| Objective:Hepatocellular carcinoma(HCC)is the most common primary malignant hepatic tumor.And it ranks third among the global cancer-related mortality.Although the diagnosis and treatment of hepatic cancer have been greatly improved,because of the asymptomatic or untypical performance usually in the early stage of hepatic cancer,when clinical symptoms appear,most liver cancer has entered the middle and advanced stages.As a result,hepatic cancer causes 1 million deaths every year.Since the blood supply of hepatic tumors comes from the hepatic artery,the blood supply of the tumor is interrupted or reduced after hepatic artery embolizes,followed with the necrosis and shrink of the tumor,achieving the effect of treating liver cancer.Therefore,transcatheter hepatic arterial chemoembolization(TACE),which is proposed based on this principle-is increasingly used clinically and can prolong the survival time of patients with liver cancer effectively.At present,the Response Evaluation Criteria In Solid Tumors(RECIST)is the main method for evaluating the efficacy of TACE.However,the high lipiodol density and the scanning process are prone to cause lipiodol artifacts,which will reduce the ability of enhanced CT to identify tumors,affecting the clinical judgment of residual or recurring tumors.Therefore,finding a molecular marker is very important to evaluate the efficacy of TACE.Non-coding RNA(ncRNA)is a kind of transcript that can be encoded by the genome but not translated into protein.Initially,ncRNA was considered to be the "noise" caused by gene transcription.Yet currently it has been proven that more than 75% of the genome encodes a large number of functional ncRNAs.And there are more and more evidences showing that ncRNAs play an important role in biological functions of the cell.According to the length of transcripts,ncRNAs can be divided into two categories: small non-coding RNA(sncRNA)and long non-coding RNA(long non-coding RNA,lncRNA).SncRNA is less than 200 nucleotides in length and includes micro RNA(miRNA),small interfering RNA(si RNA),and PIWI-interacting(pi RNA).lncRNA is a type of non-coding RNA with the length of more than 200 nucleotides.It has recently been shown that it can use multiple modes of action to regulate gene expression as well as participating in a wide range of cellular processes.More and more evidences show that lncRNA is playing a key role both in the carcinogenic and tumor suppression pathways.We have been looking for a more sensitive indicator suitable for clinical evaluation of the efficacy of TACE.In the preliminary work,we collected the serum samples of 5 patients before and after TACE treatment from 2018 to 2019 in the Interventional Department of the Second Affiliated Hospital of China Medical University.And then they were conducted under several experiments such as high-throughput sequencing and bioinformatics analysis.For the first time,it was screened and identified that lncRNA-AC018682.1 may be a key factor affecting the efficacy of TACE.lncRNA-AC018682.1 is located on human chromosome 2p21 and contains 2 exons.But there is no relevant research reporting the function of this gene.Therefore,we will further study the role of lncRNA-AC018682.1 in the process of liver cancer and explore its related mechanisms.Methods: 1.We collected 30 cases of serum samples before and after TACE treatment during 2018 to 2019 in the Interventional Department of the Second Affiliated Hospital of China Medical University.And we then detect the expression of lncRNA-AC018682.1in the serum by q RT-PCR method.QRT-PCR was used to detect the expression levels in liver cancer cell lines and normal liver cell lines.And the cellular localization of lncRNA-AC018682.1 in liver cancer cell lines was determined by fluorescence in situ hybridization(FISH).2.We cultured human hepatocarcinoma cell lines Hep3 B and HepG2 in vitro.lncRNA-AC018682.1 was down-regulated by lentivirus-mediated shRNA,and the down-regulation efficiency was verified by q RT-PCR.The proliferation of lncRNA-AC018682.1 in liver cancer cells was tested by CCK-8.The role of lncRNA-AC018682.1 in the invasion and migration of liver cancer cells was detected by Transwell experiment.3.We predicted the miRNA that can compete with lncRNA-AC018682.1 in the combination interaction and its downstream target genes through bioinformatics.And we verified the interaction between lncRNA-AC018682.1 and miR-1226-5p through the dual luciferase reporter assay.Using q RT-PCR we verified the expression level of miR-1226-5p after down-regulating lncRNA-AC018682.1.Again,the interaction between miR-1226-5p and the downstream target gene AURKA was verified by the dual luciferase reporter assay.Western blot experiment was performed to detect the protein expression levels in different liver cancer cell lines and normal liver cell lines.It was also used to detected the expression level of AURKA protein after knocking down lncRNA-AC018682.1.After down-regulating lncRNA-AC018682.1 in liver cancer cells,miR-1226-5p was also down-regulated,further detecting the effect of lncRNA-AC018682.1 in the proliferation of liver cancer cells by CCK-8.Also in this situation,the role of lncRNA-AC018682.1 in the invasion and migration of liver cancer cells was detected by Transwell experiment.And the expression level of AURKA protein was detected by Western blot experiment.4.In order to discuss the function of lncRNA-AC018682.1 in the nucleus,the dual luciferase reporter assay was used to observe its relationship with the AURKA promoter,and bioinformatics analysis was used to obtain the transcription factor KLF15 that can bind to the AURKA promoter.RNA binding protein immunoprecipitation(RIP)experiment was used to test the relationship between lncRNA-AC018682.1 and KLF15.And we used bioinformatics analysis to predict the binding site of AURKA promoter with KLF15,and analyzed the enrichment level of KLF15 on AURKA promoter by chromatin immunoprecipitation assay(CHIP).Finally,CHIP analysis was used to observe the KLF15 enrichment level of the AURKA promoter region in the hepatocarcinoma cell line after knocking down lncRNAAC018682.1.Results: 1.There was a significant difference in the expression of lncRNA-AC018682.1before and after TACE treatment.The expression level in liver cancer cell lines was significantly higher than that in normal liver cell lines,which was located in the nucleus and cytoplasm.2.Using lentivirus-mediated shRNA to down-regulate lncRNA-AC018682.1 can obviously inhibit the abilities of proliferation,migration and invasion of liver cancer cell lines.3.In the cytoplasm,lncRNA-AC018682.1 can be served as a molecular sponge attaching to miR-1226-5p,affecting the expression level of AURKA,which further affects the abilities of proliferation,migration and invasion of liver cancer cells.4.In the nucleus,lncRNA-AC018682.1 up-regulates the activity of the AURKA promoter and affect its protein expression level by recruiting the transcription factor KLF-15.Conclusion: There are significant differences in the expression of lncRNA-AC018682.1after TACE treatment,and it is closely related to the malignant biological behavior of liver cancer cells.In the cytoplasm,lncRNA-AC018682.1 affects the expression level of AURKA by adsorbing miR-1226-5p,and in the nucleus,lncRNA-AC018682.1up-regulates the activity of the AURKA promoter as well as affecting its protein expression level by recruiting the transcription factor KLF-15.These two approaches work together to promote the proliferation,migration and invasion of liver cancer cells.This study provides a new molecular target for evaluating the efficacy of TACE. |
PDF Full Text Request