Junwei Tang

HULC and Linc00152 Act as Novel Biomarkers in Predicting Diagnosis of Hepatocellular Carcinoma

Background/Aims: The alterations of long non-coding RNAs (lncRNAs) are related to multiple diseases. They can be detected in plasma as biomarkers for the diagnosis of multiple diseases. In this study, we aimed to determine the expression of circulating lncRNAs in human, which may be promising biomarkers for the diagnosis of hepatocellular carcinoma (HCC). Methods: Eight lncRNAs were chosen as candidates on the basis of the literature to evaluate the diagnostic value and accuracy of the plasma lncRNA profiling system. The candidate lncRNAs were validated by qRT-PCR arranged in the training and validation sets. Additional double-blind testing was performed in 20 patients clinically suspected of having HCC. Results: Circulating HULC and Linc00152 were significantly up-regulated in plasma samples of HCC patients during training set and validation set. Areas under the receiver operating characteristic (ROC) curves of the validated two lncRNAs signature were 0.78 and 0.85, respectively. Combination of HULC and Linc00152 possessed a moderate ability to discrimination between HCC and control with an area under ROC value of 0.87 while the combination of AFP was 0.89 with a positive correlation with tissues expression. Conclusions: Our results suggest that both plasma levels of HULC and Linc00152 achieve a fine diagnostic accuracy in diagnosing ontogenesis and metastasis of HCC and may act as novel biomarkers for HCC.

A novel biomarker Linc00974 interacting with KRT19 promotes proliferation and metastasis in hepatocellular carcinoma

Location-associated long noncoding RNA (lncRNA) was reported to interact with target protein via a cis-regulatory process especially for the Flank10kb class lncRNA. Based on this theory, we aimed to explore the regulatory mechanisms of Linc00974 and KRT19 (an lncRNA beyond the Flank10kb class with protein) when we first confirmed the aberrant expression in hepatocellular carcinoma in a previous study. Knockdown of Linc00974 resulted in an inhibition of cell proliferation and invasion with an activation of apoptosis and cell cycle arrest in vitro, which was also validated by a subcutaneous and tail vein/intraperitoneal injection xenotransplantation model in vivo. We further investigated the interaction pattern of Linc00974 and KRT19. MiR-642 was identified, by acting as the competing endogenous RNA in regulating Linc00974 and KRT19. Linc00974 was increased owing to an abnormal hypomethylation promoter, which induced the upregulation of KRT19 via ceRNA interaction, resulting in the activation of the Notch and TGF-β pathways as detected by cDNA microarray. We also discovered Linc00974F-1 stably expressed in the plasma. By the combined analysis of Linc00974F-1 with CYFRA21-1, we found that these joint indicators predicted growth and metastasis of tumor in HCC patients. In conclusion, the combination of Linc00974 and KRT19 may be novel indices for clinical diagnosis of tumor growth and metastasis in HCC, while Linc00974 may become a potential therapeutic target for the prevention of HCC progression.

LINC00152 promotes proliferation in hepatocellular carcinoma by targeting EpCAM via the mTOR signaling pathway

Hepatocellular carcinoma (HCC) is well known as the sixth most common malignant tumor and the third leading cause of cancer-related deaths globally. LINC00152 was documented as an important long non-coding RNA (lncRNA) involved in the pathogenesis of gastric cancer; however, the detailed mechanism of action of LINC00152 remains unknown. Here, based on the increased level of LINC00152 in HCC tissues, we found that LINC00152 could promote cell proliferation in vitro and tumor growth in vivo. Furthermore, microarray-based analysis indicated that LINC00152 could activate the mechanistic target of rapamycin(mTOR) pathway by binding to the promoter of EpCAM through a cis-regulation, as confirmed by Gal4-λN/BoxB reporter system. Thus, LINC00152 might be involved in the oncogenesis of HCC by activating the mTOR signaling pathway and might be a novel index for clinical diagnosis in the future.

The aberrant expression of MEG3 regulated by UHRF1 predicts the prognosis of hepatocellular carcinoma

MEG3 as a tumor suppressor has been reported to be linked with pathogenesis of malignancies including hepatocellular carcinoma (HCC). However, the mechanism of MEG3 in HCC still remains unclear. In our study, the aberrant decreased level of MEG3 in 72 tumor tissues obtained from HCC patients and cell lines was examined by using real‐time PCR. The inhibition affection in proliferation and inducing affection in apoptosis was further confirmed in vivo and vitro, we also demonstrated that MEG3 regulates HCC cell proliferation and apoptosis partially via the accumulation of p53. Besides, the hypermethylation of MEG3 in promoter region was identified by bisulfite sequencing while MEG3 increased with the inhibition of methylation. Subsequently, UHRF1, a new identified oncogene which is required for DNA methylation and recruits, was investigated. A negative correlation of MEG3 and UHRF1 expression was verified in primary HCC tissues. Down‐regulation of UHRF1 induced MEG3 expression in HCC cell lines, which could be reversed by the up‐regulation of UHRF1. In addition, up‐regulation of MEG3 in HCC cells partially diminished the promotion of proliferation induced by UHRF1. Moreover, Kaplan–Meier analysis demonstrated that the patients with low expression of MEG3 indicated worse overall and relapse‐free survivals compared with high expression of MEG3. Cox proportional hazards analyses showed that MEG3 expression was an independent prognostic factor for HCC patients. In conclusion, we demonstrated MEG3, acting as a potential biomarker in predicting the prognosis of HCC, was regulated by UHRF1 via recruiting DNMT1 and regulated p53 expression.

The long noncoding RNA lnc-EGFR stimulates T-regulatory cells differentiation thus promoting hepatocellular carcinoma immune evasion

Long noncoding RNAs play a pivotal role in T-helper cell development but little is known about their roles in Treg differentiation and functions during the progression of hepatocellular carcinoma (HCC). Here, we show that lnc-epidermal growth factor receptor (EGFR) upregulation in Tregs correlates positively with the tumour size and expression of EGFR/Foxp3, but negatively with IFN-γ expression in patients and xenografted mouse models. Lnc-EGFR stimulates Treg differentiation, suppresses CTL activity and promotes HCC growth in an EGFR-dependent manner. Mechanistically, lnc-EGFR specifically binds to EGFR and blocks its interaction with and ubiquitination by c-CBL, stabilizing it and augmenting activation of itself and its downstream AP-1/NF-AT1 axis, which in turn elicits EGFR expression. Lnc-EGFR links an immunosuppressive state to cancer by promoting Treg cell differentiation, thus offering a potential therapeutic target for HCC.

Epstein-Barr Virus-Encoded Latent Membrane Protein 2A Promotes the Epithelial-Mesenchymal Transition in Nasopharyngeal Carcinoma via Metastatic Tumor Antigen 1 and Mechanistic Target of Rapamycin Signaling Induction

ABSTRACT Epstein-Barr virus-encoded latent membrane protein 2A (LMP2A) promotes the epithelial-mesenchymal transition (EMT) of nasopharyngeal carcinoma (NPC), thereby increasing tumor invasion. Recently, the dysregulation of metastatic tumor antigen 1 (MTA1) was found to enhance tumor metastasis in a variety of cancers. A molecular connection between these two proteins has been proposed but not firmly established. In this study, we reported the overexpression of MTA1 in 29/60 (48.3%) NPC patients, and the overexpression of MTA1 significantly correlated with tumor metastasis. The overexpression of MTA1 promoted EMT via the Wnt1 pathway and β-catenin activation. We demonstrated that LMP2A reinforces the expression of MTA1 via the mechanistic target of rapamycin (mTOR) pathway to promote EMT in NPC. Furthermore, by knocking down 4EBP1 in combination with the new mTOR inhibitor INK-128 treatment, we discovered that LMP2A expression activates the 4EBP1-eIF4E axis and increases the expression of MTA1 at the translational level partially independent of c-myc. These findings provided novel insights into the correlation between the LMP2A and MTA1 proteins and reveal a novel function of the 4EBP1-eIF4E axis in EMT of nasopharyngeal carcinoma. IMPORTANCE Prevention of the recurrence and metastasis of NPC is critical to achieving a successful NPC treatment. As we all know, EMT has a vital role in metastasis of malignancies. LMP2A, an oncoprotein of Epstein-Barr virus, a well-known NPC activator, induces EMT and has been proved to exert a promoting effect in tumor metastasis. Our study demonstrated that LMP2A could induce EMT by activating MTA1 at the translational level via activating mTOR signaling and the 4EBP1-eIF4E axis. Taken together, our findings bridge the gap between the NPC-specific cell surface molecule and the final phenotype of the NPC cells. Additionally, our findings indicate that LMP2A and mTOR will serve as targets for NPC therapy in the future.

LINC01225 promotes occurrence and metastasis of hepatocellular carcinoma in an epidermal growth factor receptor-dependent pathway

The long noncoding RNAs (lncRNAs) have long been clarified to participate in hepatocellular carcinoma (HCC) as a biomarker. We carried out the present study in order to identify HCC-related lncRNAs and elucidate the functional roles in the development and progression of HCC. Our previous study has provided that LINC01225 may be an HCC-related gene. Here, we verified that LINC01225 was upregulated in HCC. Knockdown of LINC01225 resulted in inhibited cell proliferation and invasion with activated apoptosis and cell cycle arrest in vitro. Overexpression of LINC01225 in LINC01225 knockdown cells presented that attenuated cell proliferation and invasion were restored and enhanced. Subcutaneous and tail vein/intraperitoneal injection xenotransplantation model in vivo validated reduced tumor progression and metastasis. Investigation of mechanism found that LINC01225 could bind to epidermal growth factor receptor (EGFR) and increase the protein level of EGFR, and subsequently fine tune the EGFR/Ras/Raf-1/MEK/MAPK signaling pathway. Analysis with clinicopathological information suggested a high expression of LINC01225 is positively associated with poor prognosis. We also proved that LINC01225 was stably expressed in serum and can act as a novel biomarker in predicting the diagnosis of HCC. As a conclusion, LINC01225 plays a crucial role in HCC and can act as a biomarker for the diagnosis and prognosis of HCC.

Inhibition of MTA1 by ERα contributes to protection hepatocellular carcinoma from tumor proliferation and metastasis.

BackgroundAlthough expression of MTA1 inversely correlates with the nuclear localization of ERα, the effect and molecular mechanism of ERα regulation of MTA1 remain unknown.MethodsQuantitative real-time PCR and western blot analyses were used to measure levels of MTA1. The effect on HCC cell proliferation and invasion was assessed by EdU incorporation assays and Transwell, respectively. ShRNA and dual-luciferase assays were used to investigate the regulatory relationship between MTA1 and ERα in cell lines.ResultsWe found that MTA1 gene regulation by ERα may be influenced by nuclear corepressors. The MTA1 promoter has three functional ER-element half-sites that lead to decreased MTA1 transcription and expression. ERα overexpression suppressed the proliferation and invasion of hepatocellular carcinoma cells (HCC). In addition, overexpression of MTA1 attenuated ERα-mediated suppression of the proliferation and invasion of HCC cells and tumor formation in vivo. These results suggested feedback regulation between ERα and MTA1. In summary, our results demonstrated that ERα suppressed proliferation and invasion of human HCC cells through downregulation of MTA1 transcription.ConclusionsOur study is an improved description of the mechanisms of the suppressive effect of ERα on HCCs, adding understanding to the gender disparity of HCC progression.

PTPRO-Associated Hepatic Stellate Cell Activation Plays a Critical Role in Liver Fibrosis

Background/Aims: PTPRO (protein tyrosine phosphatase, receptor type O) is implicated in diverse physiological and pathological processes in cancer and hepatic ischemia/reperfusion injury, although little is known about its role in hepatic fibrosis. Methods: Here, by using genetically deficient mice, we reported that PTPRO knockout (PTPRO-/-) significantly attenuated liver injury, release of inflammatory factors, tissue remodeling, and liver fibrosis in two experimental mouse models of fibrogenesis induced by bile-duct ligation or carbon tetrachloride administration. Results: However, we proved that PTPRO expression was strongly downregulated in clinical and experimental liver fibrosis specimens. Further investigations revealed that stimulation of primary hepatic stellate cells (HSCs) and hepatocytes with specific activator platelet-derived growth factor (PDGF)-BB increased PTPRO transcription in HSCs but had the opposite effect in primary hepatocytes. More importantly, synthetic short hairpin RNA targeting PTPRO significantly neutralized PDGF-BB-induced HSC proliferation and myofibroblast marker expression through downregulated phosphorylation of extracellular signal-regulated kinase (ERK) and AKT. Conclusion: These observations confirm that PTPRO plays a critical role in liver fibrogenesis by affecting PDGF signaling in HSC activation and might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases.

Alpha1-ACT Functions as a Tumour Suppressor in Hepatocellular Carcinoma by Inhibiting the PI3K/AKT/mTOR Signalling Pathway via Activation of PTEN

Background & Aims: To investigate the expression and prognostic value of α1-ACT (Alpha1-antichymotrypsin) in patients with HCC (hepatocellular carcinoma) and identify the mechanism by which α1-ACT inhibits proliferation and promotes apoptosis of HCC. Methods: We first measured α1-ACT expression levels and determined their relationship with the clinicopathological characteristics and prognosis of patients with HCC.We then established stable HCC cell lines with both α1-ACT overexpression and knockdown and performed a functional analysis in vitro.We first examined the relationship between α1-ACT and the PTEN/PI3K/AKT/mTOR pathway using Western blotting. Then, we determined whether α1-ACT can directly bind to PTEN using co-immunoprecipitation. Finally, we measured α1-ACT expression to evaluate its correlation with the PI3K/AKT/mTOR pathway-related apoptosis proteins in a xenograft tumour mouse model using immunohistochemistry. Results: The α1-ACT expression level was significantly lower in the HCC tissues than in the paratumour tissues and was negatively positively correlated with the level of Ki67, AFP, the AJCC stage, tumour size and tumour invasion. The overexpression of α1-ACT can inhibit cell proliferation and increase cell apoptosis by activating PI3K/AKT/mTOR-mediated apoptosis via binding to PTEN and activating it in vitro. Additionally, the overexpression of α1-ACT can also increase the proportion of cells in the G0/G1 stage by increasing cyclin p21 expression and inhibiting the migration and invasion abilities of HCC cells by regulating MMP2 and MMP9. The xenotransplantation studies with nude mice also showed that overexpression of α1-ACT inhibited tumourigenesis and knockdown of α1-ACT had the opposite effect. Conclusions: Our study demonstrates that α1-ACT suppresses liver cancer development and metastasis via targeting the PTEN/PI3K/AKT/mTOR signalling pathway, which may be a potential target for therapeutic intervention in HCC.