The Role And Clinical Significance Of MiR-18a-5p Targeting Regulation Of RORA In The Proliferation Progression Of Colorectal Cancer

Background and purpose:Colorectal cancer(CRC)is a common malignant tumor of the gastrointestinal tract.Although CRC has been greatly improved in recent years in terms of screening and treatment,the 5-year survival rate of CRC patients has not been fundamentally improved,so it is urgent to explore the key molecular targets for CRC development and thus develop new targeted drugs.MicroRNAs(miRNAs)play a key role in regulating tumorigenesis and development,and can be positively regulated to promote tumor development or negatively regulated to inhibit tumor progression.It was found that microRNA-18a-5p(miR-18a-5p)has a role in regulating the development of various tumors,but the role and mechanism of miR-18a-5p in CRC is unclear.The aim of this study was to investigate the role played by miR-18a-5p and its target gene Retinoid-related orphan nuclear receptor A(RORA)in CRC and its clinical significance,so as to provide an experimental basis for the exploration of molecular targets for CRC diagnosis and treatment.Methods:1.To analyze the expression levels of miR-18a-5p and RORA in colorectal cancer and the correlation between them based on public databases.2.quantitative real-timePCR(qRT-PCR,)was used to detect the expression of miR-18a-5p and RORA mRNA in colorectal cancer tissues and paracancerous tissues;fluorescence in situ hybridization(FISH)and immumohistochemistry(IHC)methods were used to detect the expression of miR-18a-5p and RORA in colorectal cancer tissues.3.Based on the clinical data of 30 CRC patients,Chi-square test was used to analyze the relationship between the expression level of miR-18a-5p and clinicopathologic features of CRC patients.4.The expression levels of miR-18a-5p and RORA mRNA in colorectal cancer cell lines HCT116,RKO,SW620,SW480 and human colorectal epithelial cells HCoEpiC were detected by qRT-PCR.5.RKO cells were transfected with miR-18a-5p mimics(miR-18a-5p mimics),miR-18a-5p inhibitor(miR-18a-5p inhibitor)and the corresponding negative control sequences.qRT-PCR was performed to detect the expression level of miR-18a-5p in RKO cells after transfection to verify the transfection efficiency.6.Cell scratch assay and Transwell invasion assay were used to detect the migration and invasion ability of RKO cells in each stable transfection group.EDU and CFSE cell proliferation assay were used to detect the proliferation of RKO cells in each transfection group.Flow cytometry was used to detect the apoptosis rate of RKO cells in each transfection group.7.Bioinformatic prediction of miR-18a-5p and RORA gene binding site information,and detection of the targeting and regulatory effect of miR-18a-5p on RORA gene using dual luciferase gene reporter assay.8.qRT-PCR and Western blot(WB)to detect the effect of miR-18a-5p on RORA mRNA and RORA protein expression.9.Cell function rescue assay,cells were divided into miR-18a-5p mimics NC group,miR-18a-5p mimics group,miR-18a-5p mimics+oe-RORA group,oe-RORA group,to detect whether miR-18a-5p promotes the malignant phenotype of RKO cells by down-regulating RORA expression.10.Bioinformatics analysis combined with FISH and immunofluorescence staining was performed to verify the correlation of miR-18a-5p and RORA with the expression of the classical immunomolecular marker CD8A,respectively.Results:1.Through searching the online public database,it was found that miR-18a-5p and RORA were significantly high and low expressed in colorectal cancer tissues,respectively,and their expressions were significantly negatively correlated.2.Testing clinical tissue samples showed that compared to paracancer tissue,the expression of miR-18a-5p was significantly higher in colorectal cancer tissues,while the expression of RORA was lower 3.The overexpression of miR-18a-5p is closely related to the poor clinicopathological features of CRC patients 4.Compared with normal colorectal epithelial cells HCoEpiC,the endogenous expression of miR-18a-5p was significantly increased in four colon cancer cell lines(HCT116,RKO,SW620,SW480),while the endogenous expression of RORA was opposite 5 Overexpression of miR-18a-5p can significantly promote the proliferation,migration and invasion of RKO cells,while silencing the endogenous expression of miR-18a-5p can significantly inhibit the proliferation,migration and invasion of RKO cells,and induce the apoptosis ability of RKO cells.6.Dual luciferase gene reporter assay revealed that miR-18a-5p mimics significantly inhibited the fluorescence activity of wild-type sequence fluorescent vector(RORA-wt),but not its mutant sequence fluorescent vector(RORA-mut)in the 3’UTR region of RORA gene,so it was concluded that miR-18a-5p could target binding to the 3’UTR region of RORA.RT-PCR and WB results showed that mimics and inhibitor of miR-18a-5p significantly downregulated and upregulated RORA expression in RKO cells,respectively.7.Cell function rescue experiment showed that transfection of miR-18a-5p mimics significantly enhanced the invasion ability of RKO cells,while the enhanced effect of invasion was effectively reduced after transfection of mimics+oe-RORA(RORA overexpressed lentiviral vector).However,ce-RORA transfection significantly inhibited the invasion ability of RKO cells.Meanwhile,compared with the mimics transfection group,simultaneous transfection of mimics+oe-RORA significantly improved the apoptotic ability of RKO cells,and single transfection of oe-RORA could significantly induce apoptosis of RKO cells.8.Bioinformatics analysis combined with FISH and immunofluorescence staining showed that the expression of RORA in colorectal cancer tissues was significantly positively correlated with CD8+T cell infiltration and the expression of its surface marker protein CD8A,while miR-18a-5p was significantly negatively correlated with the expression of CD8A.Conclusions:miR-18a-5p plays a biological role in promoting the proliferation and invasion of colorectal cancer by targeting the regulation of RORA gene.This regulatory pathway may also play a key role in promoting the progression of colorectal cancer by regulating the infiltration of CD8+T cells in CRC tissues and reshaping the immune microenvironment of CRC.