Preliminary Study On The Mechanism Of Colorectal Cancer Occurrence And Development Promoted By Long Non-coding RNA-ZFAS1 And Its Potential Biomarkers

Background and objective:Colorectal cancer（CRC）is one of the most common cancers,and at present,the incidence of CRC has risen to the third place of malignancies.As many as 1 million new diagnoses occur every year,resulting in high morality rates worldwide.The pathogenesis of CRC is still unclear,which may be the malignant transformation and irreversible gene changes induced by long-term interaction of genetic and environmental factors and involves in key physiological functions of cells,including uncontrolled signal transduction of proliferation,apoptosis and differentiation.At present,the diagnosis of CRC depends mostly on imaging,enteroscopy,fecal occult blood test and so on.However,due to lack of symptoms at early stage,it is easy to miss and misdiagnose.Therefore,it is not only of great research value,but also of great clinical application prospects to deeply understand the genes related to the pathogenesis of CRC and use them as ideal biomarkers for CRC.Long non-coding RNAs（lncRNAs）are a class of RNA molecular with more than 200 nucleotides in length that do not encode proteins.At present,accumulating studies have shown that the abnormal expression of lncRNAs is closely related to the proliferation,migration,invasion and angiogenesis of cancers.The mechanism of lncRNAs in tumorigenesis and development has become a new hotspot in the field of molecular biology of tumors.Up to now,it has been reported that abnormal expression of lncRNAs not only plays an important role in the occurrence and development of CRC,but also plays an important role in clinical diagnosis,malignant degree judgement,curative effect observation and prognosis evaluation of CRC.In order to further study the role of lncRNA in the development of CRC,we firstly downloaded the RNA-seq data of 24 pairs of CRC tissues and matched adjacent normal tissues from The Cancer Genome Atlas（TCGA）database.A lot of differentially expressed lncRNAs were found between CRC tissues and adjacent normal tissues,and screened out lncRNA-ZFAS1from these abnormal expressions for further study.To study the mechanism of CRC in regulating CRC growth,metastasis and angiogenesis,to explore new targets for CRC treatment,and to seek biological biomarkers for minimally invasive humoral diagnosis of CRC,so as to provide a reference for the individualized diagnosis and treatment of CRC,and to provide a more reliable theoretical basis for the development of new targeted therapeutic drugs for CRC and the optimization of clinical treatment.Our study is mainly divided into the four following parts:Part I:Abnormal expression of lncRNA-ZFAS1 in CRC and its clinical significanceMethods:1.RNA-seq data of 24 CRC patients were downloaded from TCGA database,and the differential lncRNAs between CRC tissues and adjacent normal tissues were analyzed using R software.2.The expression of lncRNA-ZFAS1 was detected by qRT-PCR in 112 patients with CRC.The expression levels of ZFAS1 in CRC cell lines（HCT116,HCT8,HT29,SW480,SW620 and DLD-1）and normal colon mucosal epithelial cells（FHC）were also detected.The clinicopathological and prognostic follow-up information of the patients were collected.3.Chromatin immunoprecipitation（ChIP）and dual luciferase reporter assays were performed to determine whether SP1 could bind to the promoter region of ZFAS1.4.The expression of SP1 in HCT116 and HCT8 cells was silenced by siRNA,and the transfection efficiency was observed by qRT-PCR.5.COX regression model and Kaplan-Meier survival curve were used to analyze the correlation between ZFAS1 expression and clinicopathological parameters,prognosis of CRC patients.Results:1.The abnormally high expression of lncRNA-ZFAS1 in CRC tissues was screened from TCGA database.The high expression of lncRNA-ZFAS1 in CRC tissues and cells was further confirmed by qRT-PCR.（all p<0.05）.2.Transcription factor SP1 could bind to the promoter region of ZFAS1,meanwhile,ZFAS1 was markedly downregulated after SP1 knockdown in CRC cells（p<0.05）.3.In CRC tissues,the expression of ZFAS1 was significantly correlated with TNM stage,pT stage and distant metastasis（all p<0.05）.4.COX regression analysis indicated that the expression level of ZFAS1wasanindependentpredictorofoverallsurvivalrateof patients.Kaplan-Meier survival analysis showed that theoverall survival rate of CRC patients with high expression of ZFAS1 was lower than that of CRC patients with low expression of ZFAS1（p<0.01）.Conclusion:In CRC,SP1 induced ZFAS1 overexpression,ZFAS1 was closely associated with TNM stage,pT stage and distant metastasis of tumors,and with prognosis of patients.Part II lncRNA-ZFAS1 promotes the development of CRC by sponging miR-150-5pMethods:1.The expression of ZFAS1 in HCT116 and HCT8 cells was silenced by siRNA;the expression of ZFAS1 in HCT116 and HCT8 cells was upregulated by pcDNA3.1-ZFAS1 transfection;the transfection efficiency was verified by qRT-PCR.2.CCK-8 and colony formation assays were used to detect the effects of ZFAS1 and miR-150-5p on the proliferation of HCT116 and HCT8 cells.Wound healing and Transwell invasion assays were employed to detect the effects of ZFAS1 and miR-150-5p on the ability of migration and invasion of HCT116and HCT8 cells,respectively.HUVECs tube formation assay was performed to detect the effects of ZFAS1 and miR-150-5p on angiogenesis.3.ZFAS1 stable low expression cell lines was screened by using Puromycin,the effect was detected by qRT-PCR.4.Subcellular localization of ZFAS1 was detected by RNA fluorescence in situ hybridization（FISH）and cytoplasmic separation assays.5.RNA Immunoprecipitation（RIP）and dual luciferase reporter assays were confirmed whether ZFAS1 acts as competitive endogenous RNA（ceRNA）to sponge miR-150-5p.6.The expression of miR-150-5p in CRC tissues was detected by qRT-PCR,and the relationship between the expression of miR-150-5p and ZFAS1 in CRC tissues was analyzed.7.The effects of ZFAS1 on the growth,metastasis and angiogenesis of CRC were verified by tumor xenografts,tail vein injection and Chicken chorioallantoic membrane（CAM）experiments,respectively.Results:1.After ZFAS1 knockdown in CRC cells,the ability of proliferation,migration,invasion of CRC cells and tube formation of HUVECs were significantly decreased（all p<0.05）.After up-regulation of ZFAS1expression in CRC cells,the ability of proliferation,migration,invasion of CRC cells and tube formation of HUVECs were significantly increased（all p<0.05）.2.ZFAS1 was mainly distributed in the cytoplasm of CRC cells.3.Bioinformatic predicted showed that miR-150-5p and miR-590-3p had miRNA response elements（MREs）which could be combined with ZFAS1.And the expression of microRNA-150-5p in CRC cells obviously decreased after ZFAS1 overexpression（p<0.001）,while the expression of miR-590-3p did not change significantly（p>0.05）.4.Dual luciferase reporter and RIP assays showed that ZFAS1 could act as ceRNA to sponge miR-150-5p.5.Compared with adjacent normal tissues,miR-150-5p was significantly lower in CRC tissues（p<0.001）.The expression of ZFAS1 in CRC tissues was negatively correlated with that of miR-150-5p（r2=0.4903,p<0.001）.6.Two stable ZFAS1 knockdown CRC cell lines（HCT116 and HCT8）were constructed.7.The decreased ability of proliferation,migration,invasion of CRC cells and HUVECs tube formation caused by ZFAS1 knockdown could be reversed by inhibition of miR-150-5p（all p<0.05）.8.Tumor xenografts,tail vein injection and Chicken chorioallantoic membrane（CAM）experiments showed that the ability of CRC cells growth and metastasis,and angiogenesis were markedly reduced after ZFAS1 knockdown（all p<0.05）,while the inhibition of miR-150-5p in CRC cells could restore or at least partially store the decline ability of CRC growth,metastasis and angiogenesis induced by ZFAS1 knockdown（all p<0.05）.Conclusion:In CRC,ZFAS1 promotes tumorigenesis,and could act as ceRNA to sponge miR-150-5p.The downregulation of miR-150-5p can reverse the inhibition of growth,metastasis and angiogenesis of CRC cells induced by ZFAS1 knockdown.PartⅢThe mechanism of ZFAS1-microRNA-150-5p-VEGFA-Akt/mTOR axis regulating malignant progression of CRC Methods:1.Dual luciferase reporter assay was used to verify whether vascular endothelial growth factor A（VEGFA）is a target gene of miR-150-5p.2.qRT-PCR and western blot assay were used to detect the expression levels of VEGFA mRNA and protein in HCT116 and HCT8 cells after ZFAS1knockdown or miR-150-5p overexpression.3.Immunohistochemistry（IHC）and qRT-PCR was used to detect the expression of vascular endothelial growth factor A on protein and mRNA levels in colorectal cancer and matched adjacent normal tissues.4.Western blot was used to detect the expression of VEGFA,vascular endothelial growth factor receptor 2（VEGFR2）,phosphorylated vascular endothelial growth factor R2（p-VEGFR2）,Akt,phosphorylated Akt（p-Akt）,mTOR,phosphorylated mTOT（p-mTOR）and phosphorylated mTOT（p-mTOR）,and expression levels of epithelial-mesenchymal transition（EMT）related proteins（E-cadheirn,Vimetin,N-cadherin）in CRC cells with ZFAS1 knockdown or both ZFAS1 and miR-150-5p knockdown.Results:1.Several bioinformatics databases predicted that VEGFA might be the target gene of miR-150-5p.Then the wild and mutant luciferase reporter gene plasmids of ZFAS1（pmirGLO-VEGFA 3’UTR-WT and pmirGLO-VEGFA3’UTR-Mut）were constructed and co-transfected with agomiR-150-5p or agomiR-NC into HCT116 and 293T cells.The results confirmed that VEGFA was the target gene of miR-150-5p.2.When ZFAS1 was knocked down or miR-150-5p upregulated in HCT116 and HCT8cells,the expression levels of VEGFA mRNA and protein significantly decreased（p<0.001）.3.In CRC tissues,the expression of VEGFA protein and mRNA was positively correlated with ZFAS1（both p<0.05）,but negatively correlated with miR-150-5p（both p<0.05）.4.After ZFAS1 knockdown in HCT116 and HCT8 cells,the expression of VEGFA was downregulated,the phosphorylation of VEGFR2 was downregulated（p<0.05）,the phosphorylation of Akt and mTOR was also downregulated（p<0.05）,the expression of E-cadherin was upregulated,and the expression of Vimentin and N-cadherin was downregulated.After transfection with antagomiR-150-5p,the phosphorylation levels of VEGFR2,Akt and mTOR were significantly increased（all p<0.05）,the expression of E-cadherin was downregulated,and the expression of Vimentin and N-cadherin was upregulated（all p<0.05）.5.After treating with Ki8751,an inhibitor of vascular endothelial growth factor receptor2 in HCT116 and HCT8 cells,the phosphorylation levels of VEGFR2,Akt and mTOR were significantly decreased,the expression of E-cadherin was upregulated,the expression of Vimentin and N-cadherin was downregulated（both p<0.05）,and the ability of proliferation,migration,invasion of treated cells and tube formation ability of HUVECs cells were significantly decreased（all p<0.05）.Conclusion:ZFAS1 promotes malignant progression of CRC through the miR-150-5p mediated VEGFA/VEGFR2/Akt/mTOR pathway and EMT process.PartⅣPotential value of serum ZFAS1 in the diagnosis of colorectal cancerMethods:1.QRT-PCR was used to detect the expression of ZFAS1 in the serum of 100CRC patients and 100 healthy people.2.ROC curve analysis of ZFAS1 in serum was performed to investigate the potential value of CRC diagnosis.3.The basic data and clinicopathological data of 100 patients with CRC were collected to analyze the correlation between the expression of ZFAS1in serum and these pathological features.Results:1.The expression level of ZFAS1 in serum of patients with CRC was significantly higher than that of healthy people（p<0.001）.2.ROC curve analysis showed that when the expression of ZFAS1 in serum of CRC patients was more than 1.49(2^-??CT),the sensitivity and specificity of diagnosing CRC were 79%,91%and the area of the curve was 0.909（95%CI:0.867-0.952）.3.The expression of serum ZFAS1 was correlated with TNM stage,pT stage and distant metastasis in CRC patients（all p<0.05）and was not significantly associated with serum CEA and CA199 levels and age,sex,location of tumors,differentiation,lymph node metastasis（all p>0.05）.4.The serum ZFAS1 levels of CRC patients with TNM stage III/IV was significantly higher than that of patients with TNM stage I/II（p<0.001）.The serum ZFAS1 level of CRC patients with T3-T4 was significantly higher than that of patients with T1-T2（p<0.05）.The serum ZFAS1 level of CRC patients with distant metastasis was significantly higher than that of CRC patients without metastasis（p<0.001）.Conclusion:ZFAS1 may be a potential marker for CRC diagnosis.