| Part I Expression and clinical significance of miR-320 a and RACK1 in colorectal cancer Objective :Colorectal cancer(CRC)has a high incidence in the world and is a major disease that seriously endangers human health.The incidence of colorectal cancer in China is still on the rise.Early detection,early diagnosis and early treatment can achieve a higher cure rate and a better prognosis.Therefore,further exploration of the pathogenesis of colorectal cancer and search for new non-invasive tumor markers and therapeutic targets are of great clinical significance for early diagnosis,drug development and improvement of prognosis of colorectal cancer.As an important member of miRNA associated with colorectal cancer,miR-320 a has been reported to play a role as a tumor suppressor in colorectal cancer.The preliminary experiment of this subject combined with the prediction results of bioinformatics software suggests that,in colorectal cancer,miR-320 a may be involved in regulating the expression of activated protein kinase C1 kinase1(RACK1).The purpose of this section is to evaluate the diagnostic value of miR-320 a and RACK1 for colorectal cancer by detecting the expression of miR-320 a and RACK1 in the tumor tissues of patients with colorectal cancer,as well as the expression of miR-320 a in serum and feces.Methods:1?The expression levels of miR-320 a in cancer and adjacent tissues of colorectal cancer patients were detected by qRT-PCR,and the expression levels of miR-320 a in serum and feces samples of colorectal cancer patients and healthy subjects were detected by the same method.Differences in the expression of miR-320 a in patients with cancer and adjacent tissues,in serum samples from patients and healthy subjects,and in feces samples from patients and healthy subjects were compared.Receiver operating characteristic curve(ROC)was used to evaluate the diagnostic value of detection of miR-320 a in tissues,serum and feces samples for colorectal cancer.2?Western Blot(WB)was used to detect the expression level of RACK1 protein in colorectal cancer patients and adjacent tissues.The expression differences of RACK1 protein in cancer and adjacent tissues were compared.To evaluate the diagnostic value of detection of RACK1 protein in tissue samples for colorectal cancer.3?The clinicopathological data of colorectal cancer patients were collected to analyze the relationship between the expression of miR-320 a and RACK1 proteins in the cancer tissues and the clinicopathological characteristics of the patients.4?The expression levels of RACK1 m RNA in colorectal cancer and adjacent tissues were detected by qRT-PCR.RACK1 m RNA expression in cancer and adjacent tissues was compared.The correlation between RACK1 m RNA expression in cancer and adjacent tissues and RACK1 protein expression in cancer and adjacent tissues was analyzed.The correlation between the expression changes of miR-320 a in cancer and adjacent tissues and the expression changes of RACK1 protein in cancer and adjacent tissues was analyzed.5 ? Using online bioinformatics software(http://www.targetscan.org,Targetscan 6.0)to predict the target genes of miR-320 a.Result:1?The expression levels of miR-320 a in tumor tissues,serum and feces samples of colorectal cancer patients were significantly down-regulated(P<0.001).ROC curve analysis showed that the Area under the cure(AUC)of miR-320 a in tissue samples was 0.705,with a sensitivity of 42.9% and specificity of 71.4% for the diagnosis of colorectal cancer.The AUC of miR-320 a in serum samples was 0.844,and the sensitivity and specificity of miR-320 a in the diagnosis of colorectal cancer were 76.2% and 92.9%respectively.The AUC of miR-320 a in feces samples was 0.919,and the sensitivity and specificity of miR-320 a to the diagnosis of colorectal cancer were 81.0% and 95.2%.2?The expression level of RACK1 protein in the cancer tissues of patients with colorectal cancer was significantly upregulated(P<0.001).ROC curve analysis showed that the AUC of RACK1 protein in tissue samples was 0.909,with a sensitivity of 90.5% and specificity of 71.4% for the diagnosis of colorectal cancer.3?The expression level of miR-320 a in the tumor tissues of patients with colorectal cancer was correlated with the tumor size,degree of differentiation,depth of infiltration,presence of lymph node metastasis and vascular nerve invasion.The larger the tumor,the lower the degree of differentiation,the deeper the degree of infiltration,the presence of lymphatic metastasis and vascular nerve invasion,the lower the expression level of miR-320 a.The expression level of RACK1 protein was correlated with tumor size,degree of differentiation,depth of infiltration and presence of vascular nerve invasion.The larger the tumor,the lower the degree of differentiation,the deeper the degree of infiltration,and the presence of vascular nerve invasion,the higher the expression level of RACK1 protein.4?There was no significant difference in RACK1 m RNA expression between colorectal cancer and adjacent tissues(P=0.054).The correlation between the expression of RACK1 m RNA in cancer and adjacent tissues and the expression of RACK1 protein in cancer and adjacent tissues was very low(R=0.324,P=0.036).The expression changes of miR-320 a in cancer and adjacent tissues were negatively correlated with the expression changes of RACK1 protein in cancer and adjacent tissues(R=-0.800,P<0.001).5?The query of bioinformatics analysis software Targetscan found that there was a complementary sequence between miR-320 a and the 3'UTR of RACK1 gene,that is,RACK1 was a potential target gene of miR-320 a.Conclusion:1?The detection of miR-320 a and RACK1 proteins in colorectal tissues has certain sensitivity and specificity for the diagnosis of colorectal cancer.The detection of miR-320 a in serum and feces was highly sensitive and specific in the diagnosis of colorectal cancer,suggesting that miR-320 a could be used as a potential non-invasive tumor marker in the diagnosis of colorectal cancer.2?There was a strong negative correlation between the expression of miR-320 a and RACK1 in the tumor tissues of colorectal cancer patients,Low expression of miR-320 a and high expression of RACK1 protein were associated with the progression of colorectal cancer,combined with bioinformatics software prediction,miR-320 a may be involved in regulating the expression of RACK1 gene in colorectal cancer.Part II A preliminary study on the inhibition of proliferation,migration and invasion of colon cancer cells by miR-320 a targeting RACK1 Objective :RACK1 protein has been reported in the literature mostly as an oncogene to promote the occurrence and development of various types of tumors,and its role in colorectal cancer is still controversial.miRNAs regulate the expression of more than one third of human genes and are currently the largest family of genes that regulate the expression of genes.The clinical data analysis and bioinformatics software prediction results in the first part of this paper suggest that miR-320 a may be involved in regulating the expression of RACK1 gene in colorectal cancer.The purpose of this section is to clarify the role of RACK1 protein in the malignant biological behavior of colorectal cancer cells.To investigate the targeting and regulation of RACK1 gene by miR-320 a in colon cancer cells and its effect on the proliferation,migration and invasion of colon cancer cells.Methods:1?The expressions of miR-320 a and RACK1 proteins in various colon cancer cell lines were detected,and the typical cell lines were screened as further experimental objects.2?Short hairpin RNA(sh RNA)sh RACK1 was transfected into HCT116 cells by RNA interference(RNAi).The expression level of RACK1 protein was determined by WB after sh RACK1 transfection to determine whether the model was successfully constructed.MTT assay was used to observe the proliferation of HCT116 cells transfected with sh RACK1.Transwell ventricular migration and invasion assay were used to observe the changes of migration and invasion ability of HCT116 cells transfected with sh RACK1.3?AgomiR-320 a,a mimicry of miR-320 a,was transfected into HCT116 cells by liposome method.The expression level of miR-320 a in the cells after transfection with agomiR-320 a was determined by qRT-PCR,and the transfection efficiency was detected.The expression level of RACK1 protein was determined by WB after transfection with agomiR-320 a.The proliferation of HCT116 cells after transfection with agomiR-320 a was observed by MTT assay.Transwell ventricular migration and invasion assay were used to observe the changes of migration and invasion ability of HCT116 cells after transfection with agomiR-320 a.4?The binding effect of miR-320 a on 3'UTR of RACK1 gene was verified by double luciferase reporter method.Result:1?Among all colon cancer cell lines,the expression level of miR-320 a was the lowest in HCT116 cells,while the expression level of RACK1 was the highest.Therefore,HCT116 cells were selected for the next experiment.2?After transfection with sh RACK into HCT116 cells,the expression level of RACK1 protein in the cells was significantly down-regulated,indicating that the cell model with low expression of RACK1 protein was successfully constructed.The MTT assay showed that down-regulation of RACK1 protein expression in HCT116 cells inhibited the proliferation of tumor cells.The results of Transwell ventricular migration and invasion assay showed that down-regulation of RACK1 protein expression in HCT116 cells inhibited the migration and invasion of tumor cells.3?After transfection of agomiR-320 a into HCT116 cells,the detection results showed that the expression level of miR-320 a in the cells was significantly up-regulated,indicating that the cell model of miR-320 a overexpression was successfully constructed.The expression of RACK1 protein in the cells was significantly down-regulated,indicating that miR-320 a could inhibit the expression of RACK1 gene.The MTT results showed that upregulation of miR-320 a in HCT116 cells inhibited the proliferation of tumor cells.The results of Transwell ventricular migration and invasion assay showed that upregulation of miR-320 a in HCT116 cells inhibited the migration and invasion of tumor cells.4?The results of the dual luciferase reporter gene experiment showed that miR-320 a could specifically bind to the 3'UTR of RACK1 gene and inhibit the expression of luciferase,indicating that RACK1 was the target gene of miR-320 a.Conclusion:1?RACK1 gene exists in colorectal cancer cells as a pro-cancer factor,and its reduced expression will inhibit the proliferation,migration and invasion of tumor cells.2?miR-320 a inhibits the proliferation,migration and invasion of tumor cells in colon cancer cells by targeting and down-regulating the expression of RACK1 protein. |
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