Paracancerous Liver Tissues Research Articles

Comparative study of ultra-high field diffusion-weighted MRI imaging between hepatocellular carcinoma and paracancerous, distant cancerous, and background liver tissues

Objective: To investigate the differences in multi-b-value apparent diffusion coefficient (ADC) and exponential apparent diffusion coefficient (eADC) between hepatocellular carcinoma (HCC) and paracancerous liver tissue, distant cancerous liver tissue, and background liver tissues by ultra-high field 3.0T diffusion-weighted (DWI) MRI imaging. Methods: Sixty-eight consecutive HCC cases confirmed by surgical pathology from January 2018 to October 2021 were enrolled and divided into a cirrhosis (n=39) and a non-cirrhosis group (n=29) according to the presence or absence of cirrhosis.The average ADC and eADC of liver tissues of paracancerous (including proximal and distal), distant cancerous, and background were measured by DWI images with diffusion sensitivity factors (b) of 50, 100, 400, 600 s/mm2, and 1 000 s/mm2, respectively. The Kruskal-Wallis H test and Bonferroni method were used to test the differences between the measured values of the five tissues. The statistical differences were used to evaluate the diagnostic efficacy of the five tissues by parametric receiver operating characteristic (ROC) curve and area under the curve (AUC). Results: The comparison of average ADC and eADC among five types of tissues in the liver cirrhosis group showed that the average ADC and eADC measured at b values of 50, 100, 400, and 600 s/mm2 had statistically significant differences (adjusted P<0.005) between cancerous and proximal paracancerous, distal paracancerous, distant cancerous, and background liver tissue, as well as the average ADC measured at b=1 000 s/mm2 between cancerous and proximal paracancerous tissue. The average ADC and eADC in the non-cirrhosis group had statistically significant differences (adjusted P<0.005) between cancerous and proximal paracancerous, distant paracancerous, distant cancerous, and background liver tissue measured at b values of 50, 100, and 400 s/mm2, respectively. The average ADC and eADC measured at b=600 s/mm2 showed statistically significant differences (adjusted P<0.005) between cancerous and proximal paracancerous, distal paracancerous, and distant cancerous liver tissue, as well as the average ADC measured at b=1 000 s/mm2 between cancerous and distal paracancerous, and distant cancerous liver tissue. The average ADC and eADC in the cirrhosis group had no statistically significant difference between the proximal paracancerous and the distant cancerous, as well as the background liver tissue measured at b-values of 50, 100, 400, 600, and 1 000 s/mm2, respectively (adjusted P>0.005), while there were statistically significant differences (adjusted P<0.005) in the average ADC values in the non-cirrhosis group between the proximal paracancerous and the distant paracancerous and background liver tissues at b=50 s/mm2, as well as the average ADC and eADC values between the proximal paracancerous and the distant liver tissues at b=100 s/mm2. The average ADC and eADC values measured in the cirrhosis group and non-cirrhosis group had no statistically significant difference between the distant paracancerous, distant cancerous, and background liver tissue (adjusted P>0.005). The efficacy of average ADC and eADC in distinguishing five types of tissues (cancerous and proximal paracancerous, distant paracancerous, distant cancerous, and background liver tissue) showed that in the cirrhosis group, the diagnostic efficacy was best at b=50 s/mm2. The area under the ROC curve (AUC) of average ADC was 0.815, 0.828, 0.855, and 0.855, respectively, and the AUC of average eADC was 0.815, 0.830, 0.856, and 0.855, respectively. The diagnostic efficacy was best in the non cirrhosis group at b=100 s/mm2, with average ADC AUCs of 0.787, 0.823, 0.841, and 0.821, and average eADC AUCs of 0.836, 0.874, 0.893, and 0.873, respectively. The AUC of the average ADC in the non-cirrhosis group for distinguishing between proximal paracancerous and distant cancerous liver tissues, as well as proximal paracancerous and background liver tissues, with b=50 s/mm2, were 0.605 and 0.604, respectively. The average AUC of ADC and eADC for distinguishing between proximal paracancerous and distant liver tissues with b=100 s/mm2 were 0.619 and 0.620, respectively. Conclusion: The average ADC and eADC measured by multiple b-values are helpful in distinguishing HCC from proximal paracancerous, distal paracancerous, distant-cancerous, and background liver tissues in patients with cirrhosis and non-cirrhosis, while the average ADC and eADC at b=50 s/mm2 and 100 s/mm2 exhibit differences between the proximal paracancerous from the distant cancerous liver tissue and background liver tissue in patients with non-cirrhosis.

Long-read transcriptome landscapes of primary and metastatic liver cancers at transcript resolution

BackgroundThe liver ranks as the sixth most prevalent site of primary cancer in humans, and it frequently experiences metastases from cancers originating in other organs. To facilitate the development of effective treatments and improve survival rates, it is crucial to comprehend the intricate and diverse transcriptome landscape of primary and metastatic liver cancers.MethodsWe conducted long-read isoform sequencing and short-read RNA sequencing using a cohort of 95 patients with primary and secondary liver cancer who underwent hepatic resection. We compared the transcriptome landscapes of primary and metastatic liver cancers and systematically investigated hepatocellular carcinoma (HCC), paired primary tumours and liver metastases, and matched nontumour liver tissues.ResultsWe elucidated the full-length isoform-level transcriptome of primary and metastatic liver cancers in humans. Our analysis revealed isoform-level diversity in HCC and identified transcriptome variations associated with liver metastatis. Specific RNA transcripts and isoform switching events with clinical implications were profound in liver cancer. Moreover, we defined metastasis-specific transcripts that may serve as predictors of risk of metastasis. Additionally, we observed abnormalities in adjacent paracancerous liver tissues and characterized the immunological and metabolic alterations occurring in the liver.ConclusionsOur findings underscore the power of full-length transcriptome profiling in providing novel biological insights into the molecular mechanisms underlying tumourigenesis. These insights will further contribute to improving treatment strategies for primary and metastatic liver cancers.

Deciphering the Divergent Gene Expression Landscapes of m6A/m5C/m1A Methylation Regulators in Hepatocellular Carcinoma Through Single-Cell and Bulk RNA Transcriptomic Analysis.

RNA modifications mediated by the m6A, m1A, and m5C regulatory genes are crucial for the progression of malignancy. This study aimed to explore the expression of regulator genes for m6A/m5C/m1A methylation at the single-cell level and to validate their expression in cancerous and adjacent para-cancerous liver tissues of adult patients with HCC who underwent tumor resection. The bulk sequencing from The Cancer Genome Atlas (TCGA) database and the single-cell RNA sequencing (scRNA-seq) data obtained from the Gene Expression Omnibus (GEO) database were used to identify the dysregulated m6A/m5C/m1A genes for hepatocellular carcinoma (HCC). A real-time polymerase chain reaction (real-time PCR) was used to measure the expression of dysregulated m6A/m5C/m1A genes in collected human HCC tissues and compared with adjacent para-cancerous liver tissues. Immune cell infiltration with these significantly expressed methylation-related genes was evaluated using Timer2.0. A discrepancy in m6A/m5C/m1A gene expression was observed between bulk sequencing and scRNA-seq. The clustered heatmap of the scRNA-seq-identified dysregulated m6A/m5C/m1A genes in TCGA cohort revealed heterogeneous expression of these methylation regulators within the cancer, whereas their expression in the adjacent liver tissues was more homogeneous. The real-time PCR validated the significant overexpression of DNMT1, NSUN5, TRMT6, IGF2BP1, and IGFBP3, which were identified using scRNA-seq, and IGFBP2, which was identified using bulk sequencing. These dysregulated methylation genes are mainly correlated with the infiltration of natural killer cells. This study suggests that cellular diversity inside tumors contributes to the discrepancy in the expression of methylation regulator genes between traditional bulk sequencing and scRNA-seq. This study identified five regulatory genes that will be the focus of further studies regarding the function of m6A/m5C/m1A in HCC.

Attenuation of binuclear hepatocytes in the paracancerous liver tissue is associated with short-term recurrence of hepatocellular carcinoma post-radical surgery.

Short-term recurrence of hepatocellular carcinoma (HCC) after radical resection leads to dismal outcomes. To screen high-recurrence risk patients to provide adjuvant treatment is necessary. Herein, based on our previous research, we further focused on the changes in the abundance of binuclear hepatocytes (ABH) in the paracancerous liver tissue to discuss the relationship between the attenuation of binuclear hepatocytes and postoperative short-term recurrence, by combining with the assessment of the value of a reported independent early recurrence risk factor in HCC, protein induced by vitamin K absence or antagonist-II (PIVKA-II). A cohort of 142 paracancerous liver tissues from HCC patients who received radical resection was collected. Binuclear hepatocytes were reduced in the paracancerous liver tissues, compared with the liver tissues from normal donors. ABH was negatively correlated with clinical features such as tumor size, TNM stages, tumor microsatellite formation, venous invasion, and Alpha-fetoprotein (AFP) level, as well as the expression of E2F7 and Anillin, which are two critical regulators concerning the hepatocyte polyploidization. According to the short-term recurrence information, ABH value was laminated, and univariate and multivariate logistic regression was performed to analyze the relationship between paracancerous ABH and short-term tumor relapse. Simultaneously, the predictive effectiveness of the ABH value was compared with the preoperative PIVKA-II value. As observed, the paracancerous ABH value below 1.5% was found to be an independent risk factor for recurrence. In conclusion, the paracancerous ABH is a credible indicator of short-term recurrence of HCC patients after radical resection, and regular assessment of ABH might help to prevent short-term HCC recurrence.

M6 A reader YTHDF3 triggers the progression of hepatocellular carcinoma through the YTHDF3/m6 A-EGFR/STAT3 axis and EMT.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the fourth leading cause of tumor-related deaths worldwide. N6-methyladenosine (m6 A) mediates RNA metabolism in tumor biology. However, the regulatory role of YTHDF3, an m6 A reader, in HCC progression and its underlying mechanisms remains unclear. Therefore, this study aims to investigate the oncogenic effect of YTHDF3 on HCC progression via the epigenetic regulation of m6 A-modified mRNAs. The expression levels of YTHDF3 in HCC tissues and matched adjacent liver tissues were detected using western blot analysis, immunohistochemistry, and quantitative real-time polymerase chain reaction. The function of YTHDF3 in HCC progression and its underlying mechanisms have been studied both in vitro and in vivo. YTHDF3 expression was significantly higher in HCC tissues than in paracancerous liver tissues. YTHDF3 was also significantly upregulated in HCC with microvascular invasion (MVI) compared to that in HCC without MVI. YTHDF3 overexpression facilitated the proliferation, invasion, and migration of HCC cells both in vitro and in vivo. However, the YTHDF3 knockdown resulted in an inverse trend. Mechanistically, YTHDF3 enhanced the translation and stability of the m6 A-modified epidermal growth factor receptor (EGFR) mRNA, which activated the downstream EGFR/signal transducer and activator of transcription 3 (STAT3) and epithelial-mesenchymal transition (EMT) oncogenic pathways. YTHDF3 enhanced the stability and translation of m6 A-modified EGFR mRNA and stimulated HCC progression via the YTHDF3/m6 A-EGFR/STAT3 and EMT pathways. These findings reveal that YTHDF3 plays a significant role in regulating HCC progression, suggesting a promising and novel target for HCC treatment.

LncRNA SAMD12-AS1 Suppresses Proliferation and Migration of Hepatocellular Carcinoma via p53 Signaling Pathway.

Purpose Assessment of lncRNA SAMD12-AS1 expression in liver cancer tissues and cell lines to investigate the underlying molecular mechanisms that regulate liver cancer cell growth, development, invasion, and migration. Methods The lncRNA SAMD12-AS1 expression in tumor tissues of 32 liver cancer patients was measured by real-time PCR, and its effect on the clinicopathological manifestations and liver cancer patients' prognosis was determined. LncRNA SAMD12-AS1 overexpression and knockdown in liver cancer cell lines were established by cell transfection. The effects of lncRNA SAMD12-AS1 knockdown and overexpression on liver cancer cell growth, development, invasion, and migration were determined by MTT, Transwell, and clonogenic assays. Furthermore, its effects on the expression of E-cadherin, vimentin, p53, and p21 in hepatocellular carcinoma cells were determined by Western blot assay. Results The level of lncRNA SAMD12-AS1 expression in tumor tissues was remarkably higher than that in paracancerous liver tissues (p < 0.01). It was found that the lncRNA SAMD12-AS1 expression was largely correlated with TNM stage of tumor, vascular invasion, and hepatitis B surface (HBs) antigen in liver cancer patients (p < 0.05). Cell function experiments showed that lncRNA SAMD12-AS1 overexpression promoted liver cancer development, migration, and invasion (p < 0.05), while lncRNA SAMD12-AS1 knockdown inhibited the activity of liver cancer cells to invade and migrate (p < 0.05). Western blot analysis showed that overexpression of lncRNA SAMD12-AS1 markedly inhibited p21, p53, and E-cadherin expression and promoted vimentin expression. Conversely, knockdown of lncRNA SAMD12-AS1 significantly promoted p21, p53, and E-cadherin expression and inhibited vimentin expression (p < 0.05). Conclusion LncRNA SAMD12-AS1 is associated with the TNM stage and vascular invasion of liver cancer. It promotes liver cancer cell development, invasion, and migration by regulating p53 expression. Thus, lncRNA SAMD12-AS1 could be a novel biological target for the treatment of liver cancer.

Involvement of TRPC7-AS1 Expression in Hepatitis B Virus-Related Hepatocellular Carcinoma.

Objective To investigate the expression of transient receptor potential (TRP) superfamily genes, especially TRPC7-AS1 in hepatitis B virus- (HBV-) related hepatocellular carcinoma (HCC). Methods Three cancer samples of HBV-related HCC at phase IV and matched paracancerous liver tissues were included in the study. Total RNA was extracted, and differential expression of RNA was screened by high-throughput transcriptome sequencing. The expression of TRPC7-AS1 was detected by quantitative real-time PCR. The N6-adenosyl methylation RNA in MHCC97H, HepG2, and HL-7702 was enriched by coimmunoprecipitation with m6A antibody, and the relative level of N6-adenosyl methylation RNA in TRPC7-AS1 was detected. Results The expression of TRP family genes in cancer tissues was higher than that in paracancerous liver tissues, including TRPC7-AS1, TRPC4AP, PKD1P6, and PKD1P1. Moreover, the expression level of TRPC7-AS1 in MHCC97H and HepG2 was also significantly higher than that in L02, a normal liver cell. The methylation level of N6-adenosine of TRPC7-AS1 was lower in HepG2 cells than that in L02 cells. Conclusion TRP superfamily genes, especially TRPC7-AS1, were highly expressed in HBV-related HCC. TRPC7-AS1 could be a potential therapeutic target or diagnostic marker for HCC.

Correlation of peritumoral circWDR25 expression with the prognosis of patients with hepatocellular carcinoma after curative resection

To study the association between the expression of peritumoral circWDR25 (hsa-circRNA-0004310) secreted by hepatic stellate cells (HSCs) and the prognosis of the patients with hepatocellular carcinoma (HCC) after curative resection. HSCs cell line LX-2 was co-cultured separately with 3 liver cancer cell lines (Hep3B, SMMC-7721, and HCCLM3) in Transwell chambers to obtain tumor cell-activated HSCs. The supernatants of HSC cultures were collected to isolate the exosomes, from which total RNA was extracted to detect circRNA expression profile. We also collected specimens of paracancerous liver tissues from 288 HCC patients undergoing radical resection in our department from January, 2014 to October, 2015, and the expression levels of circWDR25 and α-SMA were detected with in situ hybridization. Log-rank test and Cox regression analysis were used for univariate and multivariate analysis of the factors affecting the patients' prognosis, respectively. Gene expression profiling revealed that the expression of circWDR25 was the most obviously up-regulated in the exosomes isolated from tumor-activated LX-2 cells. The expression of peritumoral circWDR25 was positively correlated with HSCs adjacent to the cancer loci (r=0.156, P=0.008). Multivariate analysis showed that a preoperative AST level >36 g/L, multiple tumors, a tumor diameter >5 cm, HSC>70, and circWDR25>190 were independent risk factors affecting the overall survival of HCC patients after radical resection; a preoperative AST level >36 g/L, multiple tumors, a tumor diameter >5 cm, presence of tumor thrombus, HSC>70, and circWDR25>190 were all independent risk factors for tumor-free survival in patients with liver cancer. Peritumoral circWDR25 and HSCs are factors affecting the prognosis of HCC patients after radical hepatectomy, and their high expression in the adjacent tissues is closely related to a poor prognosis of the patients.

Quantitative acetylome analysis reveals histone modifications that may predict prognosis in hepatitis B-related hepatocellular carcinoma.

Lysine acetylation (Kac) as an important posttranslational modification of histones is essential for the regulation of gene expression in hepatocellular carcinoma (HCC). However, the atlas of whole acetylated proteins in HCC tissues and the difference in protein acetylation between normal human tissues and HCC tissues are unknown. In this report, we characterized the proteome and acetyl proteome (acetylome) profile of normal, paracancerous, and HCC liver tissues in human clinical samples by quantitative proteomics techniques. We identified 6781 acetylation sites of 2582 proteins and quantified 2492 acetylation sites of 1190 proteins in normal, paracancerous, and HCC liver tissues. Among them, 15 proteins were multiacetylated with more than 10 lysine residues. The histone acetyltransferases p300 and CBP were found to be hyperacetylated in hepatitis B virus pathway. Moreover, we found that 250 Kac sites of 214 proteins were upregulated and 662 Kac sites of 451 proteins were downregulated in HCC compared with normal liver tissues. Additionally, the acetylation levels of lysine 120 in histone H2B (H2BK120ac), lysine 18 in histone H3.3 (H3.3K18ac), and lysine 77 in histone H4 (H4K77ac) were increased in HCC. Interestingly, the higher levels of H2BK120ac, H3.3K18ac, and H4K77ac were significantly associated with worse prognosis, such as poorer survival and higher recurrence in an independent clinical cohort of HCC patients. Overall, this study lays a foundation for understanding the functions of acetylation in HCC and provides potential prognostic factors for the diagnosis and therapy of HCC.

Arginase-1 and P-glycoprotein are downregulated in canine hepatocellular carcinoma

BackgroundHepatocellular carcinoma is the most common primary hepatic malignancy in humans and dogs. Several differentially expressed molecules have been studied and reported in human hepatocellular carcinoma and non-neoplastic liver lesions. However, studies on the features of canine hepatocellular carcinoma are limited, especially related to the differential characteristics of neoplastic and non-neoplastic lesions.ObjectivesThe study's objective was 1) to examine and evaluate the expression of arginase-1, P-glycoprotein, and cytokeratin 19 in canine liver tissues and 2) to investigate the differential features of hepatocellular carcinomas, liver tissue with non-neoplastic lesions, and paracancerous liver tissues in dogs.MethodsThe expression levels of three markers underwent immunohistochemical analysis in 40 non-neoplastic liver tissues, 32 hepatocellular carcinoma tissues, and 11 paracancerous liver tissues. Scoring of each marker was performed semi-quantitatively.ResultsArginase-1 and P-glycoprotein were significantly downregulated in hepatocellular carcinoma, compared with hepatic tissues with non-neoplastic diseases (p < 0.001). Expression levels of arginase-1 and P-glycoprotein were also significantly lower in hepatocellular carcinoma than in paracancerous liver tissues (arginase-1, p = 0.0195; P-glycoprotein, p = 0.047). Few cytokeratin 19-positive hepatocytes were detected and only in one hepatocellular carcinoma and one cirrhotic liver sample.ConclusionsThe results of this study suggest that downregulation of arginase-1 and P-glycoprotein is a feature of canine hepatocellular carcinoma; thus, those markers are potential candidates for use in differentiating hepatocellular carcinomas from non-neoplastic liver lesions in dogs.

CIAP2 via NF-κB signalling affects cell proliferation and invasion in hepatocellular carcinoma

AimsTo investigate the role of cIAP2 in the malignant biological behaviours of hepatocellular carcinoma (HCC) cells and determine its mechanism of action. Main methodscIAP2 protein expression was detected via immunohistochemistry (IHC) in 102 HCC specimens and 43 paracancerous liver tissues, and its relationship with clinicopathological features and patient prognosis was analysed. Then, short interfering RNA (siRNA) technology was used to knock down cIAP2 expression in BEL7402 and HepG2 cells. Cell Counting Kit-8 (CCK8) and Transwell assays were used to determine cell proliferation and invasion after knockdown of cIAP2 expression. The relationship between cIAP2 and the NF-κB pathway was explored via western blotting (WB) and a dual luciferase reporter system. Finally, nude mouse models of liver cancer were established to detect the effect of cIAP2 on tumourigenicity and the proliferation activity of orthotopic HCC cells. Key findingscIAP2 expression was significantly increased in HCC tissues and was correlated with intravascular thrombosis in HCC. High cIAP2 expression was correlated with poor patient prognosis. cIAP2 knockdown significantly reduced the proliferation and invasion of BEL7402 and HepG2 cells and the activity of the NF-κB pathway. Animal experiments showed that cIAP2 knockdown reduced the tumourigenicity of HepG2 cells in the liver of nude mice and the proliferation activity of the orthotopic HCC cells. SignificancecIAP2 plays an important role in HCC proliferation and invasion and may exert its effects via the NF-κB signalling pathway.

33-kDa ANXA3 Knockdown Suppresses Carcinogenesis, Angiogenesis and Enhances Chemoresistance of Hepatocarcinoma &lt;i&gt;via&lt;/i&gt; Inhibiting ERK, PI3K/Akt-HIF and Intrinsic Apoptosis Pathways

Background: As a member of annexin family proteins, annexin A3 (ANXA3) has 36-kDa and 33-kDa isoforms, which plays important roles in aggressiveness, tumorigenesis and drug resistance in tumors. Previous studies mainly focused on the role of total ANXA3 in cancers without distinguishing the distinction between the two isoforms, the role of 33-kDa ANXA3 in cancer remains unclear. Current work established the potential role and regulation mechanism of 33-kDa ANXA3 in hepatocarcinoma. Methods: The expression patterns of ANXA3, CRKL, Rac1, c-Myc and pAkt were analyzed in hepatocarcinoma specimens by western blotting; The biological function of 33-kDa ANXA3 in hepatocarcinoma cell growth, metastasis, apoptosis, angiogenesis, chemoresistance and the underlying molecular mechanism were investigated using gain-of-function strategyin vitro or in vivo. Findings: 33-kDa ANXA3 protein was significantly upregulated in cancerous tissues compared with paracancerous non-tumor liver tissues of hepatocarcinoma patients. Its stable knockdown decreased the in vivo tumor growing velocity and malignancy of hepatocarcinoma HepG2 cells transplanted in nude mice. The in vitro experimental results indicated 33-kDa ANXA3 knockdown suppressed proliferative, colony forming, migrative and invasive abilities of HepG2 cells through downregulating CRKL, Rap1b, Rac1, pMEK, pERK2 and c-Myc in ERK pathway; inhibited angiogenesis ability of HepG2 cells through inactivating PI3K/Akt-HIF pathway; induced apoptosis and enchanced chemoresistance of HepG2 cells through increasing Bax/decreasing Bcl-2 expressions and inactivating caspase 9/caspase 3 in intrinsic apoptosis pathway. Accordingly, CRKL, Rac1, c-Myc and pAkt were also upregulated in cancerous tissues compared with paracancerous non-tumor liver tissues of hepatocarcinoma patients. Interpretation: 33-kDa ANXA3 plays vital role in hepatocarcinoma malignancy, angiogenesis and chemoresistance. It is of potential use in prognosis and therapeutics for hepatocarcinoma. Funding Statement: This work was supported by grants from National Natural Science Foundation of China (81672737, 81272186, 31900517) and Natural Science Foundation of Liaoning (LZ2019003, 20181550168, LQ2017001). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: Tissue use and study protocol were approved by the Medical Ethics Committee of Dalian Medical University. Informed consent was obtained from each patient. All experiment methods were performed in accordance with the relevant guidelines and regulations.

Bidirectional interaction of lncRNA AFAP1-AS1 and CRKL accelerates the proliferative and metastatic abilities of hepatocarcinoma cells

Actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1), a long non-coding RNA transcribed from the antisense strand of protein coding gene AFAP1, has attracted attention in cancer research. Despite, its biological function and regulatory mechanism in hepatocellular carcinoma still unknown. The present study revealed AFAP1-AS1 mediated hepatocarcinoma progression through targeting CRKL. The bidirectional interaction of AFAP1-AS1 and oncogenic protein CRKL, and the deregulation of AFAP1-AS1 effects on Ras, MEK and c-Jun activities were investigated in depth. AFAP1-AS1 was upregulated in surgical tumorous tissues from hepatocarcinoma patients compared with the paired paracancerous non-tumor liver tissues, and in hepatocarcinoma Huh7, HCCLM3 and HepG2 cell lines compared with LO2, a normal liver cell line. AFAP1-AS1 knockdown noticeably suppressed the proliferative, migratory and invasive properties, and the epithelial-mesenchymal transition (EMT) process of HepG2 and HCCLM3 through upregulating E-cadherin and downregulating N-cadherin and vimentin. CRKL knockdown reduced AFAP1-AS1 expression levels in HepG2 and HCCLM3 cells. AFAP1-AS1 suppression impaired CRKL expression in HepG2 and HCCLM3. AFAP1-AS1 level change was positively correlated with the expression level changes of Ras, MEK and c-Jun in mediating the invasiveness of hepatocarcinoma cells. Current work demonstrated AFAP1-AS1 to be an applicable progression indicator of hepatocarcinoma. AFAP1-AS1 probably promotes the proliferation, EMT progression and metastasis of hepatocarcinoma cells via CRKL mediated Ras/MEK/c-Jun and cadherin/vimentin signaling pathways. AFAP1-AS1-CRKL bidirectional feedback signaling is worthy of further study on the monitoring, diagnosis and treatment of cancers.

Increased expression of protease-activated receptors 2 indicates poor prognosis in HBV related hepatocellular carcinoma

ObjectiveTo investigate the potential role of protease-activated receptor 2 (PAR2) in the prognosis of hepatocellular carcinoma (HCC).MethodsA total of 202 HCC patients who underwent liver resections were included. Tissue microarray was established with specimens of both HCC and paired adjacent liver tissues. PAR2 expression was detected by immunohistochemistry (IHC) assays.. A semi-quantification method was used to define the expression level of PAR2. The correlations between PAR2 expression and clinical features of patients with HCC was explored. The association of different PAR2 expressions with both overall survival and disease-free survival was analyzed.ResultsResults showed that the expression of PAR2 in HCC tissues was higher than that in paired para-cancerous liver tissues (4.12 ± 3.55 vs. 2.71 ± 2.56, P < 0.001). Higher expression of PAR2 was associated with poor differentiation (P < 0.001) and advanced tumor-node-metastasis stage (P = 0.015). Kaplan-Meier survival analysis indicated that HCC patients with high PAR2 expression had decreased overall survival (P = 0.033) and disease-free survival (P = 0.043) compared to patients with lower PAR2 expression. Multivariate analysis indicated that PAR2 expression (P = 0.032) was a significant independent prognostic factor for both overall survival and disease-free survival (P = 0.032; P = 0.032, respectively).ConclusionOur data revealed that PAR2 expression was increased in HCC. High PAR2 expression was correlated with both decreased overall survival and disease-free survival in patients with HCC. High PAR2 expression indicated a poor prognosis.

Monoacylglycerol lipase high expression as an independent indicator of survival for patients with hepatocellular carcinoma

Objective: To study the expression level of monoacylglycerol lipase (MAGL) in liver tissues of patients with hepatocellular carcinoma (HCC), and its clinical correlation. Methods: Immunohistochemistry was employed to detect MAGL protein in 353 cases with hepatocellular carcinoma (HCC) and tissue microarray (TMA) for paracancerous liver tissues. The expression levels of MAGL in TMA were quantitatively analyzed using Image-Pro plus 6.0. The difference in MAGL expression between liver cancer tissues and paracancerous liver tissues was compared. Combined with the clinical follow-up data of TMA patients, the correlation between the expression of MAGL in TMA and the degree of HCC tumors differentiation and the survival rate of 1-year and 3-year were analyzed using Logistic regression analysis. The survival curves of patients with different levels of MAGL protein was plotted and analyzed using Kaplan-Meier method. The expression of MAGL protein was analyzed by multiple linear regression analysis. COX regression was used to analyze the correlation between MAGL protein expression level and the risk of HCC death in the included patients. Results: The expression of MAGL in HCC tissues was significantly higher than paracancerous liver tissues. The expression level of MAGL was correlated to the degrees of HCC tumors differentiation (P < 0.001) and 1-year survival rate (P = 0.01), but not with 3-year survival rate (P = 0.91). Survival curve showed that the expression level of MAGL was negatively correlated with prognosis and survival of HCC patients (P = 0.001). Multiple linear regressions showed a negative correlation between MAGL expression level and overall survival time of HCC patients (P=0.010, R2=0.166, Durbin-Watson value: 1.989). COX regression showed that the expression of MAGL was a risk factor for death of patients with HCC [P = 0.004, Exp (B) = 1.000]. Conclusion: The expression level of MAGL has positive correlation with the malignant degree in HCC patients, and negative correlation with its prognosis. Therefore, MAGL may serve as a new prognostic indicator for HCC patients.

The clinical significance of the changes of Th17 cells and cytokines IL-17 and IL-6 in hepatocellular carcinoma patients

Objective To investigate the change of the percentage of Th17 cells and cytokine among hepatocellular carcinoma (HCC) tissues and para-carcinoma tissue and the liver tissue of hepatic hemangioma, and to explore its clinical significance. Methods To choose 26 of hepatocellular carcinoma patients between November 2014 and December 2016 and also include 11 cases of hepatic hemangioma as control group. The percentage of Th17cells in HCC tissue and para-carcinoma tissue and the liver tissue of hepatic hemangioma was evaluated by flow cytometric; The levels of IL-6 and IL-17 in HCC tissues, para-carcinoma tissue and the liver tissue of hepatic hemangioma were detected by RT-PCR. Results The ratio of Th17 cells to CD4 cells in peripheral blood of HCC was significantly higher than that in control group (4.07%±0.68% vs. 1.39%±0.41%), P<0.05. The ratio of Th17 cells to CD4 cells in patients with hepatocellular carcinoma was significantly higher than that in paracancerous tissues and liver tissues of hepatic hemangioma (4.76%±0.75% vs. 2.30%±0.26% vs. 0.77%±0.31%, P<0.05). Multivariate linear stepwise regression analysis of the changes of Th17 cells in patients with HCC and their correlation with clinical parameters showed that Th17 lymphocytes in peripheral blood of patients with HCC were closely related to TNM staging(YTh17=2.647+ 0.687TNM. It’s indicated that the level of IL-6 and IL-17 in liver cancer tissues was significantly higher than that of the liver hemangioma in the control group through ELISA (P<0.05). The proportion of peripheral blood Th17 cells to CD4 cells is different in different TNM stages(stage I<stage II<stage III~IV, the difference was statistically significant. RT-PCR analysis showed that the expression of IL-17, IL-6mRNA in hepatocellular carcinoma was significantly higher than that in adjacent tissues and hepatic hemangioma liver tissue (all P<0.05). Conclusion The increased proportion of Th17 cells and the increased levels of cytokines IL-6 and IL-17 may be closely correlated with occurrence and development of hepatocellular carcinoma. It can be an important target of anti-tumor therapy. Key words: Liver neoplasms; Carcinoma, hepatocellular; Th17 cells; Interleukin-6; Interleukin-17

LncRNA 00152 promotes the development of hepatocellular carcinoma by activating JAK2/STAT3 pathway.

The aim of this study was to explore the regulatory role of lncRNA 00152 and JAK2/STAT3 pathway in the pathogenesis of hepatocellular carcinoma (HCC), and to investigate the possible underlying mechanism. Expression levels of lncRNA 00152 in HCC tissues, matched para-cancerous tissues and normal liver tissues were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), respectively. The correlation between lncRNA 00152 expression and pathological characteristics of HCC patients was analyzed. Meanwhile, the expression level of lncRNA 00152 in HCC cell lines was detected by qRT-PCR. After knockdown or overexpression of lncRNA 00152 in MHCC97 or HB611 cells, the proliferative ability and cell cycle were detected by EdU assay and flow cytometry, respectively. Also, Western blot was conducted to detect the protein expression levels of JAK2 and STAT3 in MHCC97 and HB611 cells. The expression of lncRNA 00152 in HCC tissues was significantly higher than that of matched para-cancerous tissues and normal liver tissues. LncRNA 00152 expression was positively correlated with tumor stage and tumor size, whereas negatively correlated with the overall survival of HCC patients. High expression of lncRNA 00152 might be a potential hallmark for the diagnosis of HCC, with the AUC of 0.8425. Similarly, lncRNA 00152 was highly expressed in HCC cell lines when compared with that of normal liver cells. Knockdown of lncRNA 00152 in MHCC97 cells remarkably decreased the proliferative ability and arrested cell cycle. Overexpression of lncRNA 00152 in HB611 cells significantly promoted cell proliferation and cell cycle. Furthermore, Western blot results showed that lncRNA 00152 knockdown upregulated the protein expression levels of JAK2 and STAT3 in HCC cells. High expression of lncRNA 00152 promotes the development of HCC by activating the JAK2/STAT3 pathway.

MiR-409 down-regulates Jak-Stat pathway to inhibit progression of liver cancer.

To elucidate the influence of microRNA-409 and the Jak-Stat pathway on the development of liver cancer. The quantitative Real-time polymerase chain reaction (qRT-PCR) method was used to detect the expression of microRNA-409 in hepatocarcinoma, paracancerous tissues and normal liver tissues, and the correlation between its expression and clinicopathological parameters of patients was analyzed, with the area under the microRNA-409 curve (AUC) being detected. The level of microRNA-409 in different liver cancer cells was detected by qPCR. Then it was overexpressed or knock-downed in the liver cancer cells by cell transfection technique. The cell apoptosis and viability after inhibition or overexpression of microRNA-409 were evaluated by propidium iodide (PI) staining and cell counting kit-8 (CCK-8) assay. Subsequently, Jak2 and Stat3 mRNA levels were detected by qPCR in hepatocarcinoma and paracancerous tissues, with their protein levels analyzed by Western blot after microRNA-409 was inhibited or up-regulated. At last, CCK-8 assay was performed to evaluate the effect of Jak2 on cell viability. Compared with paracancerous and normal liver tissues, the level of microRNA-409 was remarkably reduced in hepatocarcinoma tissues and was negatively correlated with tumor stage, tumor size and overall survival time of patients with liver cancer. Meanwhile, microRNA-409 expression in hepatoma cell lines was also strikingly lower than that in normal liver cells. After overexpression of microRNA-409 in HHCC, cell viability significantly decreased while apoptosis increased, and opposite results were shown in HepG2 cells after miR409 was knock-downed. In liver cancer tissues, the levels of Jak2 and Stat3 were significantly higher than those in adjacent tissues. Additionally, up-regulating microRNA-409 reduced the level of Jak2 and Stat3 protein, while down-regulating it elevated them. In addition, Jak2 could reverse the inhibitory effect of microRNA-409 on the proliferation of hepatoma cells. Highly-expressed microRNA-409 can down-regulate the Jak-Stat signaling pathway and inhibit cell proliferation to slow down the progression of liver cancer.

Mitofusin-2 acts as biomarker for predicting poor prognosis in hepatitis B virus related hepatocellular carcinoma

ObjectiveTo investigate the expression of Mitofusin-2 (MFN2) in HCC tissues and its role in the development of HCC.MethodsA total of 107 HCC specimens were collected for tissue microarray analysis and immunohistochemistry (IHC) analysis. The relationship between MFN2 expression and clinical features of patients with HCC was analyzed.ResultsExpression level of MFN2 in HCC tissues was 0.92 ± 0.78, significantly lower than that of matched paracancerous liver tissues (1.25 ± 0.75). Patients with low expression of MFN2 had significantly higher rates of cirrhosis than those with high expression of MFN2 (P = 0.049). Kaplan-Meier survival analysis showed that HCC patients with low expression of MFN2 had a worse prognosis in overall survival than HCC patients with high expression of MFN2 (P = 0.027). Patients with high expression of MFN2 had a better prognosis in disease-free survival compared with HCC patients with low expression of MFN2 (P = 0.047). Vascular invasion and MFN2 expression were shown to be prognostic variables for overall survival in patients with HCC. Multivariate analysis showed that vascular invasion (P < 0.001) and MFN2 expression (P = 0.045) were independent prognostic factors for overall survival. Vascular invasion (P < 0.001) and MFN2 expression (P = 0.042) were independent risk factors associated with disease-free survival.ConclusionOur data revealed that MFN2 expression was decreased in HCC samples. High MFN2 expression was correlated with longer survival times in patients with HCC and served as an independent factor for better outcomes. Our study therefore provides a promising biomarker for the prognostic prediction of HCC and a potential therapeutic target for the disease.

Reduced expression of olfactomedin 1 was associated with poor prognosis of hepatocellular carcinoma

Objective To investigate the expression level of olfactomedin 1 (OLFM1) in hepatocellular carcinoma (HCC) and its relationship with clinicopathological characteristics and prognosis of patients with HCC. Methods The expression of OLFM1 protein in 71 cases of HCC and corresponding paracancerous liver tissues was detected by Western blotting. The relationship between the expression of OLFM1 and the clinicopathological factors, as well as prognosis after operation, in the patients with HCC was analyzed. Results The average expression level of OLFM1 protein was 0.42±0.33 in HCC tissues, and it was 1.11±0.53 in paracancerous liver tissues. The expression level of OLFM1 protein in HCC tissues was significantly lower than that in paracancerous liver tissues (P=0.001). The down-expression of OLFM1 in HCC was closely related to the malignant biological characteristics of multiple tumors, larger tumor diameter, absence of tumor capsule, vascular invasion and poor tumor differentiation. The difference was statistically significant (P=0.024, P=0.034, P=0.010, P=0.002, P=0.024, P=0.001). The recurrence-free survival and overall survival in the group of OLFM1 down-expression were significantly lower than those of OLFM1 up-expression (P=0.009, P=0.013). Conclusion The down-expression of OLFM1 is closely related to the high degree of malignancy and poor prognosis in HCC. The OLFM1 may play an important role in the development and metastasis of HCC. Key words: Olfactomedin 1; Hepatocellular carcinoma; Recurrence; Prognosis