Study On The Mechanism Of LncRNA-4045 In Hepatocellular Carcinoma And Prognosis Prediction

Background and objective: Hepatocellular carcinoma(HCC)is the most common type of Primary Hepatic carcinoma(PHC).Because HCC usually has an occult early onset,a high degree of malignancy,and is easy to metastasize and relapse,it leads to a high mortality rate of patients and poses a threat to human health all over the world.So far,the etiology,pathogenesis and molecular mechanism of HCC have not been fully elucidated,which seriously hinders the research on early diagnosis,treatment and improvement of prognosis of HCC.Long noncoding RNAs(lncRNAs)are a class of important non-coding RNAs.Since their important biological functions were proposed,lncRNAs have been proved to be related to the occurrence and development of a variety of cancers including HCC through extensive and in-depth studies by many researchers.With the extensive use of high-throughput sequencing and microarray in cancer research,many HCC-associated lncRNAs have been identified and validated by previous studies through high-throughput screening.However,due to the huge number of lncRNAs transcribed from human genomes,there are still a large number of potential HCC-related lncRNAs that have not been discovered and fully understood.In our previous study,by transcriptome microarray detection of HCC cancer tissues and adjacent normal liver tissues,one of the differentially expressed lncRNAs screened out by our research group: lncRNA-4045(ENST00000524045.6)was highly expressed in HCC cancer tissues.Further study showed that,its expression in tissue samples with expanded sample size(50pairs of HCC cancer tissues and adjacent normal liver tissues)were consistent with the microarray results according to q RT-PCR validation.The expression level analysis results of lncRNA-4045 in HCC tissues and adjacent normal liver tissues derived from the TCGA database were also consistent with the microarray results.Furthermore,no studies have reported this lncRNA in HCC or other human diseases.Therefore,lncRNA-4045 has potential research prospects for elucidating the mechanisms of occurrence and development of HCC,as well as the prevention and treatment of HCC.The purpose of this study is to further explore the mechanism of function of lncRNA-4045 in HCC and its prognostic role in HCC patients.Methods: 1.Part I:(1)Real-time fluorescence quantitative reverse transcription polymerase chain reaction,q RT-PCR)was used to detect the expression levels of lncRNA-4045 in normal liver cell lines and HCC cell lines,and to screen suitable cell lines for subsequent studies.(2)The medium was prepared according to the suitable medium formula for each cell line,and the cell lines were cultured in a 37℃ constant temperature incubator containing 5% CO2 and 95% humidified air.(3)Lentiviral vectors were used to infect cells to construct knockdown and overexpression stable transfected cell lines of lncRNA-4045,and the knockdown and overexpression efficiency was detected by q RTPCR.(4)Total RNA of the cell lines was extracted by Trizol method.(5)CCK-8assay and plate clone formation assay were used to detect cell proliferation.(6)Transwell chamber migration and invasion assay were used to detect the ability of cell migration and invasion,and scratch assay was also used to detect the ability of cell migration.(7)Flow cytometry was used to detect the proportion of apoptosis(Annexin V,PI double staining)and the distribution of cell cycle(PI single staining).2.Part II:(1)The potential target gene of lncRNA-4045 was detected by high-throughput RNA sequencing(RNA-seq).(2)The potential target genes of lncRNA-4045 and their possible mechanisms of biological function were predicted by using bioinformatics online databases such as TCGA,GEPIA,Encori,KEGG and GO.(3)Fluorescence in situ hybridization(FISH)technique was used to detect the sublocalization of lncRNA-4045 in cells to understand the location and mechanism of its possible biological function.(4)The survival prediction value of lncRNA-4045 on HCC patients was analyzed using TCGA data.The Kaplan-Meier method was used to draw the survival curves and the Logrank test was used to compare the differences in the survival curves.3.Part III:(1)AKR1B10 knockdown and overexpression transient transfection cell line were constructed by plasmid transfection on Hep3 B cells with lncRNA-4045 overexpressed,and the interference and overexpression efficiency were detected by q RT-PCR.(2)Western Blot(WB)was used to detect the changes of the expression level of the potential target protein AKR1B10 in the cell lines with knockdown and overexpression of lncRNA-4045.(3)Rescue and enhancement experiments: in Hep3 B cells with overexpression of lncRNA-4045 and knockdown or overexpression of Ak R1B10,CCK-8 method and plate clone formation assay were used to detect cell proliferation ability.4.SPSS 20.0 and R3.2 software were used for statistical analysis of data,and t test,analysis of variance,Pearson correlation and other statistical methods were used for analysis.The differential gene analysis of TCGA data was performed using the R language limma package.Results:I.The role of lncRNA-4045 in HCCQRT-PCR was performed on a normal liver cell line HL-7702 and 7 HCC cell lines: Hep G2,MHCC97-H,HCCLM3,MHCC97-L,SMCC-7721,Hep3 B and Huh-7.The results indicated that the background expression of lncRNA-4045 was significantly higher in MHCC97-H,HCCLM3 and MHCC97-L cell lines,and significantly lower expression in Hep G2 and Hep3 B cell lines,compared with the normal liver cell line HL-7702.2.Lentivirus transfection had a good interference efficiency on lncRNA-4045 in HCCLM3 and MHCC97-L cells,and achieved a good efficiency on overexpression of Hep G2 and Hep3 B cell lines.These cell lines were used in subsequent experiments.3.Knocking down lncRNA-4045 inhibited the proliferation,clonal formation,migration and invasion abilities of HCCLM3 and MHCC97-L cells,and increased the apoptosis rate,induced more cells distributed in the G0/G1 phase of the cell cycle.Overexpression of lncRNA-4045 increased proliferation,clone formation,migration and invasion ability of Hep G2 and Hep3 B cells,decreased apoptosis rate,and kept more cells distributed in the S phase of the cell cycles.Ⅱ.Screening the potential target genes of lncRNA-4045 and prognosis prediction1.In order to find potential target genes affected by lncRNA-4045,transcriptome sequencing was performed on HCCLM3 cells that knocked down lncRNA-4045 and transfected with negative control(NC)lentivirus.75 differentially expressed genes were found,suggesting that these differentially expressed genes might be affected by the alteration of lncRNA-4045.2.112 potential target genes that might be regulated by lncRNA-4045 were screened out from TCGA derived data.3.Ten micro RNAs(mi RNAs)with potential sponging effects on lncRNA-4045 were predicted by using ENCORI database.4.The results of FISH experiment showed that lncRNA-4045 was sublocalized in both cytoplasm and nucleus of HCCLM3 cells with approximately equal content,the location of the functional mechanism of lncRNA-4045 might be either of them.5.Comprehensive analysis of the results of bioinformatics prediction and experiments indicated that a gene with important function in liver was focused on:Aldo-keto reductase family 1 member B10(Ak R1B10),which has been reported to be abnormally highly expressed in HCC tissues and is closely related to the occurrence and development of HCC.6.The expression levels of lncRNA-4045 and Ak R1B10 were simultaneously detected by q RT-PCR in 43 HCC tissue samples and cell lines with interference and overexpression of lncRNA-4045,and it was found that lncRNA-4045 was positively correlated with the expression levels of Ak R1B10 in both HCC tissue samples and cell lines.Therefore,Ak R1B10 was identified as a potential target gene of lncRNA-4045 for subsequent mechanism studies.7.The overall survival(OS)and progression-free survival(PFS)of HCC patients with high lncRNA-4045 expression were shortened compared with those with low lncRNA-4045 expression.Ⅲ.Validation of the mechanism of lncRNA-4045 in HCC1.Results of WB showed that lncRNA-4045 knockdown significantly downregulated AKR1B10 protein expression in MHCC97-L cells,but no significant change of AKR1B10 protein expression was found in HCCLM3 cells;Overexpression of lncRNA-4045 resulted in significantly upregulated AKR1B10 protein expression in Hep3 B cells,but had no significant influence of AKR1B10 protein expression in Hep G2 cells.2.Rescue and enhancement experiments:(1)Rescue experiments: Hep3 B cells with overexpression of lncRNA-4045 were transfected with interference plasmid to knock down AKR1B10 gene,resulting in partially abolished cell proliferation and clone formation ability enhanced by overexpression of lncRNA-4045;(2)Enhancement experiment: Hep3 B cells with overexpression of lncRNA-4045 were transfected with AKR1B10 gene overexpression plasmids,which further enhanced the proliferation and clone formation ability of cells with overexpression of lncRNA-4045.Conclusion: 1.lncRNA-4045 plays a carcinogenic role in HCC,and its high expression causes the enhancement of malignant behaviors of HCC cells,which may be closely related to the occurrence and progression of HCC.2.lncRNA-4045 plays a role by influencing the expression of AKR1B10 protein.3.The high expression of lncRNA-4045 in HCC tissues can predict the poor prognosis of HCC patients and is a potential prognostic indicator of HCC.