MiR-17-5p as a Novel Prognostic Marker for Hepatocellular Carcinoma

Currently, the role of p53-induced RING-H2 protein (PIRH2) in the development of hepatocellular carcinoma (HCC) remains unknown. The objective of this retrospective study was to investigate the expression of PIRH2 and its relation to prognosis in patients with HCC after hepatic resection. Reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR, and Western blot analyses were used to detect expression levels of PIRH2 in 30 samples of HCC tissue and paracarcinomatous liver tissue (PCLT) and in 5 samples of normal liver tissue (NL). In addition, immunohistochemical analysis was performed on 122 HCC specimens and follow-up information data from those patients were reviewed. Both messenger RNA and protein expression levels of PIRH2 were elevated significantly in HCC tissues compared with PCLT and NL tissues. The increased PIRH2 expression was correlated with vein invasion, Edmondson-Steiner grade, TNM stage, and multiple tumor nodes (P<.05). It is noteworthy that the patients with HCC who had high PIRH2 expression had shorter overall survival and disease-free survival than the patients who had low PIRH2 expression (median survival, 280 days vs 372 days; P = .0002; median disease-free survival, 220 days vs 310 days; P = .0016). Multivariate Cox regression analysis revealed that high PIRH2 expression was an independent prognostic factor for patients with HCC (relative risk, 1.792; P = .009). The current data revealed that increased expression of PIRH2 was correlated with poor survival in patients with HCC, indicating that PIRH2 is a novel prognostic marker for HCC.

ABSTRACTObjective: MAGI1 (membrane-associated guanylate kinase, WW and PDZ domain containing 1) plays an important role in stabilization of adherens junctions and suppression of invasiveness and metastasis. However, its expression and clinical revelance in hepatocellular carcinoma (HCC) are still unknown. So, this study was performed to detect the expression and clinical significance of MAGI1 in human HCC. Methods: The mRNA and protein expression levels were examined by using reverse transcription-PCR (RT-PCR) and Western blotting in 31 paired HCC and paracarcinomatous liver tissues (PCLT). Furthermore, samples of 104 HCC patients were determined immunohistochemically for MAGI1 expression and the correlation of MAGI1 levels with prognosis was analyzed. Results: The expression levels of MAGI1 were significantly downregulated in HCCs than those in PCLTs (p < .01). Importantly, the decreased expression of MAGI1 correlated with multiple tumor nodules (p < .05), absence of capsular formation (p < .05), worse Edmondson–Steiner grade (p < .05), vein invasion (p < .01), shortened median overall survival time (25 versus 42 months; p = .013), and disease-free survival time (20 versus 40 months; p = .018) of HCC. Multivariable Cox regression analysis revealed that MAGI1 expression level was an independent factor for prognosis (p = .038). Conclusions: MAGI1 expression is decreased in HCC, which correlates with poor prognosis, suggesting MAGI1 as a novel prognostic marker for HCC.

Tumor Associated Glycoprotein-72 is a Novel Marker for Poor Survival in Hepatocellular Carcinoma

Because of its role in cell migration, the Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) 2 has been implicated in cancer metastasis. Evidence to support such a role of WAVE2 in human cancer, however, is lacking. We thus examined the expression of WAVE2 in hepatocellular carcinoma (HCC) tissues to test whether the levels of WAVE2 expression correlated to the progression of HCC. Samples of 112 HCC patients were determined immunohistochemically for WAVE2 expression and the correlation of WAVE2 levels with prognosis was analyzed. Among the 112 cases, 31 paired HCC and paracarcinomatous liver tissue specimens were analyzed for WAVE2 levels by reverse transcription-PCR and Western blotting, respectively. Among 112 cases of HCCs, the immunohistochemistry data indicated significant increase of WAVE2 expression levels in 71 cases. Importantly, the increased WAVE2 expression correlated with the multiple tumor nodules (P = 0.008), the absence of capsular formation (P = 0.035), Edmondson-Steiner grade (P = 0.009), vein invasion (P = 0.023), and a shortened median survival time (326 versus 512 days; P = 0.003). Multivariable Cox regression analysis revealed the WAVE2 expression level was an independent factor for prognosis. The immunohistochemistry data were further confirmed by results of reverse transcription-PCR and Western analysis of 31 HCC cases, in which the WAVE2 mRNA and protein in HCC tissues were significantly elevated when compared with paracarcinomatous liver tissue (P < 0.001). WAVE2 expression is elevated in HCC tissues, which correlates with a poor prognosis, suggesting WAVE2 as a candidate prognostic marker of HCC.

Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a microtubule-associated protein that impacts spindle assembly in human cells. Several studies have shown that the overexpression of TPX2 is correlated with multiple tumor types. However, the role of TPX2 in hepatocellular carcinoma (HCC) remains undetermined. TPX2 expression was detected by quantitative reverse transcription polymerase chain reaction and immunoblotting in six cell lines and 130 pairs of HCC and adjacent non-cancerous liver tissues. Matrix metalloproteinase (MMP)2 and MMP9 expression was detected by immunohistochemistry to evaluate their correlations with TXP2. TPX2 siRNA was used to knock down TPX2 expression, and cell migration and invasion were determined. Moreover, clinical significance of TPX2 in HCC was analyzed. TPX2 expression was found to be obviously higher in HCC tissues than that in non-tumor tissues. Elevated TPX2 expression was observed in HCC cell lines as compared with that in a non-transformed hepatic cell line. Clinical analysis indicated that TPX2 expression in the HCC tissues was evidently correlated with the tumor-node-metastasis stage, tumor number and tumor differentiation. TPX2 is a novel prognostic marker for predicting 5-year overall survival and disease-free survival of HCC patients. TPX2 expression was positively correlated with MMP2 and MMP9. In vitro studies found that TPX2 knockdown prominently reduced cell invasion and migration and decreased phosphorylated AKT, MMP2 and MMP9 expression in MHCC-97H cells. Overexpression of TPX2 was associated with clinicopathological characteristics and poor patient outcomes. TPX2 may serve as a novel prognostic marker for HCC.

Single-stranded DNA binding protein 1 (SSBP1) is a DNA binding protein found in mitochondria, encoded by nuclear genes. SSBP1 plays a crucial role in responding to mitochondrial DNA (mtDNA) damage and maintaining genome stability, and it is linked to cancer occurrence and progression, but its role in hepatocellular carcinoma (HCC) is still unclear. Therefore, the aim of this research was to investigate the expression of SSBP1 and its potential clinical significance in HCC. RNA-seq data and clinical information of HCC samples and normal liver samples were downloaded from The Cancer Genome Atlas (TCGA). The expression of SSBP1 in HCC and its correlation with clinical pathological indicators, prognosis, immune cells, and infiltration were analyzed using R software, while the diagnostic value of SSBP1 in HCC was evaluated. Using the R software, we conducted Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and gene set enrichment analysis (GSEA) on the SSBP1 expression in HCC. Immunohistochemistry (IHC) detected SSBP1 expression in 31 HCC tissue pairs. Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR) quantified protein and mRNA levels in 6 fresh HCC tissue. Protein expression and distribution of SSBP1 in HCC cell lines were analyzed using qRT-PCR, Western blotting, and Immunofluorescence techniques. SSBP1 mRNA expression was significantly higher in HCC tissues compared to normal tissues (P<0.001) in both matched and unmatched samples. SSBP1 expression was correlated with gender and M stage (P<0.05), but not with other factors. High SSBP1 expression was identified as an independent risk factor for overall survival (OS) in HCC patients [hazard ratio =1.713, P=0.01]. Immunocell infiltration analysis showed a negative correlation between SSBP1 expression and level of naive B cells, but a positive correlation with memory B cells and macrophages (|Spearman's r| >0.2, P<0.05). The diagnostic value of SSBP1 mRNA expression for early diagnosis prognosis of HCC (area under the curve >0.50). The GO enrichment analysis of SSBP1 revealed that it was enriched for mitochondrial biological functions. KEGG analysis showed that SSBP1 was associated with multiple DNA replication, mismatch repair and homologous recombination pathways. GSEA analysis showed that the first three pathways strongly related to the high expression of SSBP1 were DNA repair, myc-targets-v1 and reactive oxygen species signaling pathways. Validation through IHC and Western blotting high SSBP1 protein expression in HCC tissue, as well as qRT-PCR and Western blotting results showed high expression in HCC cell lines. Immunofluorescence experiments indicated the localization of SSBP1 in the mitochondria of HCC cells. High expression of SSBP1 is an independent risk factor for poor prognosis in HCC patients and has good diagnostic value.

Inhibitor of growth 4 (ING4) is a candidate tumor suppressor that plays an important role in tumor growth and angiogenesis. Here, we examined the expression of ING4 in hepatocellular carcinoma (HCC) tissues and analyzed its correlation with the progression of HCC. Specimens from 136 HCC patients were determined immunohistochemically for ING4 expression. The correlation of ING4 levels with clinicopathologic variables, prognosis, and metastatic potential was analyzed. Among the 136 cases, 36 paired HCC and paracarcinomatous liver tissue specimens were analyzed for ING4 expression levels by real-time quantitative reverse transcription-PCR and Western blotting. MVD was determined by CD34 immunostaining to test whether it correlated with ING4 protein expression level. The ING4 mRNA and protein levels were significantly lower in HCC than paracarcinomatous liver tissue from both real-time quantitative reverse transcription-PCR and Western blotting (P = 0.039 and 0.012, respectively). Importantly, the ING4 protein level correlated with the Edmondson-Steiner grade (P = 0.035), vein invasion (P = 0.015), and microvessel density (P = 0.005). Survival and metastasis analysis indicated that HCC patients with lower ING4 expression had poorer overall survival and disease-free survival than those with high expression (P = 0.0001 and 0.0065; respectively). Multivariable Cox regression analysis revealed that the ING4 expression level was an independent factor for prognosis (hazard risk, 9.63; P = 0.001). ING4 expression is down-regulated in HCC tissues. ING4 expression level correlates with prognosis and metastatic potential, which suggests that ING4 is a candidate prognostic marker of HCC.

BACKGROUNDThe kinesin superfamily protein member KIF21B plays an important role in regulating mitotic progression; however, the function and mechanisms of KIF21B in cancer, particularly in hepatocellular carcinoma (HCC), are unknown.AIMTo explore the role of KIF21B in hepatocellular carcinoma and its effect on prognosis after hepatectomy.METHODSFirst, data on the differential expression of KIF21B in patients with HCC from The Cancer Genome Atlas database was analyzed. Subsequently, the expression levels of KIF21B in HCC cell lines and hepatocytes were detected by reverse transcription-polymerase chain reaction, and its biological effect on BEL-7404 cells was evaluated by KIF21B knockdown. Immunohistochemical analysis was used to validate the differential expression of KIF21B in HCC tissues and adjacent normal tissues from 186 patients with HCC after hepatectomy. The Kaplan-Meier method was used to assess prognosis significance.RESULTSKIF21B expression levels were significantly higher in HCC tissues than in corresponding adjacent normal tissues. The expression levels of KIF21B in four HCC cell lines were higher than that in normal liver cells. Functional experiments showed that KIF21B knockdown remarkably suppressed cell proliferation and induced apoptosis. Moreover, immunohistochemistry results are consistent with The Cancer Genome Atlas analysis, with KIF21B expression levels being increased in HCC tissues compared to adjacent normal tissues. Univariate and multivariate analyses revealed KIF21B as an independent risk factor for overall survival and disease-free survival in patients with HCC after hepatectomy.CONCLUSIONTaken together, our results provide evidence that KIF21B plays an important role in HCC progression and may be a potential diagnostic and prognostic marker for HCC.

To compare kinesin family member 1B (KIF1B) expression with clinicopathologic parameters and prognosis in hepatocellular carcinoma (HCC) patients. KIF1B protein and mRNA expression was assessed in HCC and paracarcinomatous (PC) tissues from 68 patients with HCC using Western blot and quantitative real-time reverse transcription-PCR, respectively. Student's t-tests were used to analyze relationships between clinicopathologic parameters and KIF1B expression, the Kaplan-Meier method was used to analyze survival outcomes, and the log-rank test was used to compare survival differences between groups. Mean protein and mRNA levels of KIF1B were similar between HCC and PC tissues. However, HCC tissues with vein invasions had significantly lower KIF1B protein levels compared to those without vein invasions (2.30 ± 0.82 relative units vs 2.77 ± 0.84 relative units, P < 0.05). KIF1B protein levels in HCC tissues from patients with recurrence during the follow-up period were significantly lower than those without recurrence (2.31 ± 0.92 relative units vs 2.80 ± 0.80 relative units, P < 0.05). However, KIF1B protein and mRNA expression in HCC patients was not associated with other clinicopathologic parameters. Ratios of KIF1B mRNA expression in HCC tissues to those in PC tissues were correlated with overall survival (13.5 mo vs 20.0 mo, P < 0.05) and disease-free survival (11.5 mo vs 19.5 mo, P < 0.05). Downregulation of KIF1B in HCC tissues is associated with poor prognosis; additional clinical studies are needed to confirm whether KIF1B can serve as a prognostic marker.

BackgroundSESN2 plays important roles in the regulation of cell survival, cell protection, and tumor suppression. However, the relationship between SESN2 expression and the clinicopathological attributes of hepatocellular carcinoma (HCC) is barely investigated.MethodsOne-step quantitative reverse transcription PCR, Western blotting analysis in 15 fresh HCC tissues, and immunohistochemistry (IHC) analysis in a tissue microarray (TMA) containing 100 HCC cases were performed to examine SESN2 expression. Survival analyses by Cox regression method and Kaplan-Meier curve were performed to describe the overall survival of 100 HCC patients.ResultsThe SESN2 expression in HCC tissues declined dramatically compared with the corresponding noncancerous tissues, and SESN2 expression was remarkably associated with HBV infection (p = 0.019), HCV infection (p = 0.001), and lymph node metastasis (p = 0.033). Survival analysis further demonstrated that SESN2 expression could serve as an independent prognostic biomarker for overall survival in univariate (p = 0.001) and multivariate analyses (p = 0.003).ConclusionThe data are the first to indicate that SESN2 might be a novel prognostic marker for HCC and that elevated SESN2 expression predicts advantageous outcomes in HCC patients.

Background: The molecular pathways along with the clinical significance of long non-coding RNAs (lncRNAs) in hepatocellular carcinoma (HCC) remain uncertain. Our study sought to identify and characterize lncRNAs associated with HCC. Methods: LncRNA TMCO1-AS1 was identified by differential expression analysis, receiver operating characteristic (ROC) analysis, and univariate analysis using RNA sequencing and clinical information of HCC from the public database. Then clinical correlations and survival analysis were conducted to further appraise the prognostic significance of lncRNA TMCO1-AS1 in HCC. Hepatoma and adjoining normal tissues from 66 patients who received surgical operation at our center were used to verify the results of the bioinformatics analysis. A survival prognostic model was established combining TMCO1-AS1 expression and other clinical characteristics. Results: Bioinformatics analysis showed the aberrant high expression of TMCO1-AS1 in HCC tissue. TMCO1-AS1 expression was positively correlated with alpha-fetoprotein (AFP) level, vascular invasion, tumor stage, as well as tumor differentiation. Moreover, survival analysis found a significant inverse association between the expression of TMCO1-AS1 and the survival of patients with HCC. Cox analysis indicated that TMCO1-AS1 was an independent factor for HCC prognosis. Analysis of the HCC tissues from patients at our center provided results similar to those of the bioinformatics analysis. Risk models for overall survival (OS) and recurrence-free survival (RFS) incorporating TMCO1-AS1 exhibited better sensitivity and specificity than using clinical characteristics alone. Conclusion: High TMCO1-AS1 expression is significantly correlated with the unfavorable poor prognosis of HCC, indicating its potential of being a novel prognostic marker for HCC.

hMex-3A is associated with poor prognosis and contributes to the progression of hepatocellular carcinoma

PurposeThe tumor suppressor candidate 3 (TUSC3) has been considered to be closely associated with the occurrence, development and invasion of various malignant tumors. However, the expression of TUSC3 in hepatocellular carcinoma (HCC) tissues remains ambiguous. The purpose of this research was to investigate the expression of TUSC3 in HCC tissues and analyze the relationship between TUSC3 levels and clinicopathological characteristics and prognosis of HCC patients.Materials and methodsImmunohistochemistry was used to detect the expression of TUSC3 in HCC and the corresponding para-cancerous tissues from 92 samples of HCC patients. mRNA and protein expression levels of TUSC3 were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays in 25 paired HCC and corresponding adjacent nontumor tissues. Furthermore, statistical analysis was applied to evaluate the correlation between TUSC3 level and the clinicopathological features and prognosis of HCC patients.ResultsImmunohistochemical assay indicated that the expression of TUSC3 was significantly lower in HCC tissues when compared with the corresponding para-cancerous tissues (χ2=11.512, P=0.001). The analysis of clinicopathological characteristics showed that low expression of TUSC3 in HCC tissues was significantly associated with Edmondson grade, Barcelona Clinic Liver Cancer stage and tumor size (P=0.008, 0.009 and 0.020, respectively). Univariate analysis showed that the expression of TUSC3 was strongly correlated with overall survival (OS) and disease-free survival (DFS) after radical surgery in HCC patients (P<0.001, P<0.001, respectively). Multivariate analysis revealed that the TUSC3 level was an independent risk factor for OS and DFS in HCC patients (P=0.001, P<0.001, respectively). Results of qRT-PCR and Western blot assays indicated that the level of TUSC3 in HCC tissues was significantly lower than that in the corresponding adjacent noncancerous tissues (P<0.01, P<0.001).ConclusionThe expression of TUSC3 in HCC was significantly downregulated and was correlated with tumor progression and prognosis, which could be used as an independent predictor of prognosis in HCC patients.

The immunobiology of hepatocellular carcinoma in humans and mice: Basic concepts and therapeutic implications

Lenvatinib is a prevalent treatment for hepatocellular carcinoma (HCC), yet resistance to the drug significantly limits its effectiveness. This study investigates the role of FBXO32 (F-Box Protein 32) in HCC progression and lenvatinib resistance. Methods: We utilized the GSE211850 and GSE46408 datasets to identify an E3 ubiquitin ligase that is highly expressed in both lenvatinib-resistant HCC cells and HCC tissues. The expression and clinical relevance of this E3 ubiquitin ligase were further validated using lenvatinib-resistant HCC cells, online databases, and HCC clinical tissue samples. The phenotype was verified by cell and animal experiments. Techniques such as RNA sequencing, western blotting, immunofluorescence, Co-immunoprecipitation (Co‑IP), Ubiquitination, and cycloheximide (CHX) chase assay reveal the mechanism. FBXO32 is highly expressed in both lenvatinib-resistant HCC cells and HCC tissues. High FBXO32 expression correlated with increased ALT, AFP levels, larger tumors, and advanced TNM stages, serving as an independent risk factor for overall survival (OS) and recurrence-free survival (RFS). Functional assays demonstrated that FBXO32 overexpression enhanced cell proliferation, stemness, apoptosis resistance, and lenvatinib resistance, while knockdown had opposing effects. KEGG enrichment analysis indicated a link between FBXO32 and the Hedgehog signaling pathway. FBXO32-mediated degradation of SUFU, a Hedgehog pathway inhibitor, activated this pathway. Inhibiting Hedgehog signaling counteracted FBXO32's impact on HCC growth and resistance. Conclusion: FBXO32 is a critical marker for lenvatinib efficacy and HCC prognosis, suggesting that targeting FBXO32 or the Hedgehog pathway could provide innovative strategies for overcoming lenvatinib resistance in HCC.