Paired Human Hepatocellular Carcinoma Tissues Research Articles

Hsa_Circ_0098181 Suppresses Hepatocellular Carcinoma by Sponging miR-18a-3p and Targeting PPARA.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths, and its incidence is still high in China. This study aimed to investigate the circular RNAs (circRNAs) involved in the development of HCC and elucidate the mechanism. RNA sequencing found 72 downregulated circRNAs and 88 upregulated circRNAs in human HCC tissues, including hsa_circ_0098181, hsa_circ_0072309, hsa_circ_0000831, and hsa_circ_0000231. The reduction of hsa_circ_0098181 was confirmed in eight paired human HCC tissues, hepatoma cell lines, and CCL4/DEN-induced mouse HCC models by RT-qPCR. The FISH assay revealed that hsa_circ_0098181 is mainly located in the cytoplasm of hepatocytes in the paratumor tissues. Further log-rank analysis performed in 91 HCC patients demonstrated that low expression of hsa_circ_0098181 was related to poor prognosis. The plasmid and lentivirus overexpressing hsa_circ_0098181 were delivered into HCC cell lines. After hsa_circ_0098181 was upregulated, the proliferation, invasion, migration, and colony formation of HCC cell lines were inhibited, and the apoptosis was promoted. Moreover, exogenous hsa_circ_0098181 delivery mitigated the tumor formation ability of Huh7 in Balb/C nude mice. The dual-luciferase reporter assay and the RIP assay verified that hsa_circ_0098181 sponged miR-18a-3p to regulate PPARA. In addition, a rescue experiment found miR-18a-3p mimic partly reversed the suppression of hsa_circ_0098181 on proliferation, invasion, and migration of HCC cell lines. In conclusion, hsa_circ_0098181 can repress the development of HCC through sponging miR-18a-3p and promoting the expression of PPARA in vitro and in vivo, and hsa_circ_0098181 might be a therapeutic target for HCC.

Abstract 80: Pooled shRNA screening using mouse xenografts of human hepatocellular carcinoma cells identifies CDK5 as a potential mechanism mediating sorafenib resistance

Abstract Background: Sorafenib is the only approved systemic agent for hepatocellular carcinoma (HCC). However, the efficacy of sorafenib is modest for HCC with low objective response rate and short time to tumor progression, indicating that the inherent and acquired resistance underlies the limited success of sorafenib in HCC. We conducted a pooled shRNA screening in mouse xenografts of human HCC cells treated with sorafenib, and intended to identify genes that are involved in the therapeutic efficacy of sorafenib. Materials and Methods: We introduced a pool of 3920 shRNAs targeting 1960 genes, including various kinases, transcriptional factors, phosphatase and cytokine, into Huh7 HCC cells by retrovirus infection. The Huh7 cells after infection with pooled shRNAs were implanted subcutaneously in immunocompromised mice, and were treated with vehicle or sorafenib at the dose of 30mg/kg/day for 4 weeks. Genomic DNAs were extracted from HCC xenografts, PCR-amplified of the shRNA regions, and deep-sequenced of the PCR products to quantify the abundance of each shRNA. Candidate genes were identified from those shRNAs with statistically different frequencies between vehicle-treated and sorafenib-treated xenografts, and were further validated by their impacts on sorafenib-sensitivity in HCC cells. Results: By calculating the abundance frequencies of each shRNA of sorafenib- treated xenografts versus that of vehicle-treated xenografts, we found that 3 candidate sorafenib- resistance genes because their shRNA abundance was significantly decreased in sorafenib-treated xenografts. Among them, only the cyclin dependent kinase 5 (CDK5) was further investigated because its expression was confirmed to be increased in sorafenib- treated xenografts. In PLC5 and a sorafenib-resistant subline of Huh7 cells (Huh7-SR), downregulation of CDK5 by RNA interference or roscovitine, a CDK5 inhibitor, enhanced the sensitivity to sorafenib- induced growth-suppression. On the other hand, overexpression of CDK5 increased the resistance to sorafenib in Huh7 cells. Mechanistically, sorafenib combined with shRNA-mediated CDK5 downregulation augmented the induction of apoptosis, and resulted in an increased downregulation of multiple anti-apoptosis molecules such as XIAP and survivin in PLC5 and Huh7-SR cells. In 8 paired human HCC tissues, we found 3 of them have unequivocally increased expression of CDK5 in HCC cells of post-sorafenib HCC tissues compared with those of pre-treatment tissues. Conclusions: Our results suggest that increased CDK5 expression may mediate resistance to sorafenib in HCC in vivo, and targeting CDK5 is warranted further studies in the treatment of HCC. (This study was supported by grants MOST 103-2314-B-002 -181 -MY2, MOST 105-2314-B-002-194-, MOST 103-2314-B-002 -090 - & MOST 104-2314-B-002 -073). Citation Format: Tzu-Hsuan Lin, Wen-Chi Feng, Yu-Yun Shao, Li-Chun Lu, Ya-Jhen Chen, Ann-Lii Cheng, Chih-Hung Hsu. Pooled shRNA screening using mouse xenografts of human hepatocellular carcinoma cells identifies CDK5 as a potential mechanism mediating sorafenib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 80. doi:10.1158/1538-7445.AM2017-80

Abstract 2067: Tumor-infiltrating PMN-MDSCs mediate sorafenib resistance in hepatocellular carcinoma through immune suppression

Abstract Background]: The mechanisms underlying the inherent or acquired resistance to sorafenib in advanced hepatocellular carcinoma (HCC) are not fully understood. We studied the role of myeloid-derived suppressor cells (MDSCs), a subset of bone marrow cells with immune-suppressive and proangiogenic activities, in sorafenib-treated mice and patients. Materials and Methods: An orthotopic mouse liver cancer model was established by implanting BNL cells to BALB/cByJNarl mice. Liver tumor-bearing mice were treated with sorafenib (5 mg/kg/d) for 1 week or 3 weeks from the day 8 after tumor cells implantation. For combination therapy, mice were treated sorafenib and anti-IL-6 Ab or its isotype control Ab (500 μg/every 3 days starting from day 4 for 4 doses). Tumor-infiltrating leukocytes (TILs) and leukocytes of other tissues including liver, spleen, peripheral blood, and bone marrow were isolated, and characterized by flow cytometry analysis. Morphological characteristics of MDSCs were determined by cytospin and Liu staining. Pro-inflammatory cytokines were detected by ELISA or mouse inflammatory cytokines beads array (BD). Paired HCC tissues from advanced HCC patients treated with sorafenib (before and post-sorafenib progression tissues) were evaluated for the expression of CD11b, CD15, and CD66b in TILs by immunohistochemistry. Results: In mice treated with sorafenib, the CD11b+Gr1+Ly6G+ MDSCs were significantly increased in liver tumors, but not in others tissues. Morphologically, these MDSCs were segmented nuclear PMN type cells. The analysis of TILs revealed that the percentage of total immune effectors (CD4+ or CD8+ IFN-γ-expressing T cells) versus total immune suppressors (IL-10 or TGF β-expressing CD4+ T cell, FOXP3-expressing CD4+ T cells) was lower in the tumors of sorafenib-treated mice than those of the vehicle-treated mice. The T cell proliferation capability was also significantly decreased in TILs of sorafenib-treated mice versus TILs of vehicle-treated mice. Multiple inflammatory cytokines including VEGF, G-CSF, MCP-1, and IL-6 were significantly increased in mouse liver tumors treated with sorafenib. When combined with sorafenib, anti-IL-6 antibody alleviated the early tumor-infiltration of Ly6G+ MDSCs, restored the proliferative activity of TILs, decreased the levels of pro-inflammatory factors in tumors, and suppressed the liver tumor growth synergistically. In 6 paired human HCC tissues, we found that 3 exhibited higher proportion of CD11b, CD15, or CD66b+ cells in post-sorafenib progression tissues than their pre-sorafenib treatment tissues. Conclusions: Our data indicate that tumor-infiltrating PMN type MDSCs and associated immunosuppression may be an important mechanism impeding the therapeutic efficacy of sorafenib in HCC. (This works was supported by MOST 105-2314-B-002-180). Citation Format: Chun-Jung Chang, Li-Chun Lu, Cher-wei Liang, Chih-Hung Hsu, Ann-Lii Cheng. Tumor-infiltrating PMN-MDSCs mediate sorafenib resistance in hepatocellular carcinoma through immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2067. doi:10.1158/1538-7445.AM2017-2067

MTA1 modulated by miR-30e contributes to epithelial-to-mesenchymal transition in hepatocellular carcinoma through an ErbB2-dependent pathway.

MTA1 is a metastasis-associated protein, which is essential to epithelial-to-mesenchymal transition (EMT). However, information concerning its up- and downstream regulation is rare. We investigated its upstream regulation and downstream effector in human hepatocellular carcinoma (HCC). In total, 94 paired HCC and adjacent tissue samples were involved in the study, and the results indicated that decreased miR-30e levels were associated with increased MTA1 levels in human HCC. miR-30e exerted its regulation of MTA1 transcription by binding to its 3'-untranslated region, and was negatively associated with EMT. Furthermore, significantly higher expression of MTA1 was associated with overexpression of v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ErbB2) in human HCC, and MTA1 can promote transcription of ErbB2 by binding with histone deacetylase 2 (HDAC2) and acting as a promoter. The EMT promotion effect caused by MTA1 largely depended on ErbB2, and reducing the activity of ErbB2 can significantly attenuate EMT promotion by causing overexpression of MTA1 both in vitro and in vivo. In general, downregulation of miR-30e can increase levels of MTA1 in human HCC, and furthermore promote cell invasion and metastasis by promoting ErbB2.

Low expression of DCXR protein indicates a poor prognosis for hepatocellular carcinoma patients.

The aim of this study was to investigate the prognostic value of dicarbonyl/L-xylulose reductase (DCXR) in human hepatocellular carcinoma (HCC). Immunohistochemistry and tissue microarrays were used to evaluate DCXR protein expression levels. Image-Pro Plus was used to calculate the integral optic density (IOD) in each tissue sample, which represented the expression level of DCXR. DCXR proteins were found to be significantly lower in HCC tumor tissues (P<0.0001) according to immunohistochemical analysis of DCXR protein levels in 74 paired HCC tissue and peritumoral non-cancer tissues. The prognostic value of DCXR in HCC was assessed in 290 cases of the training cohort and 74 cases of the validation cohort. Shorter overall survival (OS) time and shorter time to recurrence (TTR) in both the training and validation set were found to be associated with lower expression levels of DCXR. In the training set, the expression level of DCXR in HCC was an independent prognostic factor for OS according to univariate and multivariate analyses. In conclusion, DCXR expression is an independent prognostic factor for OS and TTR of post-operative HCC patients, and low expression levels of DCXR in HCC may indicate poor outcome of HCC patients after surgical resection.

Low miR-29c expression is a prognostic marker in hepatocellular carcinoma.

A previous study has revealed that miR-29c functions as a tumor suppressor in hepatocellular carcinoma (HCC), but the clinical significance and prognostic value of miR-29c in HCC have not been investigated. Paired human HCC tissues and adjacent noncancerous tissues were obtained from 91 patients, between 2008 to 2014. Quantitative real-time PCR (qRT-PCR) was used to analyze miR-29c expression. Kaplan-Meier survival plots and log-rank tests were used to assess differences in the overall survival of different subgroups of HCC patients. It was observed that miR-29c expression was remarkably decreased in HCC tissues relative to that in normal hepatic tissues (P < 0.001). The low miR-29c level was significantly associated with histologic grade (P = 0.001), microvascular invasion (P = 0.005), and tumor stage (P < 0.001). Kaplan-Meier analysis showed that decreased miR-29c expression correlated with shorter overall survival (P = 0.002). Multivariate Cox regression analysis showed that decreased miR-29c expression (hazard ratio = 2.19, 95%CI = 1.361-6.779, P = 0.025) was independently associated with poor survival in HCC. Our findings demonstrate that miR-29c expression is significantly downregulated in HCC patients and that miR-29c can act as an independent predictor of unfavorable clinical outcome.

Promoter hypomethylation up‐regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma

CD147 is a transmembrane glycoprotein overexpressed in human hepatocellular carcinoma (HCC) which could promote HCC progression and metastasis. Promoter methylation is one of the most important processes in gene regulation. In this study, we aim to investigate CD147 promoter methylation status and the correlation with clinicopathological features and prognosis in HCC. CD147 promoter methylation statuses and expression levels in normal and HCC cell lines and 54 paired HCC and adjacent non-tumour (ANT) tissues were, respectively, examined by bisulphite genomic sequencing, methylation-specific PCR, real-time RT-PCR, Western blot and immunohistochemistry. The correlations of promoter methylation statuses with CD147 expression level and the clinicopathological features were statistically analysed in HCC patients. Significantly higher expression of CD147 and significantly lower promoter methylation level were observed in HCC cell lines compared to normal cell lines and tissues control. In vivo and in vitro analysis indicated that demethylation with 5-Aza-2′-deoxycytidine led to increased CD147 expression through enhancing Sp1 binding affinity, and methylation with methyltransferase reduced CD147 transcriptional activity through interfering Sp1 binding. CD147 promoter methylation level in HCC tissues (22.22%) was lower than that in ANT tissues (46.30%; P < 0.05). Within HCC tissues, a significant inverse correlation was observed between CD147 expression and methylation level (r=−0.615). Moreover, HCC patients with unmethylated CD147 promoter had a significantly higher recurrence rate (88.1%versus 58.3%; P < 0.05) and death rate (83.3%versus 50.0%; P < 0.05) than patients with methylated CD147 promoter. In conclusions, promoter hypomethylation up-regulates CD147 expression primarily through increasing Sp1 binding and associates with poor prognosis in HCC patients.

Prognostic Significance of Neuroepithelial Transforming Protein 1 in Hepatocellular Carcinoma

ABSTRACTObjective: Recent works have demonstrated that neuroepithelial transforming protein 1 (NET-1) gene expression is associated with proliferation, metastasis, and clinical stages of human hepatocellular carcinoma (HCC). To investigate its prognostic significance in HCC, which currently is unknown, the authors examined the correlation between NET-1 expression and prognosis in patients with HCC. Methods: Immunohistochemical staining was used to determine NET-1 expression level in 368 paired HCC and normal liver tissue (NLT) specimens. Kaplan–Meier survival and Cox regression analyses were performed to evaluate the prognosis of HCC. Results: Among 368 specimens of HCC, the positive rate of NET-1 protein expression in HCC tissues was 86.7% (319/368), and the increased NET-1 expression was correlated significantly with high Edmondson–Steiner grade (p = .02) and TNM stage (p = .01). The expression level of NET-1 in HCC tissues was associated with intrahepatic metastasis (p = .008) and portal vein infiltration (p = .007). HCC patients with the moderate–strong NET-1 positive expression had either poorer disease-free survival or poorer overall survival than patients with negative–low positive NET-1 expression (p = .001 and .002, respectively). A multivariate Cox regression analysis revealed that NET-1 protein expression (relative risk 5.8; p = .01) was an independent prognostic factor for patients with HCC. Conclusion: Taken together, our study suggests for the first time that NET-1 status may be a new predictor of survival for HCC patients and provides the rationale for developing a novel therapy of targeting NET-1 against this fatal malignancy.

Methylation of Tip30 Promoter Is Associated with Poor Prognosis in Human Hepatocellular Carcinoma

To investigate Tip30 promoter methylation status in human hepatocellular carcinoma (HCC) and the correlation with clinicopathologic features and prognosis. The methylation status of CpG islands in Tip30 promoter was examined in 15 HCC cell lines as well as 59 paired HCC and adjacent nontumor tissues. The associations between Tip30 methylation status and the survival of patients were analyzed. Tip30 promoter was hypermethylated in 6 of 10 HCC cell lines with reduced Tip30 mRNA. DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, greatly enhanced TIP30 expression and sensitized HCC cells to cytotoxic drug-induced cell death. The promoter region of Tip30 was identified and the main promoter activity was located in the -135 to -45 region sited within a CpG island. The minimal promoter element contained four Sp1 binding sites, which were hypermethylated in HCC cell-derived promoters. Moreover, analyses of Tip30 promoter methylation status in 59 paired HCC tissues showed that 47% of the cases were hypermethylated. Recurrence rate (95% versus 67%; P = 0.011) and mortality (82% versus 53%; P = 0.033) were significantly higher in patients with methylated Tip30. Disease-free survival was significantly higher in patients with unmethylated Tip30 (33.3% versus 4.5%; P = 0.036). Our results show that epigenetic silencing of Tip30 gene expression by CpG island DNA hypermethylation is associated with poor prognosis in patients with HCC.

Decreased expression of methyl methansulfonate and ultraviolet‐sensitive gene clone 81 (Mus81) is correlated with a poor prognosis in patients with hepatocellular carcinoma

Recent work has demonstrated that methyl methansulfonate and ultraviolet-sensitive gene clone 81 (Mus81) is critical in the maintenance of chromosome stability and tumor suppression in mice. To investigate its role in human hepatocellular carcinoma (HCC), which currently is unknown, the authors examined the correlation between Mus81 expression and prognosis in patients with HCC. Reverse transcriptase-polymersase chain reaction and Western blot analyses were used to determine Mus81 expression levels in 41 paired HCC and paracarcinomatous liver tissue (PCLT) specimens. Immunohistochemistry analysis was also performed on 104 HCC specimens from patients with follow-up information. Both Mus81 messenger RNA and protein expression levels were decreased significantly in HCC specimens. Moreover, lower Mus81 expression levels were detected in nodular HCC (NHCC) than in solitary large HCC (SLHCC) specimens. Among 104 specimens of HCC, the positive rate of Mus81 was significantly lower than the rate in PCLT (P = .017), and the decreased Mus81expression was correlated significantly with high Edmondson-Steiner grade, multiple tumor nodes, and venous invasion. Patients with HCC who had low Mus81 expression had either poorer disease-free survival or poorer overall survival than patients who had with high Mus81 expression (P = .0027 and P = .0001, respectively). Multivariate Cox regression analysis revealed that low Mus81 expression was an independent prognostic factor for patients with HCC (risk ratio, 5.74; P = .012). Mus81 expression levels were decreased significantly in HCC specimens, and the decreased levels were correlated with a poor prognosis in patients with HCC, implicating Mus81 as a candidate prognostic marker in HCC.

KIAA0101 (OEACT-1), an expressionally down-regulated and growth-inhibitory gene in human hepatocellular carcinoma

BackgroundOur previous cDNA array results indicated KIAA0101 as one of the differentially expressed genes in human hepatocellular carcinoma (HCC) as compared with non-cancerous liver. However, it is necessary to study its expression at protein level in HCC and its biological function for HCC cell growth.MethodWestern blot and tissue array were performed to compare KIAA0101 protein expression level in paired human HCC and non-cancerous liver tissues from the same patients. Investigation of its subcellular localization was done by using dual fluorescence image examination and enriched mitochondrial protein Western blot analysis. The in vitro cell growth curve was used for examing the effect of over-expression of KIAA0101 in HCC cells. FACS was used to analyze the cell cycle pattern in KIAA0101 expression positive (+) and negative (-) cell populations isolated by the pMACSKKII system after KIAA0101 cDNA transfection.ResultsWestern blot showed KIAA0101 protein expression was down-regulated in HCC tissues as compared with their counterpart non-cancerous liver tissues in 25 out of 30 cases. Tissue array also demonstrated the same pattern in 161 paired samples. KIAA0101 was predominantly localized in mitochondria and partially in nuclei. KIAA0101 cDNA transfection could inhibit the HCC cell growth in vitro. In cell cycle analysis, it could arrest cells at the G1 to S phase transition.ConclusionKIAA0101 protein expression was down-regulated in HCC. This gene could inhibit the HCC cell growth in vitro and presumably by its blocking effect on cell cycle.