| BackgroundColorectal cancer(CRC)is one of the most common malignant gastrointestinal tuMors in the world,with the third highest incidence of malignant tuMors and the second highest mortality rate.Therefore,to explore the pathogenesis of colorectal cancer and looking for the cause of colorectal cancer is an urgent problem.In recent years,the regulation of tuMors by long-chain non-coding RNA has attracted more and more attention,and it has become one of the emerging hotspots of cancer research.Based on clinical practical issues,this study aims to reveal the regulatory role of long-chain non-coding RNA in the pathogenesis and progression of colorectal cancer,and to explore potential markers for the diagnosis and prognosis of colorectal cancer.ObjectiveTo investigate the regulatory function and mechanism of aberrantly expressed long non-coding RNA LINC01559 on the pathogenesis and progression of colorectal cancer.MethodsThe cancer and paracancerous tissues of 6 patients in our hospital were collected.High-throughput sequencing was used to detect the expression profile of lncRNA and mRNA in colorectal cancer.The expression of LINC01559 in tissues and cells was verified by qRT-PCR.CCK-8 cell proliferation and EdU cell proliferation assay confirmed the effect of LINC01559 on the proliferation function of colorectal cancer cell lines HCT116 and SW480.Transwell invasion and metastasis experiments and scratch experiments confirmed the interference of LINC01559 on the migration function of SW480 cell line.Fluorescence in situ hybridization(FISH)experiments confirmed the localization of LINC01559 in cancer cells.The bioinformatics website(LncBase Predicted v.2 and TgrgetscanHuMan)predicted the molecular targeting relationship between miR-106b-5p and LINC01559 and PTEN.SPSS.21 was used for statistical analysis,the count data was analyzed by chi-square test,and the measurement data was analyzed by t test.p<0.05 showed significant statistical differences.Results 1.Expression characteristics of LINC01559 in colon cancer tissues and cell lines Six patients underwent high-throughput sequencing of cancer and cancer.The sequencing results showed that LINC01559 was lowly expressed in colorectal cancer tissues compared with normal intestinal mucosa,and the difference was statistically significant(p<0.05).Expanded clinical sample to verify the expression characteristics of LINC01559 in 30 patients with cancer and adjacent tissues,and found that the expression of LINC01559 in cancer tissues was lower than that in adjacent tissues,the difference was statistically significant(p<0.05);and normal intestinal mucosa with FHC Compared with cell lines,LINC01559 was significantly down-regulated in HCT116,SW480,and HT29 colon cancer cell lines,and the difference was statistically significant(p<0.05).2.The relationship between the expression characteristics of LINC01559 and the malignant phenotype of colorectal cancer patientsUnivariate analysis(chi-square test)showed that the low expression of LINC01559 was correlated with clinical TNM stage,nerve infiltration,lymphatic metastasis and distant metastasis(all p<0.05);LINC01559 low expression and patient age,sex,tuMor size,There was no correlation between tuMor location,degree of differentiation and vascular invasion(all p>0.05).3.LINC01559 can regulate the biological function of colon cancer cell linesEdU cell proliferation assay and the CCK8 proliferation assay confirmed that the proliferation of the cancer cell lines HCT116 and SW480 was enhanced after interference with the expression of LINC01559(compared to the negative control group).Transwell invasion and scratch test confirmed that the invasion and metastasis of colon cancer cell line SW480 was enhanced after interference with LINC01559 expression compared with the negative control group.Angiogenesis experiments were performed in HCT116 and SW480 cell lines,and it was confirmed that interference with LINC01559 expression was more conducive to HUVEC angiogenesis than the negative control group.4.Interference with the expression of LINC01559 on the expression of EMT-related moleculesIn the SW480 cell line,qRT-PCR confirmed that the expression of E-cad and ZO-1 was decreased,and the expression of N-cad,ZEB-1,ZEB-2,and MMP-2 was increased after interference with LINC01559(compared to the negative control group).(all p<0.05).Using GEPIA(Gene Expression Profiling Interactive Analysis)website analysis,LINC01559 and E-cad(CDH1)expression were positively correlated,and negatively correlated with TWIsT and snail-1,the difference was statistically significant(all p<0.05).Western blot analysis showed that si-LINC01559 inhibited the expression of cadherin E-cadherin in HCT116 cell line compared with the negative control group;in SW480 cell line,si-LINC01559 inhibited cadherin E-cadherin and ZO-1 expression promotes ZEB-1 expression.5.LINC01559 competitively binds miR-106b-5p through ceRNA principle,regulates PTEN/AKT signaling pathway and affects colorectal cancer progressionThe bioinformatics prediction(LncBase Predicted v.2)found that the LINC01559 sequence and the miR-106b-5p sequence have targeting sites.Fluorescence in situ hybridization(FISH)experiments confirmed that LINC01559 is present in the cytosol.Dual luciferase reporter assays demonstrated that LINC01559 can target miR-106b-5p.The bioinformatics website predicted(TgrgetscanHuMan)found that miR-106b-5p and PTEN have targeted binding sequences.In the HCT116 and SW480 lines,we performed an invasive transfer remediation experiment.The results showed that after the interference of LINC01559,the colon cancer cell invasion and metastasis ability was enhanced.After the addition of miR-106b-5p inhibitor,si-LINC01559 attacked colon cancer.The enhancement effect of the transfer function is weakened.Indirectly,LINC01559 affects the progression of colorectal cancer by regulating miR-106b-5p.ConciusionLINC01559 is down-regulated in colorectal cancer tissues and is closely related to the malignant clinical phenotype of tumors.LINC01559 regulates PTEN/AKT signaling pathways in colorectal cancer progression by competitively binding to miR-106b-5p. |
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