The LncRNA PTOV1-AS2/miR-145-5p/FSCN1 Axis Affects The Biological Properties Of Colorectal Cancer Cells And Its Mechanism

IntroductionThe incidence and mortality rate of colorectal cancer in China are increasing year by year.Most sporadic colorectal cancers are formed by the slow development of precancerous lesions.Early screening combined with effective treatment can reduce morbidity and mortality.Currently,surgical resection,chemotherapy,radiotherapy,immunotherapy and other treatments have side effects,such as postoperative infection,radiotherapy toxicity,immune adverse reactions,and high recurrence rates.Therefore,early diagnosis and effective treatment of colorectal cancer are particularly important.Biomarkers are less invasive and more accurate in predicting disease,which help to achieve precise targeting of tumors.LncRNAs have shown great potential in cancer research as prognostic markers for diagnosis and treatment,with dual roles in promoting and inhibiting cancer development.lncRNAs play an important role in tumor development by sponging miRNAs and thereby inhibiting the effects of miRNAs on target genes and altering the expression levels of target genes.In this study,we investigated the role of lncRNA PTOV1-AS2 in the development of colorectal cancer,explored the interrelationship between PTOV1-AS2-binding miRNAs and target genes,and revealed its intrinsic regulatory mechanism to provide new targets for the diagnosis,treatment and prognosis of colorectal cancer.Part Ⅰ:PTOV1-AS2 expression and clinical relevance in colorectal cancerObjectives1.Detection of PTOV1-AS2 expression in colorectal cancer tissues and cells2.To investigate the association of PTOV1-AS2 expression with patient clinicopathology and overall survival.Methods1.To detect the expression of PTOV1-AS2 in colorectal cancer tissues and cell lines by qRTPCR and to analyze the differences in expression with normal tissues and cells.2.To count the information of clinicopathological parameters of colorectal cancer patients and analyze their correlation with PTOV1-AS2 expression levels.3.To analyze the survival curves of colorectal cancer patients with different expression levels of PTOV1-AS2 based on ENCORI.Results1.The expression of PTOV1-AS2 was significantly higher in colorectal cancer tissues than in normal colorectal tissues(P<0.01).The expression of PTOV1-AS2 was significantly higher in different human colorectal cancer cell lines than in normal human intestinal epithelial cells(all P<0.01).2.PTOV1-AS2 expression was significantly correlated with tumor size,vascular invasion,lymph node metastasis and TNM analysis in colorectal cancer patients(all P<0.05).3.Patients with colorectal cancer with high PTOV1-AS2 expression had shorter survival(HR=1.56,P=0.028).Conclusion1.PTOV1-AS2 is highly expressed in colorectal cancer tissues and cells.2.PTOV1-AS2 expression was correlated with the clinicopathology and survival of colorectal cancer patients.Part Ⅱ:Effect of PTOV1-AS2 expression on the biological function of colorectal cancer cellsObjectives1.Investigating the effect of PTOV1-AS2 on the function of colorectal cancer cellsMethods1.To establish PTOV1-AS2 overexpression and PTOV1-AS2 silencing colorectal cancer cell lines HCT116 and SW620 cell models.2.To detect the expression efficiency of PTOV1-AS2 in each model group of cells by qRT-PCR.3.The effects of overexpression and silent expression of PTOV1-AS2 on proliferation,migration,invasion and apoptosis of colorectal cancer cell lines were measured by CCK8 assay,Transwell assay and flow cytometry assay.Results1.The relative expression of PTOV1-AS2 was significantly higher in the overexpression group(all P<0.05);the relative expression of PTOV1-AS2 was significantly lower in the silencing group(all P<0.05).2.The OD 450 nm values of the control group were significantly higher in the PTOV1-AS2 overexpression group at 48,72 and 96 hours(P<0.05),while the PTOV1-AS2 silencing group was significantly lower(P<0.05).3.The number of cell migration and invasion was significantly higher in the PTOV1-AS2 overexpression group and lower in the PTOV1-AS2 silencing group compared with the control group(both P<0.05).4.The total apoptotic rate was significantly lower in all PTOV1-AS2 overexpression groups and significantly higher in all PTOV1-AS2 silencing groups when compared with the control group(all P<0.05).Conclusion1.PTOV1-AS2 overexpression promoted the proliferation,migration and invasion of colorectal cancer cells and inhibited apoptosis.2.PTOV1-AS2 silent expression inhibited the proliferation,migration and invasion of colorectal cancer cells,and promoted apoptosis.Part Ⅲ:Regulation of FSCN1 by PTOV1-AS2 in colorectal cancer through targeting miR-145-5pObjectives1.Predictive validation of PTOV1-AS2 target binding to miR-145-5p and its target gene FSCN12.Detection of miR-145-5p and its target gene FSCN1 expression in colorectal cancer tissues3.To Clarity the regulatory role of PTOV1-AS2 on miR-145-5p/FSCN1Methods1.The binding miRNA miR-145-5p and its target gene FSCN 1 were predicted by bioinformatics analysis of PTOV1-AS2,and the binding effect was verified by dual luciferase assay.2.qRT-PCR was used to detect the expression levels of miR-145-5p and its target gene FSCN1 in colorectal cancer tissues.3.Based on the overexpression of PTOV1-AS2 colorectal cancer cell line model,miR-145-5p mimic was transfected simultaneously,and the effect of PTOV1-AS2/miR-145-5p on FSCN1 expression was detected by Western blot technique.Results1.PTOV1-AS2 targets binding to miR-145-5p and miR-145-5p targets the 3’-UTR region of gene FSCN 1.2.miR-145-5p was lowly expressed in colorectal cancer tissues and FSCN1 was highly expressed in colorectal cancer tissues(both P<0.05).3.Elevated FSCN1 protein expression caused by overexpression of PTOV1-AS2 could be partially reduced by miR-145-5p(both P<0.05).Conclusion1.PTOV1-AS2 upregulates FSCN1 expression by targeting miR-145-5pPart Ⅳ:Impact of PTOV1-AS2/miR-145-5p/FSCN1 on the biological function of colorectal cancerObjectives1.To detect the effect of PTOV1-AS2/miR-145-5p/FSCN1 axis on the biological function of colorectal cancer cells2.To investigate the effect of PTOV1-AS2/miR-145-5p/FSCN1 axis on the tumorigenic ability of colorectal cancer cells in nude miceMethods1.Based on silent expression of PTOV1-AS2 colorectal cancer cell line model with simultaneous transfection of miR-145-5p inhibitor or overexpression of FSCN1 vector.2.To measure the effect of PTOV1-AS2/miR-145-5p/FSCN1 axis on proliferation,migration,invasion and apoptosis of colorectal cancer cell lines by CCK8 assay,Transwell assay and flow cytometry assay.3.To construct different groups of colorectal cancer cells in a subcutaneous tumorigenic model in nude mice.4.To record the volume and weight of tumor in rats.5.The expression of Ki67 in each group of tumour tissues was detected by immunohistochemistry.6.Detection of FSCN1 expression in each group of tumor tissues based on Western blot method.Results1.Silencing of PTOV1-AS2 expression resulted in reduced proliferation,migration,and invasion ability and increased apoptosis of colorectal cancer cells,which could be partially reversed by silencing miR-145-5p or overexpression of FSCN1(both P<0.05).2.Silencing PTOV1-AS2 expression resulted in a significant decrease in tumorigenic volume and weight of colorectal cancer cells(P<0.05);and could be partially reversed by silencing miR-145-5p or overexpression of FSCN1(both P<0.05).3.Silencing PTOV1-AS2 resulted in a significant decrease in ki67 and FSCN1 expression in tumorigenic tumors of colorectal cancer cells(P<0.05);and could be partially reversed by silencing miR-145-5p or overexpression of FSCN1(both P<0.05).Conclusion1.Silencing miR-145-5p or overexpressing FSCN1 partially reversed the effect of silencing PTOV1-AS2 expression on the function of colorectal cancer cells.2.Silencing of PTOV1-AS2 expression inhibited the tumorigenic ability of colorectal cancer cells.Inhibition of miR-145-5p or FSCN1 overexpression partially reversed the tumor suppressive effect of silencing PTOV1-AS2.