LINC01419 Promotes Cell Proliferation And Metastasis In Hepatocellular Carcinoma By Enhancing The Promoter Activity Of NDRG1

BackgroundLiver cancer was predicted to be the fifth most commonly diagnosed cancer and the third most leading cause of tumor-related mortalities in 2018 around the world.Data showed that there were 782 000 deaths from liver cancer annually worldwide,with China alone accounting for approximately50% of all deaths.Hepatocellular carcinoma(HCC)is the most frequent highly aggressive primary liver cancer,comprising 75%-85% of all cases.Nowadays,standard treatments for HCC comprise surgical resection,local ablation,liver transplantation,chemotherapy,and targeted therapy,however with limited efficacy.In addition to poor treatment availability,up to 70%of patients undergoing resection or local ablation will experience cancer recurrence within 5 years.Therefore,we urgently need targets for early diagnosis or precise treatment of HCC.HCC is caused by the accumulation of somatic genomic alterations in passenger and driver genes as well as epigenetic modifications,which explains its considerable molecular heterogeneity.Emerging evidence has demonstrated that dysfunction of long non-coding RNAs(lnc RNAs)play essential roles in the initiation and progression of hepatocellular carcinoma.Currently,accumulating evidence shows that that deregulation of lnc RNAs,including lnc SOX4,LINC001138,lnc-UCID,and TSLNC8,are associated with uncontrolled cell proliferation,apoptosis,metastasis,invasion,epithelial-to-mesenchymal transition,active angiogenesis,and resistance of HCC,and also affect the stemness in HCC cells.Over the last decade,the development of high-throughput technologies,such as metagenomics next generation sequencing,have allowed an in-depth examination of the human genome with unprecedented resolution and scale.Studies using high-throughput technologies have shown an upregulation of LINC01419 in HCC.However,the biological functions and the mechanisms of LINC01419 in HCC tumorigenesis and progression have not been studied to date.This study includes four parts to demonstrate that LINC01419 is clinically,functionally,and mechanistically oncogenic gene in HCC.LINC01419 could,therefore,be a promising therapeutic target for HCC.Objective1.To explore the expression of LINC01419 in human HCC tissues and HCC cells,and to study the correlation between the pathological characteristics of HCC patients and the expression of LINC01419 in their HCC tissues,thus providing a new direction for the early diagnosis and clinical treatment of HCC.2.To investigate the role of LINC01419 in HCC cell proliferation,migration,and invasion in vitro.3.To clarify the molecular mechanism of LINC01419 affecting HCC cell proliferation,migration,and invasion.4.To further verify the effects of LINC01419 on tumorigenic and tumor metastasis of HCC and its molecular mechanism in vivo.Methods1.Analyzing the microarray data on the differentially expressed lnc RNAs in HCC from the TCGA database by bioinformatics.QRT-PCR(quantitative reverse transcription polymerase chain reaction)analysis was used to investigate the LINC01419 expression levels in 40 HCC tumor tissues and paired adjacent non-tumor tissues,as well as in several HCC cell lines,such as Huh7,SMMC7721,SK-Hep1,Hep G2 and Hep G3 B,and the immortalized normal human hepatocyte cell line MIHA.The relationship between the clinicopathological data and LINC01419 expression levels of the 40 HCC patients was statistically analyzed.Analysis of the protein encoding capability of LINC01419 with the use of online protein coding potential assessment software,the Coding Potential Assessment Tool(CPAT),and the Coding Potential Calculator(CPC).Cytoplasmic and nuclear fractions of the HCC cells were extracted according to the instructions of the Minute? Cytoplasmic and Nuclear Extraction Kit,and then the localization of LINC01419 in HCC cells was detected by q RT-PCR,and it was verified with lnclocator software.2.The HCC cell lines stably overexpressing LINC01419 was constructed,and lentiviral sh RNA vectors were used to specifically and stably knock down the endogenous expression of LINC01419 in the cells.Their efficiencies were confirmed by q RT-PCR.MTS,Ed U,colony formation and flow cytometry assays were used to detect the effect of LINC01419 overexpression or interference on the proliferation of HCC cells.To determine the effects of LINC01419 on the migration and invasion of HCC cells,wound healing and transwell assays(with/without matrigel)were performed with HCC cells.3.Through analyses of transcriptome sequencing data,the gene expression profiles of HCC cells with knocked down LINC01419 expression were explored.Then the differentially expressed genes which functions related to HCC progression and metastasis were selected to confirm the correction of microarray analysis by q RT-PCR.The most differentially expressed gene after knockdown of LINC01419 in HCC cells was the target gene which we studied.QRT-PCR and western blot assays were performed to analyze the effects of LINC01419 on the expression of m RNA and protein levels of NDRG1 in HCC cells.QRT-PCR,western blot and immunohistochemistry(IHC)analysis were used to investigate the LINC01419 expression levels in the HCC tumor tissues and paired adjacent non-tumor tissues,as well as in several HCC cell lines and MIHA.The expression correlation between LINC01419 and NDRG1 in 40 pairs of tissues from HCC patients was analyzed by q RT-PCR.Overexpression of NDRG1 plasmid was constructed,and combined transfections were performed.MTS,Ed U,colony formation,wound healing and transwell assays(with/without matrigel)were performed with HCC cells to clarify the effect of NDRG1 overexpression on the proliferation,migration and invasion of HCC cells,as well as to explore the role of NDRG1 overexpression and LINC01419 interference in HCC cells.Four p GL3?basic vectors containing different truncated lengths of the NDRG1 promoter region(full-length,-1500 to +1,-1000 to +1,-500 to +1)were constructed.Subsequently,dual?luciferase reporter assays were performed to determine the effects of LINC01419 on the activity of each region of NDRG1 promoter in 293 T and HCC cells,and to detect the active region.4.LINC01419 overexpression and interference stable HCC cells were used to construct nude mice subcutaneous tumorigenesis and liver in situ metastasis models.For the subcutaneous tumor experiment,tumor volumes and mice weights were measured every 4 days.All mice were sacrificed 28 days after implantation,and the tumor volumes and tumor weights for each group were measured.The excised tumors were photographed and partially analyzed the expressions of NDRG1 and Ki67 by western blot and IHC analysis.Eight weeks later,the nude mice in the metastasis models were sacrificed to observe the tumor metastasis in the livers and lungs.The number of liver nodules was calculated.The metastatic tissues were photographed,then analyzed by hematoxylin-eosin(H&E)staining analysis.Results1.LINC01419 was overexpressed in HCC tissues and cells and that it was positively correlated with large tumor size(P =0.025),more vascular invasion(P =0.022)and advanced TNM stage(P =0.027)of the 40 HCC patients.However,no correlation was observed between LINC01419 expression and patient's age,sex,HBV antigen,HCV antigen,serum AFP level,liver cirrhosis and so on(P >0.05).LINC01419 was unlikely to encode any protein product,and it was mainly distributed in the cytoplasm of HCC cells.2.LINC01419-overexpression promoted HCC cell proliferation,migration,and invasion in vitro,whereas knockdown of LINC01419 had the opposite effects.3.Through analyses of transcriptome sequencing data in LINC01419 knockdown HCC cells,14 dysregulated genes with log2 Fold Change >1,p Value <0.05,and their functions related to HCC progression and metastasis were selected.NDRG1 was one of the most downregulated genes after knockdown of LINC01419 in HCC cells.NDRG1 was overexpressed in HCC tissues and cells,and it was positively correlated with LINC01419 in 40 HCC tumor tissues.Overexpression of NDRG1 promoted HCC cell proliferation,migration and invasion,whereas cotransfection of p CDH-NDRG1 and sh LINC01419 blocked such promoting effects.LINC01419 could enhance NDRG1 promoter activity,and the region-1000 to-500 bp from the NDRG1 transcription start site.4.In the subcutaneous tumor models,the volume and weight of subcutaneous tumors in the overexpressed LINC01419 group were significantly increased compared with the control group,and the protein expression levels of NDRG1 and Ki67 in HCC tissues were significantly increased,however the interference LINC01419 group got the opposite experimental results.For the orthotopic implanted intrahepatic metastatic models,tumor liver-lung metastases in the overexpression LINC01419 group were significantly more than those in the control group,and the interference LINC01419 group obtained the opposite experimental results.Conclusion1.LINC01419 is a long intergenic non-coding RNA,and it was clinically oncogenic in HCC.2.Enforced expression of LINC01419 significantly promoted HCC cell proliferation,migration and invasion in vitro.3.Mechanistic investigations revealed that LINC01419 may function by interacting with the promoter region of NDRG1 to transactivate its transcription and finally promote HCC initiation and progression.4.Enforced expression of LINC01419 significantly promoted HCC proliferation and metastasis in vivo.LINC01419 by upregulating NDRG1 expression acts as an oncogenic driver in the initiation and progression of HCC.The results of this study provided a theoretical basis for the treatment of LINC01419 targeting HCC.