,and it is negatively regulated with the KRAS gene. |
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