LncRNA NKILA Inhibits Hbv Replication And The Development Of Associated Hepatocellular Carcinoma By Downregulating The NF-κB Signaling Pathway

Background and Aim:Hepatitis B,caused by the Hepatitis B virus(HBV),is a worldwide health problem that threatens health and life.Persistent HBV infection can induce liver lesions,leading to cirrhosis and hepatocellular carcinoma(HCC).To date,approximately 257 million people have been infected with HBV worldwide.China is a major region affected by HBV infections and HCC.The mortality rate of liver cancer is the third highest among malignant tumors,and liver cancer caused by HBV infection accounts for>90%of the total number of patients.Currently,the clinical treatment of HCC mainly consists of surgical resection of cancer tissues or minimally invasive interventional therapy combined with antiviral therapy using nucleotide analogues and interferon(IFN).However,nucleotide analogues and interferons are insufficient for the functional cure of chronic hepatitis B.Moreover,HCC patients with chronic hepatitis B have poor prognosis and high mortality.Therefore,it is urgent to conduct in-depth studies on HBV replication and the pathogenesis and development of HBV-related HCC to provide new strategies for clinical treatment.NF-κB signaling pathway plays a regulating and controlling role in the replication of HBV and the occurrence and development of liver cancer.Although host-limiting factors inhibit HBV replication by regulating the status and activation of NF-κB signaling pathway and thereby regulating the accumulation of cccDNA in host cells,the role of the NF-κB signaling pathway is particularly prominent in the promotion of HBV replication.Pre-S,Core,and HBx encoded in the process of HBV replication depend on the activation of NF-κB signaling pathway to promote viral replication,including the regulation of HBx activation of this signaling pathway to promote viral replication and the occurrence and development of HBV-related HCC.These results indicate that the activation of the NF-κB signaling pathway in human homeostasis has a bidirectional regulatory effect on the replication of HBV.As an essential signaling pathway for HBV replication,it has received extensive attention,and antiviral strategies based on the inhibition of this signaling pathway require further study.NF-KB-interacting long non-coding RNA(NKILA),as a star molecule,is a negative regulator of NF-κB signaling pathway and forms stable complexes by interacting with NF-κB/IκB.It binds to two different sites of the P65 subunit of homologous dimer of NF-κB,directly masking the phosphorylation of IκB,thereby inhibiting the κBα phosphorylation and NF-κB activation induced by IKK.NKILA have also been shown to inhibit HCC growth.However,the relationship among NKILA,HBV replication,and HBV-associated HCC remains unclear.The purpose of this study was to determine whether NKILA can inhibit HBV replication and whether it is affected by HBV replication,to assess whether NKILA can inhibit the occurrence and development of HBV-related HCC,and to determine the effect of NKILA on the formation of HBx/NF-κB complex and NF-κB signal transduction.In addition,we aimed to determine the regulatory effect of NKILA on HBx-induced HCC growth to further explore the mechanism of NKILA.Materials and methods:HEK 293T cells and four liver cancer cell lines were used in this study:The function of NKILA in HepG2,Huh-7,HepG2.2.15(HepG2 integrates HBV double-copy genomes),and HepAD3 8(HBV genomes controlled by tetracycline regulated promoters were constructed from hepatoblastoma cell lines)was investigated.RNA was extracted from PBMC of clinical hepatitis B patients and the samples of HBV-related HCC tissue,and the content of NKILA was detected using reverse transcription-quantitative polymerase chain reaction(RT-qPCR).The expression of the HBV virus proteins HBeAg and HBsAg was assessed using the enzyme-linked immunosorbent assay(ELISA)after overexpression or knockdown of NKILA expression,and the effect of NKILA on HBV replication was determined.Western Blot was used to detect the expression of target proteins.Simultaneously,stable expression of NKILA or the control cell line pLVX puro-HepG2 was constructed.Four different transfected HepG2 cell lines(stable negative control vector pLVX puro,stable negative control vector pLVX-NKILA,stable negative control vector pLVX puro transfected with HBx,and stable pLVX-NKILA transfected with HBx)were used to detect the proliferative ability of hepatoma cells after different treatments using cell proliferation analysis.The migratory ability of NKILA knockout HCC cells was detected by scratch assay.The effect of NKILA knockout on the invasive ability of HCC cells was further detected using the Transwell cell assay.HepG2 cells overexpressing NKILA were injected into nude mice to prepare mouse HCC models.Immunohistochemistry was used to detect the expression of P65 to determine the effect of NKILA overexpression on tumor cells and whether it was related to NF-κB activity.Results:(1)In this study,HEK 293T and HCC cell lines(HepG2,HepG2.2.15,and HepAD38)were used to overexpress or knock down the expression of NKILA,and ELISA,RT-qPCR,Western Blot and other experimental techniques were used to prove that NKILA had inhibitory effect on the expression of HBV antigen protein.Dose gradient dependence was discovered.(2)HBV expression vectors or empty vectors were transfected into HepG2 and Huh-7 cells,and NKILA levels were measured 48 h later.The expression of NKILA in HepG2,HepG2.2.15,and HepAD38 cells was compared,and the expression levels of NKILA in PBMC of hepatitis B patients and HBV-related HCC tissues,and paracancer tissues were determined by RT-qPCR.The above experimental results showed that HBV replication can inhibit the NKILA expression,while its regulatory mechanism requires further study.(3)Nuclear and cytoplasmic NKILA in HepG2 and Huh-7 cells are mainly present in the cytoplasm.Through stable expression of NKILA compared with control HepG2 cells,overexpression of NKILA can inhibit the entry of P65 from cytoplasm into nucleus and inhibit the phosphorylation of IκBα.The results indicate that NKILA can inhibit the activation of NF-κB signaling pathway.The results of the nude mouse HCC animal model showed that the growth volume of liver cancer cells overexpressing with NKILA was significantly smaller than the control group,indicating that NKILA inhibited the occurrence and development of liver cancer.(4)The HBx protein encoded by HBV can bind to the P65 protein to form a complex and activate the NF-κB signaling pathway to promote HBV replication and the development of related liver cancer,which is an important regulatory factor in the development of HBV and HBV-related HCC.To confirm the relationship between HBV,NKILA,and HCC,this study focused on the effects of NKILA on the interaction between HBx and P65.The results showed that overexpression of NKILA can inhibit the NF-κB activity induced by HBx in HepG2 cells,and assessment of the NKILA wild type and truncated type on the NF-κB activity induced by HBx in HepG2 cells showed that the NKILA inhibited the NF-κB activity induced by HBx in HepG2 cells depended on the integrity of its own structure.Co-IP test results showed that overexpression of NKILA could inhibit the formation of HBx-NF-kB P65 complex.This study demonstrated that NKILA can inhibit the activation of HBX-dependent NF-κB signaling pathway,suggesting that NKILA may realize its role in the development of HBV-related HCC by inhibiting the activity of HBX-induced NF-κB signaling pathway.(5)To further determine the effect of NKILA on NF-κB and hepatoma cell growth.The results showed that the non-functional truncated NKILA lost the inhibition of NF-κB activation as well as the inhibition of HBV replication.HepG2 cells were transfected with four vectors(stable negative control vector pLVX puro,stable pLVX-NKILA,stable negative control vector pLVX puro transfected with HBx,and stable pLVX-NKILA transfected with HBx).The results demonstrated that NKILA inhibited the proliferation,migrations and invasion of HepG2 cells,and regulated the growth of HCC cells induced by HBx.Conclusion:The present study demonstrated that NKILA inhibits HBV replication and HBV-related HCC development by down-regulating the NF-κB signaling pathway.This finding provides a new theoretical basis for anti-HBV drug research and development while also shedding light on the mechanism of action of LncRNA NKILA in the replication of other types of viruses.This discovery offers a promising universal target for anti-tumor/antiviral drug research and development.