The Clinical Value Or Significance And Molecular Mechanisms Of Long Non-coding RNA UCA1 In Colorectal Cancer

Background: Colorectal cancer(CRC)is one of the most common gastrointestinal tumors in the world.In recent years,the incidence and mortality of Chinese CRC patients keep increasing.As CRC is prone to recurrence and metastasis,the 5-year survival rate of CRC patients is less than 20%.Therefore,searching for novel biomarkers for CRC early diagnosis will become a notable priority in CRC clinical practice. Lnc RNAs are defined as long RNA transcripts,which are longer than 200 nucleotides in length and cannot be translated into proteins.Studies have found that lnc RNAs are not only abnormally expressed in tumors,but also widely involved in the regulation of tumorigenesis through multiple mechanisms.Lnc RNAs are important biomarkers and targets for tumor diagnosis and treatment.In the early stage of this study,a carcinogenic lnc RNA-UCA1 was screened and the clinical feature was primarily determined through bioinformatics analysis.This paper intends to further explore the clinical significance and molecular mechanism of UCA1 in CRC through clinicopathological sample analysis,high-throughput RNA sequencing and cell function experiments.Objective: To explore the expression level and clinical significance of UCA1 in colorectal cancer,and explore the mechanism of UCA1 promoting the malignant biological phenotype of CRC cells via targeting GLUT1.Methods:(1)Based on the CRC datasets from GEO database and bioinformatics analysis tools,the expression of UCA1 in CRC and its correlation with clinicopathological features were further analyzed;(2)After inhibiting the expression of endogenous UCA1 in CRC cells through si RNA technique,the effects of si-UCA1 on the biological function of CRC cells were detected by CCK8 assay,plate clone formation assay,wound healing assay,Transwell migration assay,flow cytometry and nude mouse tumorigenicity assay;(3)The co-expression related pathways and co-expression genes of UCA1 in CRC were analyzed by bioinformatics analysis and GLUT1 was screened out.(4)89 pairs of clinical CRC tissues and matched normal mucosal tissues were collected,and the UCA1 expression was detected by in situ hybridization while the GLUT1 expression detected by immunohistochemistry.The correlation between clinicopathological characteristics of CRC patients and UCA1 or GLUT1 expression was respectively analyzed,and the correlation between clinicopathological characteristics of CRC patients and the co-positive expression of UCA1 and GLUT1 was also analyzed.(5)After inhibiting the expression of endogenous UCA1 in CRC cells through si RNA technique,RNA-seq was used to detect the variation of downstream gene expression profile of UCA1;(6)After inhibiting the expression of endogenous UCA1 in CRC cells through si RNA technique,the m RNA and protein expression level of GLUT1 were detected by q RT-PCR and Western blot assay;(7)The potential regulatory relationship between UCA1 and GLUT1 was explored by bioinformatics analysis,nucleocytoplasmic RNA separation assay,q RT-PCR and Western blot assay.Results:(1)Public database analysis showed that UCA1 was highly expressed in CRC tissues,and the high expression of UCA1 was positively correlated with the poor prognosis of CRC patients(p<0.05);(2)The results from CCK8 assay,plate clone formation assay,wound healing assay,Transwell migration assay,flow cytometry and nude mouse tumorigenicity assay showed that,compared with si NC group,the proliferation,invasion and metastasis of CRC cells in si-UCA1 group were significantly inhibited,while the level of apoptosis was significantly increased(p<0.05);(3)Bioformatics analysis showed that the expression of UCA1 in CRC was closely related to multiple signaling pathways,especially tumor metabolic signaling pathways.Correlation analysis by GEPIA showed that UCA1 was positively correlated with the expression of GLUT1,a key molecule in tumor metabolic signaling pathway(p<0.05);(4)Immunohistochemical and in situ hybridization results showed that UCA1 and GLUT1 were highly expressed in CRC tumor tissues,and the UCA1 expression was correlated to GLUT1 expression(p<0.05).The positive expression of UCA1 was associated with tumor N stage,the positive expression of GLUT1 was associated with tumor T stage and differentiation,and the co-positive expression of UCA1 and GLUT1 was associated with tumor N stage(p<0.05);(5)RNA-seq assay screened 613 differentially expressed genes after si-UCA1 process,including 322 up-regulated genes and 291down-regulated genes.GLUT1 was significantly down-regulated after si-UCA1;(6)The results of q RT-PCR and Western blot assay showed that the m RNA and protein expression levels of GLUT1 were inhibited(p<0.05);(7)Two mi RNAs,mi R-143-3p and mi R-873-5p,which could simultaneously bind to UCA1 and GLUT1,were screened out by bioformatics tools.Then results of q RT-PCR and Western blot showed that the m RNA and protein expression of GLUT1 was inhibited when mi R-143-3p/mi R-873-5p was overexpressed(p<0.05).The transfection of mi RNA inhibitor after si-UCA1 treatmen could reverse the decline of GLUT1 expression caused by si-UCA1(p<0.05).Conclusion:1.Both UCA1 and GLUT1 were highly expressed in CRC,and the co-positive expression of UCA1 and GLUT1 was associated with tumor N stage,which indicated that UCA1 and GLUT1 could be potential diagnostic markers of CRC;2.UCA1 may be involved in the regulation of the CRC tumorigenesis by binding to mi R-143-3p/mi R-873-5p and promoting the expression of GLUT1,suggesting that the UCA1-GLUT1 regulatory axis is expected to become a new target for CRC targeted therapy.