Sirolimus improves the prognosis of liver recipients with hepatocellular carcinoma: A single-center experience.

BACKGROUND: Tumor recurrence after liver transplantation (LT) for selective patients diagnosed with hepatocellular carcinoma (HCC) in the setting of cirrhosis is the greatest challenge effecting the prognosis of these patients. The aim of this study was to evaluate the efficacy of sirolimus on the prognosis for these recipients. METHODS: The data from 193 consecutive HCC patients who had undergone LT from January 2015 to December 2019 were retrospectively analyzed. These patients were divided into the sirolimus group [patients took sirolimus combined with calcineurin inhibitors (CNIs) (n = 125)] and non-sirolimus group [patients took CNI-based therapy without sirolimus (n = 68)]. Recurrence-free survival (RFS) and overall survival (OS) were compared between the two groups. The prognostic factors and independent risk factors for RFS and OS were further evaluated. RESULTS: Non-sirolimus was an independent risk factor for RFS (HR = 2.990; 95% CI: 1.050-8.470; P = 0.040) and OS (HR = 3.100; 95% CI: 1.190-8.000; P = 0.020). A higher proportion of patients beyond Hangzhou criteria was divided into the sirolimus group (69.6% vs. 80.9%, P = 0.030). Compared with the non-sirolimus group, the sirolimus group had significantly better RFS (P < 0.001) and OS (P < 0.001). Further subgroup analysis showed similar results. CONCLUSIONS: This study demonstrated that sirolimus significantly decreased HCC recurrence and prolonged RFS and OS in LT patients with different stage of HCC.

EHMT2 promotes the pathogenesis of hepatocellular carcinoma by epigenetically silencing APC expression.

BACKGROUND: Hepatocellular carcinoma (HCC), the second leading cause of cancer death worldwide, alone accounts for over half (466,100) of new cancer cases and 422,100 deaths based on the average year incidence rates of 2009 to 2011 in China. Due to unclear and complex underlying mechanisms for HCC development, effective therapy for HCC is still unavailable. The Wnt-ß-catenin pathway is a critical contributor of HCC pathogenesis: 40-70% of HCCs from patients harbor the nuclear accumulation of ß-catenin protein. However, the mechanisms for ß-catenin activation are not fully understood. METHODS: The deletion of EHMT2 in Hep3B and Huh1 cells was achieved by transiently transfecting cells with pX459 plasmids, which carry EHMT2 specific small guide RNA (sgRNA) sequences for Cas9 protein. All experiments were performed in triplicate and repeated more than three times. RESULTS: In the present study, we observed that EHMT2 (but not EHMT1) mRNA and protein levels were significantly elevated in HCC compared with normal controls. Next, the results of Ki67 staining, as well as MTT, soft-agar and xenograft assays, in wild-type and EHMT2-/- Hep3B and Huh1 cancer stem cells collectively revealed that the elevation of EHMT2 expression is required for the tumorigenesis of HCC. Meanwhile, we found that elevated EHMT2 expression contributes to the activation of Wnt-ß-catenin signaling: deletion of EHMT2 in Hep3B or Huh1 cells promoted the cytoplasmic localization of ß-catenin and restrained the expression of Wnt-ß-catenin signaling targets such as Myc, CCND1, MMP-7, etc. We demonstrated that EMHT2 directly mediates the H3K9me2 methylation of the APC promoter to epigenetically silence its expression. More intriguingly, our findings also showed that UNC0642, a specific inhibitor of EHMT2, exhibits anti-tumorigenesis effects in HCC both in vitro and in vivo, which were largely abolished by deletion of EHMT2 or overexpression of APC in Hep3B and Huh1 cells. CONCLUSION: Altogether, our observations emphasize that the EHMT2-APC axis is a critical contributor to Wnt-ß-catenin pathway activation in HCC, and UNC0642 may be a potential candidate for target drug treatment of HCC.

[Effect of electroacupuncture on cognitive functions, hippocampal HIF-1α and neurons apoptosis in postoperative cognitive dysfunction rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on learning and memory ability, hippocampal hypoxia inducible factor-1α(HIF-1α) and apoptosis in postoperative cognitive dysfunction(POCD) rats, and to investigate its mechanism underlying improvement of POCD. METHODS: A total of 90 aged male SD rats were randomized into a sham-operation group, a model group and an EA group, 30 rats in each group, which were further divided into 3 time-point subgroups (1, 3 and 7 days after intevention, 10 rats in each subgroup). In the model group and the EA group, left hepatectomy was adopted to establish the model of POCD. In the sham-operation group, the skin was sectioned and no hepatectomy was operated. In the EA group, EA was applied at "Siguan" ["Hegu" (LI 4) and "Taichong" (LR 3)] with dilatational wave, 2 Hz/100 Hz in frequency, 1 mA in intensity, 20 min each time, once a day. Morris water maze test was adopted to observe the cognitive functions. Real-time PCR and Western blot were used to measure the hippocampal level of HIF-1α. TUNEL method was used to evaluate the hippocampal level of neurons apoptosis. Double immunofluorescence labeling was used to detect the colocalization of HIF-1α and apoptosis in the EA group. RESULTS: Compared with the sham-operation group, the escape latency was prolonged and the frequency of platform leaping was reduced in the model group (P<0.05) after 1, 3, 7 days of intervention. Compared with the model group,the escape latency was shortened and the frequency of platform leaping was increased in the EA group (P<0.05) after 1, 3, 7 days of intervention. After 3 days of intervention, compared with the sham-operation group, the expressions of HIF-1α mRNA and protein, the level of apoptosis were increased in the model group (P<0.05); compared with the model group,the expressions of HIF-1α mRNA and protein, the level of apoptosis were decreased in the EA group (P<0.05). The colocalization of HIF-1α and apoptosis was observed in same cells in the EA group. CONCLUSION: Electroacupuncture improves cognitive functions in postoperative cognitive dysfunction rats, which may be related to its effect in down-regulating the expression of hippocampal HIF-1α and inhibiting the neurons apoptosis.

Plasma circular RNA panel to diagnose hepatitis B virus-related hepatocellular carcinoma: A large-scale, multicenter study.

To explore whether plasma circular RNAs (circRNAs) can diagnose hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), microarray and qPCR were used to identify plasma circRNAs that were increased in HBV-related HCC patients compared to controls (including healthy controls, chronic hepatitis B and HBV-related liver cirrhosis). A logistic regression model was constructed using a training set (n = 313) and then validated using another two independent sets (n = 306 and 526, respectively). Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy. We identified a plasma circRNA panel (CircPanel) containing three circRNAs (hsa_circ_0000976, hsa_circ_0007750 and hsa_circ_0139897) that could detect HCC. CircPanel showed a higher accuracy than AFP (alpha-fetoprotein) to distinguish individuals with HCC from controls in all three sets (AUC, 0.863 [95% confidence interval, CI: 0.819-0.907] vs. 0.790 [0.738-0.842], p = 0.036 in training set; 0.843 [0.796-0.890] vs. 0.747 [0.691-0.804], p = 0.011 in validation set 1 and 0.864 [0.830-0.898] vs. 0.769 [0.728-0.810], p < 0.001 in validation set 2). CircPanel also performed well in detecting Small-HCC (solitary, ≤3 cm), AFP-negative HCC and AFP-negative Small-HCC.

A novel HBx genotype serves as a preoperative predictor and fails to activate the JAK1/STATs pathway in hepatocellular carcinoma.

BACKGROUND & AIMS: Genetic variability in the hepatitis B virus X gene (HBx) is frequently observed and is associated with hepatocellular carcinoma (HCC) progression. However, a genotype classification based on the full-length HBx sequence and the impact of genotypes on hepatitis B virus (HBV)-related HCC prognosis remain unclear. We therefore aimed to perform this genotype classification and assess its clinical impact. METHODS: We classified the genotypes of the full-length HBx gene through sequencing and a cluster analysis of HBx DNA from a cohort of patients with HBV-related HCC, which served as the primary cohort (n = 284). Two independent HBV-related HCC cohorts, a validation cohort (n = 171) and a serum cohort (n = 168), were used to verify the results. Protein microarray assay analysis was performed to explore the underlying mechanism. RESULTS: In the primary cohort, the HBx DNA was classified into 3 genotypes: HBx-EHBH1, HBx-EHBH2, and HBx-EHBH3. HBx-EHBH2 (HBx-E2) indicated better recurrence-free survival and overall survival for patients with HCC. HBx-E2 was significantly correlated with the absence of liver cirrhosis, a small tumor size, a solitary tumor, complete encapsulation and Barcelona Clinic Liver Cancer (BCLC) stage A-0 tumors. Additionally, HBx-E2 served as a significant prognostic factor for patients with BCLC stage B HCC after hepatectomy. Mechanistically, HBx-E2 is unable to promote proliferation in HCC cells and normal hepatocytes. It also fails to activate the Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3)/STAT5 pathway. CONCLUSION: Our study identifies a novel HBx genotype that is unable to promote the proliferation of HCC cells and suggests a potential marker to preoperatively predict the prognosis of patients with BCLC stage B, HBV-associated, HCC. LAY SUMMARY: We classified a novel genotype of the full-length hepatitis B virus X gene (HBx), HBx-E2. This genotype was identified in tumor and nontumor tissues from patients with hepatitis B virus-related hepatocellular carcinoma. HBx-E2 could preoperatively predict the prognosis of patients with intermediate stage hepatocellular carcinoma, after resection.

Overactivated neddylation pathway in human hepatocellular carcinoma.

Dysregulation of the neddylation pathway is related to various cancers. However, the specific role of the neddylation pathway in human hepatocellular carcinoma (HCC) remains largely unclear. In this study, the neddylation pathway in HCC and adjacent noncancerous liver (ANL) tissues was evaluated by immunohistochemistry (IHC), Western blotting, and qRT-PCR (quantitative real-time polymerase chain reaction). The results showed that the entire neddylation pathway, including NEDD8 (the IHC staining of NEDD8 represents the global-protein neddylation), E1 NEDD8-activating enzymes (NAE1 and UBA3), E2 NEDD8-conjugating enzymes (UBE2F and UBE2M), E3 NEDD8-ligases (MDM2, RBX1 and RNF7), and deneddylation enzymes (COPS5, UCHL1 and USP21), was overactivated in HCC. Furthermore, the upregulation of NEDD8 in HCC was correlated with aggressive characteristics and was an independent risk factor for overall survival (OS) and recurrence-free survival (RFS) in patients with HCC after hepatectomy. The upregulation of NAE1, UBE2M, and UCHL1 in HCC was associated with aggressive characteristics and poor OS and RFS in patients with HCC after hepatectomy. In conclusion, our research reveals that the entire neddylation pathway is overactivated in HCC and associated with clinical characteristics and prognosis of patients with HCC.

IL-17A promotes the invasion-metastasis cascade via the AKT pathway in hepatocellular carcinoma.

We previously demonstrated that interleukin-17A (IL-17A) is associated with the progression of hepatocellular carcinoma (HCC). However, its role in the invasion-metastasis cascade of HCC and the efficacy of IL-17A-targeting therapeutics in HCC remain largely unknown. In this study, we found that IL-17A promoted intrahepatic and pulmonary metastasesis of HCC cells in an orthotopic implant model. Moreover, our results showed that IL-17A induced epithelial-mesenchymal transition (EMT) and promoted HCC cell colonization in vitro and in vivo, and the role of IL-17A in invasion-metastasis was dependent on activation of the AKT pathway. Remarkably, combined therapy using both secukinumab and sorafenib has better inhibition on tumour growth and metastasis compared to sorafenib monotherapy. Additionally, the combination of intratumoral IL-17A+ cells and E-cadherin predicted the outcome of patients with HCC at an early stage after hepatectomy based on tissue microarray and immunohistochemistry. In conclusion, our studies reveal that IL-17A induces early EMT and promotes late colonization of HCC metastasis by activating AKT signalling. Secukinumab is a promising candidate for clinical development in combination with sorafenib for the management of HCC.

Circular RNA cSMARCA5 inhibits growth and metastasis in hepatocellular carcinoma.

BACKGROUND & AIMS: In recent years, circular RNAs (circRNAs) have been shown to have critical regulatory roles in cancer biology. However, the contributions of circRNAs to hepatocellular carcinoma (HCC) remain largely unknown. METHODS: cSMARCA5 (a circRNA derived from exons 15 and 16 of the SMARCA5 gene, hsa_circ_0001445) was identified by RNA-sequencing and validated by quantitative reverse transcription PCR. The role of cSMARCA5 in HCC progression was assessed both in vitro and in vivo. circRNAs in vivo precipitation, luciferase reporter assay, biotin-coupled microRNA capture and fluorescence in situ hybridization were conducted to evaluate the interaction between cSMARCA5 and miR-17-3p/miR-181b-5p. RESULTS: The expression of cSMARCA5 was lower in HCC tissues, because of the regulation of DExH-Box Helicase 9, an abundant nuclear RNA helicase. The downregulation of cSMARCA5 in HCC was significantly correlated with aggressive characteristics and served as an independent risk factor for overall survival and recurrence-free survival in patients with HCC after hepatectomy. Our in vivo and in vitro data indicated that cSMARCA5 inhibits the proliferation and migration of HCC cells. Mechanistically, we found that cSMARCA5 could promote the expression of TIMP3, a well-known tumor suppressor, by sponging miR-17-3p and miR-181b-5p. CONCLUSION: These results reveal an important role of cSMARCA5 in the growth and metastasis of HCC and provide a fresh perspective on circRNAs in HCC progression. LAY SUMMARY: Herein, we studied the role of cSMARCA5, a circular RNA, in hepatocellular carcinoma. Our in vitro and in vivo data showed that cSMARCA5 inhibits the growth and migration of hepatocellular carcinoma cells, making it a potential therapeutic target.

METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N6 -methyladenosine-dependent primary MicroRNA processing.

N6 -Methyladenosine (m6 A) modification has been implicated in many biological processes. However, its role in cancer has not been well studied. Here, we demonstrate that m6 A modifications are decreased in hepatocellular carcinoma, especially in metastatic hepatocellular carcinoma, and that methyltransferase-like 14 (METTL14) is the main factor involved in aberrant m6 A modification. Moreover, METTL14 down-regulation acts as an adverse prognosis factor for recurrence-free survival of hepatocellular carcinoma and is significantly associated with tumor metastasis in vitro and in vivo. We confirm that METTL14 interacts with the microprocessor protein DGCR8 and positively modulates the primary microRNA 126 process in an m6 A-dependent manner. Further experiments show that microRNA 126 inhibits the repressing effect of METTL14 in tumor metastasis. CONCLUSION: These studies reveal an important role of METTL14 in tumor metastasis and provide a fresh view on m6 A modification in tumor progression. (Hepatology 2017;65:529-543).

Paraoxonase 3 inhibits cell proliferation and serves as a prognostic predictor in hepatocellular carcinoma.

Paraoxonase 3 (PON3) exerts prominent anti-inflammation and anti-oxidation properties mainly at the cellular level, and is primarily expressed in the liver. However, its role in HCC remains unexplored. Here, we investigated the expression pattern, clinical significance, and function of PON3 in HCC. PON3 mRNA and protein levels were respectively determined in two large cohorts using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) of tissue microarray. We found that PON3 was downregulated in most HCCs. Kaplan-Meier and log-rank test showed that PON3 downregulation predicted shorter recurrence-free survival (RFS) and overall survival (OS) time in all HCC patients, especially early-stage HCC patients. Cox regression analysis revealed that the PON3 downregulation was an independent risk factor for RFS and OS. Gain- and loss-of-function experiments revealed that PON3 suppressed cell proliferation in vivo and in vitro, which was attributed to its cell-cycle arrest effect. In addition, microarray analysis showed that some pro-proliferative genes were elevated when PON3 was knockdown, and these genes possibly involved in the underlying mechanisms. In conclusion, our studies reveal the cell proliferation inhibitory function of PON3 and offer a potential prognostic predictor and therapeutic target for HCC.

Overexpression of CHKA contributes to tumor progression and metastasis and predicts poor prognosis in colorectal carcinoma.

Aberrant expression of choline kinase alpha (CHKA) has been reported in a variety of human malignancies including colorectal carcinoma (CRC). However, the role of CHKA in the progression and prognosis of CRC remains unknown. In this study, we found that CHKA was frequently upregulated in CRC clinical samples and CRC-derived cell lines and was significantly correlated with lymph node metastasis (p = 0.028), TNM stage (p = 0.009), disease recurrence (p = 0.004) and death (p < 0.001). Survival analyses indicated that patients with higher CHKA expression had a significantly shorter disease-free survival (DFS) and disease-specific survival (DSS) than those with lower CHKA expression. Multivariate analyses confirmed that increased CHKA expression was an independent unfavorable prognostic factor for CRC patients. In addition, combination of CHKA with TNM stage was a more powerful predictor of poor prognosis than either parameter alone. Functional study demonstrated that knockdown of CHKA expression profoundly suppressed the growth and metastasis of CRC cells both in vitro and in vivo. Mechanistic investigation revealed that EGFR/PI3K/AKT pathway was essential for mediating CHKA function. In conclusion, our results provide the first evidence that CHKA contributes to tumor progression and metastasis and may serve as a novel prognostic biomarker and potential therapeutic target in CRC.

Long noncoding RNA DANCR increases stemness features of hepatocellular carcinoma by derepression of CTNNB1.

UNLABELLED: Tumor cells with stemness (stem-cell) features contribute to initiation and progression of hepatocellular carcinoma (HCC), but involvement of long noncoding RNAs (lncRNAs) remains largely unclear. Genome-wide analyses were applied to identify tumor-associated lncRNA-DANCR. DANCR expression level and prognostic values of DANCR were assayed in two HCC cohorts (China and Korea, n = 135 and 223). Artificial modulation of DANCR (down- and overexpression) was done to explore the role of DANCR in tumorigenesis and colonization, and tumor-bearing mice were used to determine therapeutic effects. We found that lncRNA-DANCR is overexpressed in stem-like HCC cells, and this can serve as a prognostic biomarker for HCC patients. Experiments showed that DANCR markedly increased stemness features of HCC cells to promote tumorigenesis and intra-/extrahepatic tumor colonization. Conversely, DANCR knockdown attenuated the stem-cell properties and in vivo interference with DANCR action led to decreased tumor cell vitality, tumor shrinkage, and improved mouse survival. Additionally, we found that the role of DANCR relied largely on an association with, and regulation of, CTNNB1. Association of DANCR with CTNNB1 blocked the repressing effect of microRNA (miR)-214, miR-320a, and miR-199a on CTNNB1. This observation was confirmed in vivo, suggesting a novel mechanism of tumorigenesis involving lncRNAs, messenger RNAs, and microRNAs. CONCLUSIONS: These studies reveal a significance and mechanism of DANCR action in increasing stemness features and offer a potential prognostic marker and a therapeutic target for HCC.

Long noncoding RNA, the methylation of genomic elements and their emerging crosstalk in hepatocellular carcinoma.

The epigenetic mechanism that incorporates DNA methylation alterations, histone modifications, and non-coding RNA expression has been identified as a major characteristic in distinguishing physiological and pathological settings of cancers including hepatocellular carcinoma (HCC), the third leading cause of mortality related cancer. The advance in methylation modification of chromatin elements (for both genomic DNA and histone tails) and the emerging roles of long noncoding RNA (lncRNA) have given us a better understanding of molecular mechanisms underlying HCC. Recently, methods like genome-wide lncRNA profiling and histone hallmark detection were reported to discover mass tumor-associated lncRNAs epigenetically deregulated by differential chromosome modification, mainly by genomic DNA and histone methylation. Therefore, aberrant methylation modification of certain particular lncRNA genes could be crucial events correlating with unfavorable outcomes in HCC. In addition, amount of lncRNAs could act as a manipulator for DNA methylation or a scaffold for histone modification to affect key signaling pathways in hepatocarcinogenesis. This suggests that methylation modification of chromatin elements may have functional crosstalk with lncRNA. Here, we aim to outline the emerging role of the methylation and lncRNA, and their crosstalk of molecular mechanism.

Aldolase B inhibits metastasis through Ten-Eleven Translocation 1 and serves as a prognostic biomarker in hepatocellular carcinoma.

BACKGROUND: Downregulation of Aldolase B (ALDOB) has been reported in hepatocellular carcinoma. However, its clinical significance and its role in pathogenesis of HCC remain largely unknown. METHODS: We analyzed the expression of ALDOB and its clinical features in a large cohort of 313 HCC patients using tissue microarray and immunohistochemistry. Moreover, the function of stably overexpressed ALDOB in HCC cells was explored in vitro and in vivo. Gene expression microarray analysis was performed on ALDOB-overexpressing SMMC7721 cells to elucidate its mechanism of action. RESULTS: ALDOB downregulation in HCC was significantly correlated with aggressive characteristics including absence of encapsulation, increased tumor size (>5 cm) and early recurrence. ALDOB downregulation was indicative of a shorter recurrence-free survival (RFS) and overall survival (OS) for all HCC patients and early-stage HCC patients (BCLC 0-A and TNM I stage patients). Multiple analyses revealed that ALDOB downregulation was an independent risk factor of RFS and OS. Stable expression of ALDOB in HCC cell lines reduced cell migration in vitro and inhibited lung metastasis, intrahepatic metastasis, and reduced circulating tumor cells in vivo. Mechanistically, we found that cells stably expressing ALDOB show elevated Ten-Eleven Translocation 1 (TET1) expression. Moreover, ALDOB expressing cells have higher levels of methylglyoxal than do control cells, which can upregulate TET1 expression. CONCLUSION: The downregulation of ALDOB could indicate a poor prognosis for HCC patients, and therefore, ALDOB might be considered a prognostic biomarker for HCC, especially at the early stage. In addition, ALDOB inhibits the invasive features of cell lines partly through TET1 expression.