Comprehensive Analysis of Cellular Senescence-Related Genes in Prognosis, Molecular Characterization and Immunotherapy of Hepatocellular Carcinoma.

BACKGROUND: Cellular senescence is a tumor suppressive response in which the cell cycle is in a state of permanent arrest and can inhibit tumor cell proliferation. In recent years, induction of cellular senescence has been shown to be important for antitumor therapy, and the link between cellular senescence and clinical prognosis and immunotherapy of hepatocellular carcinoma is still unknown. METHODS: We performed enrichment analysis of genes in three cellular senescence gene sets, screened for gene sets significantly enriched in hepatocellular carcinoma and extracted genes from them. Signature were constructed using senescence-related genes, and their expression was verified at the protein and RNA levels. Survival, clinical staging and grading, immune infiltration, immunotherapy, and drug sensitivity were also analyzed between risk groups. RESULTS: The q-PCR and immunohistochemistry results revealed significant differences in the expression of the signature genes between normal and tumor tissues. Significant differences in clinicopathological features, prognosis and immune infiltration were observed between risk groups. In the low-risk group, better OS and lower TMB scores were demonstrated, while the high-risk group had higher immune checkpoint expression, as well as lower risk of immune escape. In addition, we found that the High-risk group was more sensitive to sorafenib. CONCLUSION: In summary, the signature constructed using aging-related genes can reliably predict patient prognosis and immunotherapy efficacy, providing a new idea for immune system therapy of hepatocellular carcinoma.

Deubiquitinating enzyme USP46 suppresses the progression of hepatocellular carcinoma by stabilizing MST1.

The deubiquitinating enzyme USP46 (ubiquitin-specific protease 46) is implicated in various cancers. However, its role and regulatory mechanism in HCC (hepatocellular carcinoma) are still unknown. In this study, we showed that USP46 is downregulated in HCC tissues and that low USP46 levels are associated with poor prognosis in HCC patients. In functional experiments, overexpression of USP46 impaired proliferation and metastasis of HCC cells, whereas knockdown of USP46 enhanced cell proliferation and invasiveness in vitro and in vivo. Furthermore, we found that USP46 suppresses HCC cell proliferation and metastasis by inhibiting YAP1. Ectopic expression of YAP1 rescued the inhibition of cell proliferation and metastasis caused by USP46 overexpression. Mechanistically, USP46 promotes the degradation of YAP1 by increasing expression of MST1, and the increase in MST1 protein antagonizes YAP1 to suppress HCC progression. Finally, we demonstrated that USP46 stabilizes the MST1 protein by directly binding to it and decreasing its ubiquitination. Taken together, our results demonstrated that USP46 may be a novel tumor suppressor in HCC. Moreover, USP46 acts as a deubiquitinating enzyme of MST1 to potentiate MST1 kinase activity to suppress tumor growth and metastasis, indicating that USP46 activation may represent a potential treatment strategy for HCC.

Silencing of long non-coding RNA FOXD2-AS1 inhibits the progression of gallbladder cancer by mediating methylation of MLH1.

Evidence has documented the tumor-promoting properties of long non-coding RNA (lncRNA) FOXD2 adjacent opposite strand RNA 1 (FOXD2-AS1) in many cancers. However, little is known about its role in gallbladder cancer. Here, we aimed to characterize the functional relevance of lncRNA FOXD2-AS1 in gallbladder cancer and the possible mechanisms associated with methylation of MutL homolog-1 (MLH1). Initially, microarray-based gene expression profiling of gallbladder cancer was employed to identify differentially expressed lncRNAs. Next, the expression of lncRNA FOXD2-AS1 was examined, and the cell line presenting with the highest lncRNA FOXD2-AS1 expression was selected for subsequent experimentation. Then, the interaction between lncRNA FOXD2-AS1 and MLH1 was identified. The effect of lncRNA FOXD2-AS1 on proliferation, migration, invasion, and apoptosis as well as tumorigenicity of transfected GBC-SD cells was examined with gain- and loss-of-function experiments. We found that lncRNA FOXD2-AS1 was highly expressed, while MLH1 was poorly expressed in gallbladder cancer cells. Besides, lncRNA FOXD2-AS1 could promote MLH1 methylation by recruiting DNMT1 to the MLH1 promoter, and consequently inhibit MLH1 transcription. Silencing of lncRNA FOXD2-AS1 or overexpression of MLH1 inhibited gallbladder cancer cell proliferation, invasion, and migration, while facilitating cell apoptosis in vitro as well as retarding tumor growth in vivo. Thus, silencing of lncRNA FOXD2-AS1 suppressed the progression of gallbladder cancer via upregulation of MLH1 by inhibiting MLH1 promoter methylation. These findings present lncRNA FOXD2-AS1 knockdown as a potential candidate for the treatment of gallbladder cancer.

Long noncoding RNA LEF1-AS1 acts as a microRNA-10a-5p regulator to enhance MSI1 expression and promote chemoresistance in hepatocellular carcinoma cells through activating AKT signaling pathway.

Long noncoding RNAs (lncRNAs) contribute to the development of hepatocellular carcinoma (HCC), which could regulate various HCC biological characteristics. Here, the study seeks to investigate the role of lncRNA LEF1-AS1 in HCC cell chemoresistance by regulating microRNA (miR)-10a-5p and Musashi1 (MSI1). The microarray-based analysis was employed to identify the HCC-related lncRNA-miRNA-gene regulatory network. Expression patterns of LEF1-AS1, miR-10a-5p, and MSI1 in the HCC cell lines, tissues were accessed by means of reverse transcription-quantitative polymerase chain reaction. Next, the interaction among LEF1-AS1, miR-10a-5p, and MSI1 in HCC was accessed by bioinformatics and dual-luciferase reporter gene assay. Then, the cell line resistant to cisplatin was established, which was then treated with sh/oe-lncRNA LEF1-AS1, miR-10a-5p-mimic, and oe/sh-MSI1 vectors alone or in combination. Afterward, the effect of LEF1-AS1, miR-10a-5p, and MSI1 on HCC cell chemoresistance, proliferation, and apoptosis was assessed. At last, in vivo experiments confirmed the role of MSI1 in tumor growth and chemoresistance in HCC. LEF1-AS1 might potentially affect the growth and chemoresistance of HCC cells by regulating miR-10a-5p and MSI1. LEF1-AS1 and MSI1 expression patterns were elevated, while miR-10a-5p was repressed in HCC tissues and cell lines. LEF1-AS1 combined to miR-10a-5p and regulated MSI1, thereby activating the protein kinase B (AKT) signaling pathway. Knockdown of LEF1-AS1 and MSI1 or elevation of miR-10a-5p compromised the proliferation of Huh7 cell line resistant to DDP and promoted its chemosensitivity and apoptosis. At last, these in vitro findings were also confirmed in vivo. Our results unraveled LEF1-AS1 acts as a miR-10a-5p modulator to promote chemoresistance of HCC cells by stimulating MSI1 and activating the AKT signaling pathway, which might provide a novel therapeutic target for HCC.

The imbalance of biliary microflora in hepatolithiasis.

BACKGROUND: The imbalance of microbial flora is thought to be associated with many diseases. However, the characteristics of the biliary microflora and its relation to in hepatolithiasis are unknown. METHODS: This study included 40 patients with hepatolithiasis and 10 control patients. Bile samples were taken during hepatectomy surgeries and 16S rRNA sequencing was performed. The sequencing results were analyzed by operational taxonomic unit (OTU) clustering, species annotation and abundance analyses, sample complexity analyses, diversity analyses, and environmental factor correlation analyses. RESULTS: There were significant differences in bile microflora between the hepatolithiasis group and the control group. We found that the abundance of microflora in the bile of patients with hepatolithiasis was relatively high (52.4% versus 40.2% and 42.1% versus 29.6%). The diversity of microflora in the bile of patients with hepatolithiasis decreased significantly (Shannon (P = 0.004), Observed species (P = 0.001), PD-whole-tree (P = 0.001)). These differences are mainly associated with Enterococcus(P＜0.001), Enterobacter(P = 0.003). In addition, we found that there were intra-group differences in hepatolithiasis, but the differences in the hepatolithiasis group were generally smaller than the differences in the non-hepatolithiasis group. CONCLUSION: There is an imbalance of microflora in the bile duct of patients with hepatolithiasis. The imbalance of biliary flora may be associated with hepatolithiasis pathogenesis.

Long noncoding RNA LINC00324 exerts protumorigenic effects on liver cancer stem cells by upregulating fas ligand via PU box binding protein.

Hepatocellular carcinoma (HCC) represents a major cause of cancer death, but the molecular mechanism for its development has not yet been well characterized. Long noncoding RNAs (lncRNAs) are involved in a wide range of biological processes via their roles as oncogenes or tumor suppressor genes. The present study aimed to elucidate the role of LINC00324 in HCC through its interaction with Fas ligand (FasL). Initially, microarray-based gene expression profiling of HCC was employed to identify differentially expressed genes. Next, the expression of LINC00324 in HCC tissues and liver cancer stem cell (LCSC) lines was examined using RT-qPCR. Then, the interaction among LINC00324, PU box binding protein (PU.1) and FasL was identified with RIP, ChIP and dual-luciferase reporter gene assays. The effect of LINC00324 on viability, proliferation, migration, invasion, and apoptosis as well as the tumorigenesis of transfected cells was examined with gain- and loss-of-function experiments. LINC00324 and FasL were highly expressed in HCC. LINC00324 regulated FasL expression via interaction with PU.1. Silencing of LINC00324 or FasL suppressed expression of stemness-related genes, cell viability, proliferation, migration, invasion, self-renewal, and tumorigenesis, but enhanced cell apoptosis. Taken together, LINC00324 promotes the expression of FasL through the recruitment of PU.1, which ultimately maintains the biological properties of LCSCs, thus, highlighting LINC00324 as a promising therapeutic candidate for HCC.

Upregulated microRNA-194 impairs stemness of cholangiocarcinoma cells through the Rho pathway via inhibition of ECT2.

Cholangiocarcinoma (CCA) is devastating for its delayed presence, difficulty in diagnosis, and high mortality. Other studies have supported the important role of microRNAs (miRNAs) in the pathogenesis of CCA, and the role of miR-194 was investigated in several human cancers, though, the molecular mechanism of miR-194 in CCA stem cells remains largely unknown. We aimed to identify the functional significance of miR-194 in CCA. The microarray-based analysis was applied to detect the epithelial cell transforming sequence 2 (ECT2) expression and predict the miRNA-regulated ECT2, followed by the identification of relationship between ECT2 and obtained miRNA by dual-luciferase reporter gene assay. The effects of depletion or ectopic expression of miR-194 on Rho pathway and the biological characteristics of CCA were assessed by reverse transcription quantitative polymerase chain reaction, immunoblotting, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, scratch test, Transwell, and flow cytometry. Lastly, tumor growth was assessed by xenograft tumor in nude mice. ECT2 was highly expressed while miR-194 was poorly expressed in CCA stem cells, and the targeting relation between ECT2 and miR-194 was proved. More important, the elevated expression of miR-194 or ECT2 silencing inhibited the Rho pathway, and further promoted the apoptosis and suppressed the stem cell proliferation, migration, and invasion of CCA in vitro. miR-194 inhibited the tumor growth in vivo. In a word, miR-194 inhibits ECT2 and blocks the activation of Rho signaling pathway, thus promoting apoptosis, inhibiting proliferation and migration of CCA stem cells, and suppressing tumor growth. The mechanism can be regarded as a target for treating CCA.

LncRNA FTX represses the progression of non-alcoholic fatty liver disease to hepatocellular carcinoma via regulating the M1/M2 polarization of Kupffer cells.

BACKGROUND: The effect of lncRNA FTX on non-alcoholic fatty liver disease (NAFLD) conversion to hepatocellular carcinoma (HCC) is unclear. METHODS: In our study, C57BL/6 mice was fed with high fat diet for obtaining NAFLD mouse model, and diethylnitrosamine induced the formation of HCC tumor. The expression of iNOS and CD206 in tissues were examined using immunohistochemistry. In addition, qRT-PCR was implemented to detect the expression of FTX and mRNAs. The percentage of M1 and M2 Kupffer cells (KCs) were determined using flow cytometry. The pathological change in liver tissues was displayed by H&E staining. Besides, immunofluorescence assay was performed to ensure the primary KCs through labeling F4/80. RESULTS: Here, we found that the expression of FTX and the ratio of M1/M2 KCs in liver tissues from NAFLD-transformed HCC (NAFLD-HCC) patients lower than in liver tissues from NAFLD patients. Subsequently, we revealed that the expression of FTX and M1/M2 KCs ratio were downregulated during NAFLD conversion to HCC. Importantly, increasing of FTX inhibited HCC tumor growth, improved liver damage and promoted M1 polarization of KCs during NAFLD conversion to HCC, while these effects of FTX were reversed by inactivating of KCs. Finally, in vitro experiments, our data indicated that FTX facilitated the M1 polarization of KCs. CONCLUSION: In conclusion, our results demonstrated that upregulation of FTX suppressed NAFLD conversion to HCC though promoting M1 polarization of KCs. Our findings presented a new regulatory mechanism for NAFLD conversion to HCC, and provided a new biomarker for inhibiting this conversion.

Long noncoding LINC01551 promotes hepatocellular carcinoma cell proliferation, migration, and invasion by acting as a competing endogenous RNA of microRNA-122-5p to regulate ADAM10 expression.

Hepatocellular carcinoma (HCC) is a severe disease with high mortality in the world. It has been shown that long noncoding RNA (lncRNA) might play a role in HCC. The aim of the present study was to identify the role of long intergenic noncoding RNA 01551 (LINC01551) in the HCC development and explore the underlying mechanism of LINC01551/miR-122-5p/ADAM10 axis. The differentially expressed lncRNAs associated with HCC were screened out by a microarray analysis. The expression of LINC01551, miR-122-5p, and ADAM10 was determined in HCC tissues and cells. The potential miRNA (miR-122-5p) regulated by LINC01551 was explored, and the target relationship between miR-122-5p and ADAM10 was confirmed. To evaluate the effect of LINC01551 and miR-122-5p on proliferation, migration, invasion, and apoptosis of HCC, different plasmids were delivered into MHCC97-H cells. High expression of LINC01551 and ADAM10 yet low-expression of miR-122-5p were revealed in HCC tissues and cells. Overexpression of miR-122-5p could downregulate ADAM10. Biological prediction websites and fluorescence in situ hybridization assay verified that LINC01551 was mainly expressed in the cytoplasm. Silencing LINC01551 reduced HCC cell viability, proliferation, migration, invasion, and cell cycle entry yet induce cell apoptosis. Upregulation of LINC01551 increased its ability of competitively binding to miR-122-5p, thus reducing miR-122-5p and upregulating ADAM10 expression, as well as promoting the proliferative, migrative, and invasive ability. Taken together the results, it is highly possible that LINC01551 functions as an competing endogenous RNA (ceRNA) to regulate the miRNA target ADAM10 by sponging miR-122-5p and therefore promotes the development of HCC, highlighting a promising competitive new target for the HCC treatment.

Intrahepatic bile duct exploration lithotomy is a useful adjunctive hepatectomy method for bilateral primary hepatolithiasis: an eight-year experience at a single centre.

BACKGROUND: To evaluate the perioperative and long-term results of intrahepatic bile duct exploration lithotomy (IHBDIL) combined with hepatectomy for patients with complicated bilateral primary hepatolithiasis. METHODS: A study was conducted involving 56 patients with complicated bilateral primary hepatolithiasis who underwent IHBDIL combined with hepatectomy at our hospital from January 2006 to December 2014. The perioperative and long-term outcomes that were retrospectively analysed included the stone clearance rate, operative morbidity and mortality, and stone recurrence rate. Patients with a preoperative diagnosis of cholangiocarcinoma were excluded. RESULTS: In all 56 patients, hepatic duct stones were located in the bilateral IHBD. The surgical method was IHBDIL combined with hepatectomy. Postoperative complications occurred in 15 patients (26.8%), 14 patients responded to conservative management, and there was 1 case of postoperative mortality because of hepatic failure. The overall initial success rate of stone clearance was 85.7%, and the final clearance rate was 92.9% following postoperative choledochoscopic lithotripsy. The stone recurrence rate was 13.5%, and the occurrence of postoperative cholangitis was 10.9% during the follow-up period. CONCLUSION: IHBDIL combined with hepatectomy is a safe, effective, and promising treatment for patients with complicated bilateral primary hepatolithiasis. The perioperative and long-term outcomes are satisfactory for complicated bilateral primary hepatolithiasis.

PSMD11 promotes the proliferation of hepatocellular carcinoma by regulating the ubiquitination degradation of CDK4.

BACKGROUND: The 26S proteasome non-ATPase regulatory subunit 11 is a multiprotein complex involved in the ATP-dependent degradation of ubiquitinated proteins, and PSMD11 plays a key role in the regulation of embryonic stem cell proteasome activity. However, the role of PSMD11 in hepatocellular carcinoma has not been studied. In this study, it was found that the expression of PSMD11 in HCC tissues was significantly higher than that in para-cancerous tissues, and was associated with poor prognosis. The results of in vitro experiments showed that PSMD11 knockdown could effectively inhibit the proliferation and apoptosis of hepatoma cell lines, and flow cytometry showed that the G0/G1 phase was significantly prolonged. Through protein spectrometry, immunoprecipitation and in vitro experiments, it was found that PSMD11 can promote the proliferation of hepatocellular carcinoma through regulating the ubiquitination of CDK4 and enhancing its protein stability. This study explores the mechanism of action of PSMD11 in hepatocellular carcinoma and provides new insights for the treatment of hepatocellular carcinoma.

The Value of Chemokine and Chemokine Receptors in Diagnosis, Prognosis, and Immunotherapy of Hepatocellular Carcinoma.

Background: Chemokines and chemokine receptors (CCRs) are involved in a variety of anti-tumour and pro-tumour immune processes in vivo, such as angiogenesis, metastasis, proliferation and invasiveness, and influence patient prognosis and response to therapy. Methods: CCRs differentially expressed in HCC and associated with prognosis were extracted from TCGA and GEO databases, and the obtained CCRs were then used to construct signature genes, and the signature gene were selected for expression validation as well as functional experiments to explore the role of CCRs in the treatment and prognosis of HCC. Results: We constructed a prognostic model including five CCRs (CCL20, CCL23, CCR3, CCR10, and CXCR3) and validated the expression of signature genes. The model's risk score is an independent prognostic factor for HCC. We have also developed prognostic model nomograms for clinical use. In addition, we validated that CCR3 expression is associated with poor prognosis in HCC, and the proliferation and migration ability of HCC cells was significantly inhibited after interfering with the expression of CCR3 in MHCC-LM3. We also looked at differences in pathway enrichment, immune infiltration and immune checkpoints. Finally, we found that risk scores were also correlated with drug sensitivity, the high-risk group had a better sensitivity to sorafenib. Conclusion: The CCRs-related gene signature may better assess HCC prognosis and response to immunotherapy and tyrosine kinase inhibitors such as sorafenib in HCC, providing prospective solutions for diagnosis and treatment.

PAFAH1B3 is a KLF9 target gene that promotes proliferation and metastasis in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies. Uncontrolled cell proliferation, invasion and migration of pancreatic cancer cells are the fundamental causes of death in PDAC patients. Our previous studies showed that KLF9 inhibits the proliferation, invasion and migration of pancreatic cancer cells. However, the underlying mechanisms are not fully understood. In this study, we found that platelet-activating factor acetylhydrolase IB3 (PAFAH1B3) is highly expressed in pancreatic cancer tissues and cells. In vitro and in vivo studies showed that overexpression of PAFAH1B3 promoted the proliferation and invasion of pancreatic cancer cells, while downregulation of PAFAH1B3 inhibited these processes. We found that KLF9 expression is negatively correlated with PAFAH1B3 expression in pancreatic cancer tissues and cells. Western blotting revealed that KLF9 negatively regulates the expression of PAFAH1B3 in pancreatic cancer tissues and cells. Rescue experiments showed that overexpression of PAFAH1B3 could partially attenuate the suppression of pancreatic cancer cell proliferation, invasion and migration induced by KLF9 overexpression. Finally, chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were carried out, and the results showed that KLF9 directly binds to the promoter of PAFAH1B3 and inhibits its transcriptional activity. In conclusion, our study indicated that KLF9 can inhibit the proliferation, invasion, migration and metastasis of pancreatic cancer cells by inhibiting PAFAH1B3.

Laparoscopic hepatectomy is associated with a higher incident frequency in hepatolithiasis patients.

PURPOSES: The primary concern regarding laparoscopic hepatectomy in hepatolithiasis patients is surgical safety, which may be high in current practice. METHODS: Hepatolithiasis patients who underwent laparoscopic and laparotomic hepatectomies were retrospectively studies after being matched for age, location of gallstones, liver resection and underlying liver conditions at a ratio of 1:1 (n = 44 in each group). The rates of intraoperative incidents and postoperative complications were examined using validated classification and grading systems. The primary outcome measure was the procedure-related complication/mortality rate. RESULTS: Laparoscopy was converted to open surgery in three patients (6.8 %). The length of the operation for laparoscopic hepatectomy was significantly longer than that for laparotomic hepatectomy (277.5 min [range, 190-410 min] vs. 212.5 min [140-315 min], P < 0.001). The two groups had similar intraoperative blood loss (367.5 mL [150-1200 mL] vs. 392.5 mL [200-1400 mL], P > 0.05) and transfusion frequencies (13.6 vs. 18.2 %, P > 0.05). The laparoscopy group had a higher percentage of patients with at least one intraoperative incident compared with the laparotomy group (22.7 vs. 6.8 %; P < 0.05). Vascular events occurred in nine patients (20.5 %) undergoing laparoscopy and two patients (4.5 %) undergoing laparotomy (OR 5.4 [95 %CI, 1.1-26.7], P < 0.05). CONCLUSIONS: Laparoscopic hepatectomy is associated with a higher risk of intraoperative vascular incidents in hepatolithiasis patients compared wit laparotomy.

Protective strategy for the caudate lobe bile duct during left hemihepatectomy based on imaging data analysis.

Purpose: This study was performed to analyze the rule of confluence of the caudate lobe bile duct (CLD) into the left hepatic duct (LHD) and to discuss the protective strategy during left hemihepatectomy. Methods: MRI of 400 patients and T-tube angiography images of 100 patients were collected, and the imaging rules of the confluence of the CLD into the LHD were summarized. The clinical data of 33 patients who underwent left hemihepatectomy using the protective strategy were analyzed. Results: MRI and T-tube angiography images showed that the length from the confluence point of the CLD into the LHD to the confluence of the left and right hepatic ducts was 1.19 ± 0.40 cm and 1.26 ± 0.39 cm, respectively. The average angle between the longitudinal axis of the 2 bile ducts was 68.27° ± 22.59° and 66.58 ± 22.88°, respectively. Coronal and cross-sectional images showed that inflow from the foot side to the cranial side was noted in 79.8% and 82.0% of patients, respectively, and inflow from the dorsal to the ventral side was observed in 84.5% and 88.0%, respectively. Based on these imaging rules, the safe transection length and plane were summarized, and the CLD was effectively protected in 33 cases of left hemihepatectomy. Conclusion: In left hemihepatectomy, the LHD should be transected at least 1.5 cm away from the confluence of the left and right hepatic ducts, and the plane of transection should be oblique to the dorsal side at an angle of 45° with the LHD, these parameters represent an effective strategy to protect the CLD.

Signature construction and molecular subtype identification based on immune-related genes for better prediction of prognosis in hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) immunotherapy is a focus of current research. We established a model that can effectively predict the prognosis and efficacy of HCC immunotherapy by analyzing the immune genes of HCC. METHODS: Through the data mining of hepatocellular carcinoma in The Cancer Genome Atlas (TCGA), the immune genes with differences in tumor and normal tissues are screened, and then the univariate regression analysis is carried out to screen the immune genes with differences related to prognosis. The prognosis model of immune related genes is constructed by using the minimum absolute contraction and selection operator (lasso) Cox regression model in the TCGA training set data, The risk score of each sample was calculated, and the survival was compared with the Kaplan Meier curve and the receiver operating characteristic (ROC) curve to evaluate the predictive ability. Data sets from ICGC and TCGA were used to verify the reliability of signatures. The correlation between clinicopathological features, immune infiltration, immune escape and risk score was analyzed. RESULTS: Seven immune genes were finally determined as the prognostic model of liver cancer. According to these 7 genes, the samples were divided into the high and low risk groups, and the results suggested that the high-risk group had a poorer prognosis, lower risk of immune escape, and better immunotherapy effect. In addition, the expression of TP53 and MSI was positively correlated in the high-risk group. Consensus clustering was performed to identify two main molecular subtypes (named clusters 1 and 2) based on the signature. It was found that compared with cluster 1, better survival outcome was observed in cluster 2. CONCLUSION: Signature construction and molecular subtype identification of immune-related genes could be used to predict the prognosis of HCC, which may provide a specific reference for the development of novel biomarkers for HCC immunotherapy.

Molecular subtype identification and signature construction based on Golgi apparatus-related genes for better prediction prognosis and immunotherapy response in hepatocellular carcinoma.

Introduction: The Golgi apparatus (GA) is the center of protein and lipid synthesis and modification in normal cells and is involved in regulating various cellular process as a signaling hub, the dysfunction of which can lead to the development of various pathological conditions, including tumors. Mutations in Golgi apparatus-related genes (GARGs) are prevalent in most tumors, and their mutations can make them pro-tumor metastatic. The aim of this study was to analyze the predictive role of GARGs in the prognosis and immunotherapeutic outcome of hepatocellular carcinoma. Methods: We used TCGA, GEO and ICGC databases to classify hepatocellular carcinoma samples into two molecular subtypes based on the expression of GARGs. Signature construction was then performed using GARGs, and signature genes were selected for expression validation and tumor phenotype experiments to determine the role of GARGs in the prognosis of hepatocellular carcinoma. Results: Using the TCGA, GEO and ICGC databases, two major subtypes of molecular heterogeneity among hepatocellular carcinoma tumors were identified based on the expression of GARGs, C1 as a high-risk subtype (low survival) and C2 as a low-risk subtype (high survival). The high-risk subtype had lower StromalScore, ImmuneScore, ESTIMATEScore and higher TumorPurity, indicating poorer treatment outcome for ICI. Meanwhile, we constructed a new risk assessment profile for hepatocellular carcinoma based on GARGs, and we found that the high-risk group had a worse prognosis, a higher risk of immune escape, and a higher TP53 mutation rate. Meanwhile, TME analysis showed higher tumor purity TumorPurity and lower ESTIMATEScore, ImmuneScore and StromalScore in the high-risk group. We also found that the high-risk group responded more strongly to a variety of anticancer drugs, which is useful for guiding clinical drug use. Meanwhile, the expression of BSG was experimentally found to be associated with poor prognosis of HCC. After interfering with the expression of BSG in HCC cells SMMC-7721, the proliferation and migration ability of HCC cells were significantly restricted. Discussion: The signature we constructed using GARGs can well predict the prognosis and immunotherapy effect of hepatocellular carcinoma, providing new ideas and strategies for the treatment of hepatocellular carcinoma.

BarH-Like Homeobox 2 Suppresses Cell Proliferation, Invasion, and Angiogenesis in Hepatocellular Carcinoma by Activating N-Acetylgalactosaminyltransferase 4.

BarH-like homeobox 2 (BARX2) has been identified to play a key role in the development of multiple cancers. Meanwhile, BARX2 may be an independent prognostic biomarker for patients suffering from hepatocellular carcinoma (HCC). Nevertheless, the regulatory role of BARX2 in HCC is still unclear and needs to be unveiled. In this study, the expressions of BARX2 and N-acetylgalactosaminyltransferase 4 (GALNT4) were evaluated by quantitative real-time PCR (qRT-PCR) as well as western blot. Besides, the abilities of cells to proliferate, migrate, invade, and angiogenesis were assessed with CCK-8, colony formation, wound-healing, Transwell, and tube formation assays, separately. Cell apoptosis was determined by flow cytometry analysis. The binding relationship between BARX2 and GALNT4 was predicted by JASPAR website and verified using Chromatin immunoprecipitation (ChIP) and luciferase report assay. It was discovered that BARX2 was reduced in HCC cell lines, while its overexpression greatly repressed cell proliferation, migration, invasion, and angiogenesis and promoted cell apoptosis in HuH7 and MHCC97-H cells. BARX2 could bind to GALNT4 promoter and positively regulate GALNT4 expression. In addition, GALNT4 deficiency partly abolished the inhibitory effects of BARX2 on the progression of HCC. In summary, this study highlights that BARX2 may hold promise for serving as a potential therapeutic target, facilitating the development of a novel therapeutic strategy against HCC.

Pancancer analysis of oncogenic BARX2 identifying its prognostic value and immunological function in liver hepatocellular carcinoma.

The transcription factor BarH-like homeobox 2 (BARX2), a member of the Bar-like homeobox gene family, is involved in cell proliferation, differentiation, immune responses and tumorigenesis. However, the potential role of BARX2 in the development of liver hepatocellular carcinoma (LIHC) remains unclear. Therefore, we aimed to study the biological role of BARX2 in hepatocellular carcinoma. Through the UALCAN, GTEx PORTAL, TIMER 2.0, LinkedOmics, SMART, MethSurv, Metascape, GSEA and STRING public databases, the BARX2 mRNA level, prognostic value, coexpressed genes, associated differentially expressed genes, DNA methylation and functional enrichment of LIHC patients were studied. The relationships between BARX2 expression and various clinical or genetic parameters of LIHC patients were determined using data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and BEAT LIHC databases. In addition, the biological function of BARX2 in LIHC was studied in vitro. Through large-scale data mining, our study showed that BARX2 was differentially expressed between different normal and tumour tissues.BARX2 expression in LIHC tissues was significantly lower than that in corresponding controls, especially in patients with T2-4 stage disease. In patients with LIHC, overexpression of BARX2 was an independent poor prognostic factor associated with poor cytogenetic risk and gene mutations. Genomic hypermethylation of the BARX2 gene was associated with upregulated BARX2 expression and poor overall survival (OS) in LIHC. Functional enrichment analysis showed that BARX2 had an immunomodulatory role and was involved in the inflammatory response in LIHC occurrence. In conclusion, the oncogene BARX2 may serve as a new biomarker and prognostic factor for patients with LIHC. The immunomodulatory function of BARX2 deserves further validation in LIHC.

Comprehensive Analysis of Prognostic Value and Immune Infiltration of Ficolin Family Members in Hepatocellular Carcinoma.

Objective: Ficolin (FCN) family proteins are part of the innate immune system, play a role as recognition molecules in the complement system, and are associated with tumor development. The mechanism of its role in immunotherapy of hepatocellular carcinoma (HCC) is unclear. Methods: In this study, we used the TCGA database, HPA database, Gene Expression Profile Interaction Analysis (GEPIA), Kaplan-Meier plotter, TCGAportal, cBioPortal, GeneMANIA, TIMER, and TISIDB to analyze Ficolin family proteins (FCN1, FCN2 and FCN3, FCNs) in patients with hepatocellular carcinoma for differential expression, prognostic value, genetic alterations, functional enrichment, and immune factor correlation analysis. Results: The expression levels of FCN1/2/3 were significantly reduced in patients with HCC. Among them, FCN3 showed significant correlation with Overall Survival (OS), Progressive Free Survival (PFS) and Relapse Free Survival (RFS) in HCC. FCN1 and FCN3 may be potential prognostic markers for survival in patients with HCC. In addition, the functions of differentially expressed FCNs were mainly related to complement activation, immune response, apoptotic cell clearance and phagocytosis. FCNs were found to be significantly correlated with multiple immune cells and immune factors. Expression of FCN1 and FCN3 differed significantly in the immune and stromal cell component scores of HCC. analysis of the tumor mutation burden (TMB) and microsatellite instability (MSI) of FCNs with pan-cancer showed that FCN3 was significantly correlated with both. Conclusions: Our study provides new insights into the link between the FCN family and immunotherapy for HCC, and FCN3 may serve as a prognostic biomarker for HCC.