Study On The Mechanism Of LncRNA CYTOR,miRNA378a And FEN1 Genes In The Regulation Of Malignant Biological Behavior Of Hepatocellular Carcinoma Cells

Up-regulation of FEN1 is Associated with the Tumor Progression and Prognosis of Hepatocellular CarcinomaBackgroundStudies show that patients with hepatocellular carcinoma(HCC)have poor prognosis,particularly when patients are diagnosed at late stages of the disease development.The flap endonuclease 1(FEN1)gene is over-expressed in multiple malignant tumors and may promote tumor aggressiveness.However,its expression profile and functional roles in HCC are still unclear.Here,we evaluated the molecular mechanisms of FEN 1 in HCC.ObjectiveBy studying the mRNA and protein levels of FEN1 in multiple hepatocellular carcinoma cohorts,the role of FEN 1 in the progression of HCC was explored,and a possible marker for the diagnosis and prognosis of HCC was provided.MethodsThe expression of FEN1 in HCC was evaluated using HCC mRNA expression data from TCGA and GEO databases.The expression of FEN 1 was also confirmed by immunohistochemistry(IHC)using a tissue microarray(TMA)cohort with a total of 396 HCC patients.Kaplan-Meier analysis,univariate and multivariate Cox regression analysis were used to determine the correlation between FEN1 expression and survival rate of HCC patients.The molecular mechanism and biological functions of FEN1 in HCC were predicted using functional and pathway enrichment analysis in vitro experiments.ResultsFEN1 was over-expressed in multiple HCC cohorts at both mRNA and protein levels.The receiver operating characteristic(ROC)curve showed that FEN1 can serve as a diagnostic predictor of HCC.Meanwhile,patients with high FEN1 expression levels showed lower overall survival(OS)and relapse-free survival(RFS)rates than those with low FEN1 expression.More importantly,we found that FEN1 elevation was an independent prognostic factor for OS and RFS in HCC patients based on univariate and multivariate analyses,indicating that FEN1 might be a potential prognostic marker in HCC.Furthermore,knocking down FEN1 resulted in suppressed cell proliferation and migration in vitro.This could have been due to regulation expressions of c-Myc,Survivin and Cyclin D1 genes,indicating that FEN1 may function as an oncogene through its role in the cell cycle and DNA replication pathway.ConclusionOur study indicated that high FEN1 expression might function as a biomarker for diagnosis and prognosis.In addition,the study confirms that FEN1 is an oncogene in HCC progression.Effects of Long non-coding RNA cytoskeleton regulator onFEN1 gene expression,apoptosis and autophagy in human hepatoma cellsBackgroundsMore than 95%of tumor genomes are noncoding RNAs,including microRNAs(miRNAs)and long noncoding RNAs(LncRNAs).LncRNAs-miRNAs-mRNAs form a complex network relationships,which jointly affect the transcription,translation of DNA molecules and expression of protein functions.They play an important role in a variety of diseases,especially in the proliferation,differentiation,invasion,metastasis,apoptosis and autophagy of tumors.With the development of the research,the role of LncRNAs in tumorigenesis and development has been gradually recognized.Through high-throughput technologies such as gene chip,whole genome sequencing and tumor metabonomics,we can screen out a variety of LncRNAs differential expressions closely related to tumors.Through luciferase report experiments,we can verify that LncRNAs target miRNAs or mRNAs,and through a variety of automatic biological enrichment information technology,we can explore the main biological functions in cells and provide a more complete "response chain" for the pathogenesis of diseases.Long noncoding RNA cytoskeleton regulator(CYTOR),also known as linc00152,was first found in the study of hepatocarcinogenesis.It mainly plays the role of promoting cancer,enhancing proliferation activity of hepatocarcinoma cells and inhibiting the process of apoptosis.In human hepatocarcinoma tissues,the up-expression is often related to the increase of clinical TNM stage,early lymph or blood metastasis,increase of radiochemotherapy resistance and poor survival prognosis.CYTOR is also expressed abnormally in many other kinds of malignant tumors,such as gastric cancer,breast cancer,colorectal cancer,etc.It plays different roles in different tumors.It may be related to different tissue characteristics and target genes.In the first part of this study,it has been pointed out that the up-regulation of FEN1 gene expression in hepatoma cells and tissues is directly related to the enhancement of tumor malignancy and worse clinical prognosis.Therefore,we speculate that LncRNA CYTOR may affect the expression of FEN1 gene,regulate cell apoptosis and autophagy activity,and then participate in the process of hepatocarcinogenesis.ObjectiveThrough the experiment of human tumor tissue and cells in vitro,it was verified that up-expression of CYTOR can affect the expression of FEN 1 gene,apoptosis and autophagy activity of hepatoma cells and tissues,providing theoretical basis for the pathogenesis of hepatoma and gene targeted therapy.MethodsFirstly,the expression levels of CYTOR and FEN1 gene were detected by RT-qPCR,Pearson test was used to analyze the correlation.Then,we constructed up-regulation,down-regulation and empty cypor plasmids and transfected them into Hep-G2 cell line in vitro.RT-qPCR was used to detect the transfection efficiency.After 48 hours of culture,the expression of FEN1 gene was detected by RT-qPCR,apoptosis rate was detected by flow cytometry,the levels of caspase-9,Bcl-2 and Bax proteins were detected by Western blot,number of autophagic body was detected by immunofluorescence,and mRNA levels of beclin-1 and LC3 were detected by RT-qPCR.ResultsThe expression levels of CYTOR and FEN1 in tumor tissue were significantly higher than those in normal tissue.Pearson test showed that expression level of CYTOR and FEN1 were positively correlated(P<0.05).Compared with empty plasmid transfection group,the expression level of CYTOR and FEN1 in up-regulation CYTOR group increased significantly,apoptosis rate decreased,expression levels of caspase-9,Bcl-2 and Bax proteins decreased,number of autophagic body decreased,expression levels of beclin-1 and LC3 mRNAs also decreased(P<0.05),However,in down-regulation group,the expression levels of CYTOR and FEN1 decreased significantly,apoptosis rate increased,expression levels of caspase-9,Bcl-2 and Bax proteins increased,number of autophagy body increased,expression levels of beclin-1 and LC3 mRNAs also increased(P<0.05).ConclusionThe increased expression of LncRNA CYTOR in liver cancer tissue and cells may affect the expression of FEN1 gene,then inhibit apoptosis and autophagy activity of cells,and participate in the development of liver cancer.The mechanism of microRNA-378a influences FEN1 gene expression in human hepatocarcinoma cells and regulates cell malignant biological behavior and related signal pathwayBackgroundsMicroRNAs,as a kind of non-coding RNA widely distribute in eukaryotes,have been proved to play an important role in the expression of targeted silencing transcription genes and participate in a variety of physiological and pathological changes,especially closely related to the occurrence and development of tumor.The high-throughput gene sequencing showed that microRNA-378a(miR-378a)was up-regulated in liver cancer tissues and cells,which was directly related to several clinical features of tumor,such as TNM stage,lymph node and blood metastasis,tissue differentiation grades,chemotherapy resistance and survival outcome.MiR-378a may be involved in the regulation of malignant biological behavior by targeting silencing one or more transcription genes.We speculated that miR-378a might be related to the expression of FEN1 gene.ObjectiveTo explore the relationship between miR-378a expression in hepatoma cells and the target regulation of FEN1 gene,whether it affects cell proliferation and cycle distribution,expression of inflammatory factors and angiogenesis,as well as the activation mechanism of transforming growth factor(TGF)-β and nuclear transcription factor(NF-κB)signaling pathway.MethodsWe selected hepatoma Hep-3b and Hep-G2 cell lines,knocked out miR-378a gene accurately by CRISPR/Cas9 gene editing technology,constructed PX458-miR-378a-sg RNA1 and PX459-miR-378a-sg RNA2 recombinant plasmids,and co-transfected them into Hep-3b and Hep-G2 cells,and cultured them to obtain stable monoclonal cell lines.Gene sequencing and real-time fluorescence quantitative PCR(RT-qPCR)were used to detect miR-378a expression.The study divided into Hep-3b and Hep-G2 cell normal culture group(control group),miR-378a gene knock-out transfection Hep-3b and Hep-G2 cell group(miR-378a knock-out group).After 48 hours of culture in each group,the expression of FEN1 gene was detected by RT-qPCR method,cell proliferation rate was detected by MTT method,cell cycle distribution was detected by flow cytometry,inflammatory factors IL-6 and tumor necrosis factor(TNF)-a were detected by ELISA method Western blot was used to detect the expressions of hypoxia inducible factor(HIF)-αand vascular endothelial growth factor(VEGF),and Western blot was used to detect the expressions of TGF-β and NF-κB.ResultsAfter co-transfection of PX458-miR-378a-sg RNA1 and PX459-miR-378a-sg RNA2 recombinant plasmids into Hep-3b and Hep-G2 cells,the expression of miR-378a decreased significantly confirmed by gene sequencing and RT-qPCR(P<0.05).The aim of gene knockout was achieved,and the stable and low expression monoclonal cell lines were screened out.After 48 hours of culture,compared with Hep-3b and Hep-G2 control group,the expression of FEN1 gene,cell proliferation rate,percentage of G2/M phase in cell cycle,expressions of IL-6 and TNF-α,expressions of HIF-α and VEGF,expressions of TGF-β and NF-κB in signal pathway increased significantly in miR-378a knockout group(P<0.05).There were no differences of above indexes between Hep-3b and Hep-G2 control groups,and between miR-378a knockout groups of Hep-3b and Hep-G2(P>0.05).ConclusionThe increased expression of miR-378a may be involved in development of liver cancer.By targeting and silencing the expression of FEN 1 gene in cells,it may affect cell proliferation activity,division cycle distribution,expression of inflammatory factors and angiogenesis,and may mediate the activation of TGF-β and NF-κB signaling pathways.MiR-3 78a may be an important potential site for targeted therapy of liver cancer.The relationship between FEN1 gene expression and insulin resistance,oxidative stress,energy metabolism and senescence in primary hepatocarcinomaBackgroundsIn our previous studies,we have explored the abnormal expression of FEN1 gene in primary liver cancer,which may form a complex network with LncRNA and miRNA,regulate cell proliferation,apoptosis,autophagy,invasion and metastasis.The functional expression of gene needs to be implemented in the process of cell growth and metabolism.Hepatocytes are the main place of substance and energy metabolism.More and more studies have pointed out that there is a close relationship between the occurrence of liver cancer and type 2 diabetes mellitus,which can interact with each other and promote the occurrence of diseases.High glucose environment can increase the occurrence of insulin resistance,expression of many kinds of cancer promoting factors,direct toxic effect of glycosylation products on cells and strong oxidative stress response.Based on this,this study mainly discusses how the FEN1 gene affects the growth and metabolism of liver cancer cells.ObjectiveTo analyze the relationship between expression of FEN 1 gene and the occurrence of insulin resistance,oxidative stress,energy metabolism and senescence in primary liver cancer,and to further elaborate the internal relationship between abnormal expression of FEN1 gene and the occurrence of liver cancer.MethodsHep-G2 cell line was cultured in vitro,and the experiment was divided into three groups:blank control group,low concentration glucose intervention group(1mmol/L),medium concentration group(5mmol/L)and high concentration group(10mmol/L).After 72 hours coculture,FEN1 gene expression was detected by RT-qPCR,western blot was used to detect insulin resistance related protein including insulin-like growth factor binding proteins(IG-FBP)and advanced glycation end products(AGEs),oncogenesis related proteins including vascular endothelial growth factor(VEGF)and transforming growth factor(TGF),senescence related proteins includingβ-galactosidase(β-Gal)and hypoxia inducible factor(HIF)-1α,iron death pathway protein glutathione peroxidase 4(GPX4)in cells,oxidative stress indicators including reactive oxygen species(ROS),superoxide dismutase(SOD),malondialdehyde(MDA)and reduced glutathione(GSH)were measured by radioimmunoassay,energy metabolism indicators including the activities of hexokinase(HK)and pyruvate kinase(PK),adenosine triphosphate(ATP)content were measured by spectrophotometry.ResultsCompared with control and low concentration groups,the expression of FEN1 gene in middle and high concentration groups were significantly higher,what’s more,the expressions of IG-FBP and AGEs,VEGF and TGF,β-Gal and HIF-1α,GPX4 proteins,ROS,SOD,MDA and GSH levels,HK and PK activities and ATP content were significantly increased,too(P<0.05).Furtherly,the above indicators in high concentration group were also more higher than middle concentration group(P<0.05).However,there were no differences of above indicators between control and low concentration groups(P>0.05).ConclusionHigh glucose environment can induce the expression of FEN1 gene,affect insulin resistance,induce tumorigenesis,regulate the mechanism of cell senescence,affect energy metabolism,oxidative stress and iron related metabolism,which provides an important basis for the occurrence mechanism and targeted intervention of liver cancer.