N-glycosylation of Wnt3 regulates the progression of hepatocellular carcinoma by affecting Wnt/β-catenin signal pathway

BACKGROUND Wnt/FZD-mediated signaling pathways are activated in more than 90%of hepatocellular carcinoma(HCC)cell lines.As a well-known secretory glycoprotein,Wnt3 can interact with FZD receptors on the cell surface,thereby activating the Wnt/β-catenin signaling pathway.However,the N-glycosylation modification site of Wnt3 and the effect of this modification on the biological function of the protein are still unclear.AIM To investigate the effect of Wnt3 N-glycosylation on the biological function of HCC cells.METHODS Site-directed mutagenesis was used to verify the Wnt3 N-glycosylation sites,actinomycin D treatment was used to detect the stability of Wnt3 after site-directed mutation,the binding of the N-glycosylation site-directed mutant Wnt3 to FZD7 was observed by laser confocal microscopy,and the effects of the N-glycosylation site-directed mutation of Wnt3 on the Wnt/β-catenin signaling pathway and the progression of HCC cells were detected by western blot and cell function experiments.RESULTS Wnt3 has two N-glycosylation-modified sites(Asn90 and Asn301);when a single site at amino acid 301 is mutated,the stability of Wnt3 is weakened;the binding ability of Wnt3 to FZD7 decreases when both sites are mutated simultaneously;and the level of proteins related to the Wnt/β-catenin signaling pathway is downregulated.Cell proliferation,migration and invasion are also weakened in the case of single 301 site and double-site mutations.CONCLUSION These results indicate that by inhibiting the N-glycosylation of Wnt3,the proliferation,migration,invasion and colony formation abilities of liver cancer cells can be weakened,which might provide new therapeutic strategies for clinical liver cancer in the future.

Association of MBOAT7 rs641738 polymorphism with hepatocellular carcinoma susceptibility:A systematic review and meta-analysis

BACKGROUND The MBOAT7 rs641738 single-nucleotide polymorphism(SNP)has been proven to influence various liver diseases,but its association with hepatocellular carcinoma(HCC)susceptibility has been debated.To address this discrepancy,we conducted the current systematic review and meta-analysis.AIM To perform a systematic review and meta-analysis on association of MBOAT7 SNP and HCC susceptibility.METHODS We performed a systematic review in PubMed,Web of Science,Scopus,and EMBASE;applied specific inclusion and exclusion criteria;and extracted the data.Meta-analysis was conducted with the meta package in R.Sensitivity and subgroup analyses were also performed.This meta-analysis was registered in PROSPERO(CRD42023458046).RESULTS Eight studies were included in the systematic review,and 12 cohorts from 6 studies were included in the meta-analysis.Our meta-analysis revealed an association between the MBOAT7 SNP and HCC susceptibility in both the dominant[odds ratio(OR):1.14,95%confidence interval(95%CI):1.02-1.26,P=0.020]and recessive(OR:1.21,95%CI:1.05-1.39,P=0.008)models.Subgroup analysis revealed that stratification of the included patients by geographical origin showed a significant association in Asia(OR:1.20,95%CI:1.03-1.39).CONCLUSION This meta-analysis underscores the contribution of the MBOAT7 rs641738 SNP to hepatocarcinogenesis,especially in Asian populations,which warrants further investigation.

新补骨脂异黄酮通过caspase-3/GSDME通路诱导肝细胞癌Huh-7细胞焦亡

目的:探讨新补骨脂异黄酮(NBIF)对肝细胞癌(HCC)Huh-7细胞焦亡的影响及其分子机制。方法:体外培养Huh-7细胞,用CCK-8法检测不同浓度的NBIF处理48 h时对细胞存活率的影响,光学显微镜下观察NBIF处理后Huh-7细胞的形态变化,乳酸脱氢酶(LDH)释放实验检测细胞的LDH释放量,WB实验检测细胞中GSDME、caspase-3的蛋白水平变化。采用si RNA干扰Huh-7细胞中caspase-3、GSDME表达后,CCK-8法检测NBIF处理对细胞存活率的影响,WB实验检测GSDME蛋白表达水平,观察NBIF处理对细胞形态的影响,并检测细胞LDH释放量。结果:60μmol/L以上的NBIF均能显著抑制Huh-7细胞的增殖(均P<0.01),光学显微镜下观察到NBIF处理后的细胞出现肿胀、吐泡现象,且LDH释放增加(P<0.01);WB实验结果表明,NBIF能够激活caspase-3蛋白并切割GSDME蛋白,增加GSDME-N的表达(均P<0.01)。干扰caspase-3、GSDME表达后,NBIF对细胞的抑制作用减弱(均P<0.01),GSDME-N蛋白表达受到抑制(P<0.01),显微镜下细胞肿胀、吐泡现象几乎消失,LDH释放明显减少(P<0.05)。结论:NBIF能够通过caspase-3/GSDME途径诱导Huh-7细胞发生焦亡,从而抑制HCC细胞的增殖,为HCC的治疗提供一种新思路。

8-bromo-7-methoxychrysin-induced apoptosis of hepatocellular carcinoma cells involves ROS and JNK

AIM:To investigate whether the apoptotic activities of 8-bromo-7-methoxychrysin(BrMC) involve reactive oxygen species(ROS) generation and c-Jun N-terminal kinase(JNK) activation in human hepatocellular carcinoma cells(HCC).METHODS:HepG2,Bel-7402 and L-02 cell lines were cultured in vitro and the apoptotic effects of BrMC were evaluated by flow cytometry(FCM) after propidium iodide(PI) staining,caspase-3 activity using enzymelinked immunosorbent assay(ELISA),and DNA agarose gel electrophoresis.ROS production was evaluated by FCM after dichlorodihydrofluorescein diacetate(DCHFDA) probe labeling.The phosphorylation level of JNK and c-Jun protein was analyzed by Western blotting.RESULTS:FCM after PI staining showed a dose-dependent increase in the percentage of the sub-G1 cell pop-ulation(P < 0.05),reaching 39.0% ± 2.8% of HepG2 cells after 48 h of treatment with BrMC at 10 μmol/L.The potency of BrMC to HepG2 and Bel-7402(32.1% ± 2.6%) cells was found to be more effective than the lead compound,chrysin(16.2% ± 1.6% for HepG2 cells and 11.0% ± 1.3% for Bel-7402 cell) at 40 μmol/L and similar to 5-flurouracil(33.0% ± 2.1% for HepG2 cells and 29.3% ± 2.3% for Bel-7402 cells) at 10 μmol/L.BrMC had little effect on human embryo liver L-02 cells,with the percentage of sub-G1 cell population 5.4% ± 1.8%.Treatment of HepG2 cells with BrMC for 48 h also increased the levels of active caspase-3,in a concentration-dependent manner.z-DEVD-fmk,a caspase-3specific inhibitor,prevented the activation of caspase-3.Treatment with BrMC at 10 μmol/L for 48 h resulted in the formation of a DNA ladder.Treatment of cells with BrMC(10 μmol/L) increased mean fluorescence intensity of DCHF-DA in HepG2 cells from 7.2 ± 1.12 at 0 h to 79.8 ± 3.9 at 3 h and 89.7 ± 4.7 at 6 h.BrMC did not affect ROS generation in L-02 cells.BrMC treatment failed to induce cell death and caspase-3 activation in HepG2 cells pretreated with N-acetylcysteine(10 mmol/L).In addition,in HepG2 cells treated with BrMC(2.5,5.0,10.0 μmol/L) for 12 h,JNK activation was observed.Peak JNK activation occurred at 12 h post-treatment and this activation persisted for up to 24 h.The expression of phosphorylated JNK and c-Jun protein after 12 h with BrMC-treated cells was inhibited by N-acetylcysteine and SP600125 pre-treatment,but GW9662 had no effect.SP600125 substantially reduced BrMC-induced cell death and caspase-3 activation of HepG2 cells.N-acetylcysteine and GW9662 also attenuated induction of cell death and caspase-3 activation in HepG2 cells treated with BrMC.CONCLUSION:BrMC induces apoptosis of HCC cells by ROS generation and sustained JNK activation.

Oncolytic adenovirus-mediated MDA-7/IL-24 overexpression enhances antitumor activity in hepatocellular carcinoma cell lines

BACKGROUND: Melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24 (IL-24) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells. We investigated the effect of the replication-competent oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24, both expressing human MDA-7/IL-24 on the hepatocellular carcinoma cell lines HepG2, Hep3B, SMMC-7721, HCCLM3, and the normal liver cell line L02. METHODS: Hepatocellular carcinoma cell lines and the normal liver cell line were infected with SG600-IL24 and Ad.IL-24. The mRNA and protein expression of MDA-7/IL-24 in infected cells was confirmed by RT-PCR, ELISA, and Western blotting. MTT assay was used to investigate the proliferation effect. Hoechst staining and Annexin-V and PI staining were performed to study the MDA-7/IL-24 gene expressed in HCC cell lines and the normal liver cell line. Flow cytometry was used to analyse the cell cycle. RESULTS: RT-PCR, ELISA and Western blotting confirmed that the exogenous MDA-7/IL-24 gene was highly expressed in cells infected with SG600-IL24. MTT and apoptosis detection indicated that SG600-IL24 induced growth suppression, promoted apoptosis, and blocked cancer cell lines in the G2/M phase in hepatocellular carcinoma cell lines but not in the normal liver cell line. CONCLUSIONS: SG600-IL24 selectively induces growth suppression and apoptosis in hepatocellular carcinoma cell lines in vitro but not in the normal liver cell line L02. Compared with Ad.IL-24, SG600-IL24 dramatically enhances antitumor activity in hepatocellular carcinoma cell lines. (Hepatobiliary Pancreat Dis Int 2010; 9:615-621)

Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B

BACKGROUND: Melanoma differentiation associated gene-7 (mda-7) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of various intracellular signaling pathways. In this study, we investigated the potential effect of mda-7 on human hepatocellular carcinoma (HCC) in vitro. METHODS: Cells from the human HCC cell line Hep3B and the human liver cell line L-02 were assigned to three groups. One was cultured in Dulbecco's modified Eagle's medium without serum (control). The others were transfected with adenovirus expressing the mda-7 gene (Ad.mda-7) or adenovirus vector serving as negative control (Ad.vec). The expression of MDA-7 and Bcl-2 proteins in Hep3B and L-02 cells was confirmed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The methyl thiazolyl tetrazolium colorimetric assay and flow cytometry were used to assess tumor cell proliferation and the cell cycle. Hoechst and Annexin-V/propidium iodide staining were used to study mda-7 gene expression in Hep3B and L-02 cells. The expression of MDA-7, Bcl-2 and Bax proteins were detected by Western blotting. RESULT S: The mda-7 gene was expressed in Hep3B and L-02 cells. The protein concentrations of MDA-7 in supernatants were 790 and 810 pg/ml, respectively. mda-7 induced Hep3B growth suppression and apoptosis, compared with Ad.mda-7 and control (P<0.01). In addition, cell block in G2/M was identified by exposure of HCC cells to secreted MDA-7 protein, but this was not found in L-02. The gene expression of Bcl-2 was markedly decreased in Hep3B but not in L-02. CONCLUSIONS: mda-7 selectively induces growth inhibition and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the antiapoptosis protein Bcl-2. It could be an ideal gene for gene therapy in HCC.

Expression of B7-H4 and hepatitis B virus X in hepatitis B virus-related hepatocellular carcinoma

AIM: To investigate the expression and clinical significance of B7-H4 and hepatitis B virus X(HBx) protein in hepatitis B virus-related hepatocellular carcinoma(HBV-HCC).METHODS: The expression of B7-H4 in the human HCC cell lines Hep G2 and Hep G2.2.15 were detected by western blot, flow cytometry, and immunofluorescence. The expression of B7-H4 and HBx in 83 HBV-HCC was detected by immunohistochemistry, and the relationship with clinicopathological features was analyzed. Paraffin sections were generated from 83 HBV-HCC patients(22 females and 61 males) enrolled in this study. The age of these patients ranged from 35 to 77 years, with an average of 52.5 ± 11.3 years. All experiments were approved by the Ethics Committees of the Second Affiliated Hospital, Zhejiang University School of Medicine.RESULTS: B7-H4 was significantly upregulated in Hep G2.2.15 cells compared to Hep G2 cells. Specifically, the protein expression of B7-H4 in the lysates of Hep G2 cells was more than that in Hep G2.2.15 cells. In addition, HBx was expressed only in Hep G2.2.15 cells. Similar data were obtained by flow cytometry. The positive rates of B7-H4 and HBx in the tissues of 83 HBV-HCC patients were 68.67%(57/83) and 59.04%(49/83), respectively. The expression of HBx was correlated with tumor node metastases(TNM) stage, and the expression of B7-H4 was positively correlated with HBx(rs = 0.388; p < 0.01). The expression level of B7-H4 in HBx-positive HBV-HCC tissues was substantially higher than that in HBx-negative HBV-HCC tissues. The expression level of B7H4 was negatively related to tumor TNM stage.CONCLUSION: Higher expression of HBx and B7-H4 was correlated with tumor progression of HBV-HCC, suggesting that B7-H4 may be involved in facilitating HBV-related hepatocarcinogenesis.

富丝氨酸/精氨酸剪接因子7的表达在肝细胞癌中的意义

目的:探讨富丝氨酸/精氨酸剪接因子7(SRSF7)表达肝细胞癌(HCC)中的临床意义及生物学功能。方法:首先通过TCGA数据库中HCC转录组数据和临床数据分析SRSF7的表达水平、临床病理特征、功能和通路富集情况。再利用临床HCC样本、Huh7、Hep3B细胞株进行验证;将细胞分为对照组(control组)、过表达组(SRSF7OE组)和敲低组(SRSF7KD组)。细胞计数试剂盒(CCK-8)实验、TUNEL实验检测其增殖和凋亡能力;伤口愈合实验、Transwell迁移实验和Transwell侵袭实验检测其迁移和侵袭能力;蛋白质免疫印迹法(western blotting)实验检测上皮—间质转化(EMT)相关标志物的蛋白水平;实时荧光定量PCR(RT-qPCR)实验检测SRSF7下游调控基因的mRNA水平。结果:生信分析结果显示,SRSF7在HCC肿瘤组织中呈高表达并且与肿瘤分级、分期和患者预后不良有关(P<0.05)。Western blotting和免疫组化结果显示,SRSF7在肿瘤组织中呈高表达(P<0.05)。与对照组相比,过表达SRSF7能促进HCC细胞增殖、迁移侵袭以及EMT进程;敲低SRSF7能抑制HCC细胞增殖、促进细胞凋亡,抑制HCC细胞迁移侵袭和EMT进程(P<0.05)。结论:SRSF7在HCC中呈高表达,其高表达可能通过调控PAK5、RTL1等下游靶基因的表达来促进HCC细胞增殖,抑制细胞凋亡,并通过促进EMT进程来促进细胞迁移和侵袭,进而促进HCC的进展。

Melanoma differentiation-associated gene-7, MDA-7/IL-24, selectively induces growth suppression, apoptosis in human hepatocellular carcinoma cell line HepG2 by replication-incompetent adenovirus vector

瞄准：调查在人的肝细胞在肿瘤生长和 apoptosis 上表示 MDA-7/IL-24 的复制无能的侵入人体气管粘膜的病菌向量的效果癌(HCC ) 房间线 HepG2 和正常的肝房间衬里 L02。方法：我们构造了 recombinant 复制无能的 Ad.mda-7 病毒向量并且感染了它进人的 HCC 房间线 HepG2 和正常的肝房间线 L02。RT-PCR 被执行检测在房间表示的 mRNA。被 ELISA 用来在文化上层清液检测 MDA-7/IL-24 蛋白质表示。Ad.mda-7 导致的 apoptosis 的效果被 Hoechst 与染色的 Annexin-V 和 PI 染色和流动血细胞计数试金证实。MTT 试金被用来决定 HepG2 细胞的生长抑制，并且房间周期和 hypodiploidy 分析被流动血细胞计数执行。结果：Recombinant 复制有缺陷者病毒表示 MDA-7/IL-24 成功地被构造。RT-PCR 证明 Ad.mda-7 能调停进 HepG2 和 L02 的外长的基因 MDA-7/IL-24 的表示。在上层清液的 MDA-7/IL-24 蛋白质的集中分别地在 Ad.mda-7-infected L02 和 HepG2 房间是 130 pg/mL 和 110 pg/mL。 Ad.mda-7 感染显然导致了 apoptosis (从2.60%+/-0.72%到33.6%+/-13.2%， P=0.00012 )并且在 HepG2 (抑制比率IR=68%)的生长抑制和在房间的 G2/M 阶段的特定的癌症房间类型的百分比的增加骑车(从6.44%～32.29%， P【0.01 )，然而并非在 L02 房间。结论：这些结果有选择地在人的肝细胞癌细胞线 HepG2 与复制无能的侵入人体气管粘膜的病菌向量由 mda-7/IL-24 基因证实 apoptosis 和生长抑制的正式就职。

Polymorphisms of UGT1A7 and XRCC1 are Associated with an Increased Risk of Hepatocellular Carcinoma in Northeast China

Objective： Hepatocellular carcinoma （HCC） is a complex disease which associates with both environmental and genetic factors. The purpose of this study was to investigate whether the genetic polymorphisms of UDP-glucuronosyltransferase（UGT1A7）, an important phase II biotransformation enzyme, and X-ray repair cross-complementing group 1（XRCC1）, a pivotal DNA-repair gene, were related to the risk of HCC in Northeast China. Methods： One hundred and thirty six HCC patients and one hundred and thirty six frequency-matched controls were included in this hospital-based case-control study. Genotypes of UGT1A7 and XRCC1 were determined using allele-specific polymerase chain reaction （AS-PCR） and PCR-restriction fragment length polymorphism （RFLP）, and for which the odds ratio （OR） with 95% confidence interval （95% CI） were calculated. Results： The proportion of UGT1A7 low enzymatic allele （＊2 or ＊3） was higher in HCC patients than those in controls. The UGT1A7＊1/＊2 and ＊3/＊3 genotypes were associated with higher HCC risk （OR=2.09, 95%CI： 1.10-3.97; OR=5.67, 95%CI： 1.76-18.30, respectively）. The XRCC1 codon 399 Arg/Gln genotype could also elevate HCC risk （OR=2.16, 95% CI 1.29-3.61）. In addition to polymorphisms of UGT1A7 and XRCC1, multivariate logistic regression analysis demonstrated that other significant independent factors associated with HCC were HBV infection （OR=68.07, 95%CI： 28.03-165.26）, HCV infection （OR=30.97, 95%CI： 8.06-118.94） and family history of HCC （OR=10.62, 95%CI： 2.22-50.77）. Conclusion： The study shows that the polymorphisms of UGT1A7 and XRCC1 are associated with HCC risk. Determination of the polymorphisms of UGT1A7 and XRCC1 may provide an important clue to preventive measure against HCC.

FBW7 increases chemosensitivity in hepatocellular carcinoma cells through suppression of epithelial-mesenchymal transition

BACKGROUND: FBW7 is a tumor suppressor which regulates a network of proteins with central roles in cell division, cell growth and differentiation. This study aimed to evaluate the role of FBW7in chemosensitivity and epithelial-mesenchymal transition (EMT) in different hepatocellular carcinoma (HCC) cell lines and to investigate the relevant underlying mechanisms.METHODS: Different human HCC cell lines (Hep3B, Huh-7,and SNU-449) were cultured. The cell viability was evaluated by cell counting kit-8, and FBW7 mRNA transcription and protein expression were quantitated by real-time PCR and Western blotting. Expressions of vimentin (mesenchymal biomarker)and E-cadherin (epithelial biomarker) were evaluated by Western blotting and immunocytochemistry. Cell invasion was assayed by Transwell migration, and FBW7 plasmid or siRNA was used to evaluate the effect of FBW7 overexpression or silencing on cell chemosensitivity.RESULTS: FBW7 expression affected tumor cell chemosensitivity to doxorubicin and tumor cell invasive capacity in different HCC cell lines. FBW7hi (high FBW7 expression) Hep3B and FBW7mi (median FBW7 expression) Huh-7 cells were more sensitive to doxorubicin and lower in invasive capacity than FBW7lo (low FBW7 expression) SNU-449 cells. Silencing of FBW7in Huh-7 and Hep3B cells induced the resistance to doxorubicin and enhanced cell invasion, whereas overexpression of FBW7in SNU-449 cells restored the sensitivity to doxorubicin andsignificantly reduced invasive capacity. Furthermore, doxorubicin induced EMT toward mesenchyme in HCC cells. Downregulation of FBW7 in Huh-7 and Hep3B cells or upregulation of FBW7 in SNU-449 cells altered the direction of EMT.CONCLUSIONS: The level of FBW7 expression impacted the tumor resistance to doxorubicin and the invasion capability of HCC cells. FBW7 therefore may be a potential target for the chemotherapy of HCC through the regulation of EMT.

Recombinant adenovirus vector-mediated human MDA-7 gene transfection suppresses hepatocellular carcinoma growth in a mouse xenograft model

Hepatocellular carcinoma is one of the most common tumors in the world. The purpose of the present study was to investigate the inhibitory effects of adenoviral transduction of human melanoma differentiation-associated gene-7 （MDA-7） gene on hepatocellular carcinoma, so as to provide a theoretical basis for gene therapy of the disease. The human MDA-7 gene was cloned into replication-defective adenovirus specific to HepG2 cells us- ing recombinant virus technology. RT-PCR and Western blotting assays were used to determine the expression of human MDA-7 mRNA and MDA-7 protein in HepG2 cells in vitro. Induction of apoptosis by overexpression of the human MDA-7 gene was determined by flow cytometry. In-vivo efficacy of adenoviral delivery of the hu- man MDA-7 gene was assessed in nude mice beating HepG2 cell lines in vivo by determining inhibition of tumor growth, VEGF and CD34 expression, and microvascular density （MVD）. The results showed that AdGFP/MDA- 7 induced apoptosis of HepG2 cells in vitro and significantly inhibited tumor growth in vivo （P 〈 0.05）. The intra- tumoral MVD decreased significantly in the treated tumors （P 〈 0.05）. We conclude the recombination adenovirus AdGFP/MDA-7 can effectively express biologically active human MDA-7, which leads to inhibition of hepatocel- lular carcinoma growth.

金盏花苷E通过自噬途径下调GPX4和SLC7A11抑制肝癌细胞的增殖和迁移

目的探讨金盏花苷E抑制肝癌细胞增殖和迁移的分子机制。方法金盏花苷E处理肝癌细胞,CCK-8检测细胞活力;Western blotting检测GPX4、SLC7A11、LC3、P62的表达以及Akt/mTOR的磷酸化。自噬抑制剂LY294002和激活剂Rapamycin与金盏花苷E联合处理肝癌细胞,EdU和Transwell实验分别检测肝癌细胞的增殖和迁移能力。TCGA数据库分析GPX4和SLC7A11在肝癌及正常肝组织中的表达水平,以及与肝癌患者存活之间的关系。Western blotting和qPCR分别检测GPX4和SLC7A11在肝癌细胞和正常肝细胞中的表达水平。结果金盏花苷E能够显著抑制肝癌细胞的存活(P<0.05);GPX4和SLC7A11在肝癌组织和肝癌细胞中均显著高表达;GPX4和SLC7A11的表达与肝癌患者存活呈显著负相关(P<0.001);金盏花苷E显著抑制GPX4和SLC7A11蛋白的表达,激活Akt-mTOR通路,增强LC3Ⅱ的表达(P<0.01);自噬激活剂Rapamycin显著增强金盏花苷E对GPX4和SLC7A11的抑制作用,而自噬抑制剂LY294002则明显逆转了金盏花苷E对GPX4和SLC7A11的抑制作用(P<0.05);抑制自噬途径能够逆转金盏花苷E对肝癌细胞增殖和迁移的抑制作用,增强自噬途径则发生相反的变化(P<0.01)。结论金盏花苷E经自噬途径下调GPX4和SLC7A11抑制肝癌细胞的增殖和迁移。

Clinical significance of serum sB7-H3 and IL-1 in patients with hepatitis B related hepatocellular carcinoma

Objective: To explore the clinical significance of serum sB7-H3 and IL-1 in patients with hepatitis B associated hepatocellular carcinoma. Methods: 122 cases of patients with hepatitis B related liver diseases admitted to our hospital from January 2015 to June 2018 were selected, including 47 cases of patients with hepatitis B related hepatocellular carcinoma, 41 cases of patients with hepatitis B related cirrhosis and 34 cases of patients with hepatitis B virus, and 45 cases of healthy persons in the same period as normal control group. The serum sB7-H3, IL-1α, IL-1β levels were compared among the four groups. The relationship between serum sB7-H3, IL-1α, IL-1β and clinicopathological characteristics was analyzed, and the correlation of sB7-H3, IL-1α and IL-1β was analyzed by Pearson method. The efficacy of serum sB7-H3, IL-1α, IL-1β in early diagnosis and prognostic evaluation for hepatitis B associated hepatocellular carcinoma were analyzed by ROC and Logistic regression analysis. Results: The serum sB7-H3, IL-1α, IL-1β levels in the four groups were in order from high to low: hepatitis B related hepatocellular carcinoma>hepatitis B related cirrhosis>hepatitis B virus>normal control group, all above had statistical difference (P<0.05). The high levels of serum sB7-H3, IL-1α, IL-1β were significantly correlated with TNM stage, alpha fetoprotein level and lymph node metastasis (P<0.05). Spearman correlation analysis results showed that the serum level of sB7-H3 was positively correlated with IL-1α and IL-1β(r=0.837, 0.756;P<0.05), the serum level of IL-1α was positively correlated with IL-1β(r=0.734, P<0.05). The ROC curve and Logistic regression analysis showed that the AUC of sB7-H3, IL-1α, IL-1β and combined detection for the diagnosis of hepatitis B associated hepatocellular carcinoma was 0.893, 0.887, 0.881, 0.961 (P<0.05), respectively;the AUC of sB7-H3, IL-1α, IL-1β and combined detection for the prognostic evaluation for hepatitis B associated hepatocellular carcinoma was 0.843, 0.837, 0.834, 0.917, respectively. Conclusion:Serum sB7-H3, IL-1α, IL-1β levels could all be used for the early diagnosis and prognostic evaluation of hepatitis B associated hepatocellular carcinoma, and the combined detection is more effective, which has important clinical significance.

FBXW7/MCM7信号轴介导肝癌细胞增殖的研究

目的 探讨FBXW7靶向调控MCM7表达对肝癌细胞增殖的影响。方法 采用免疫共沉淀法(CoIP)和免疫荧光染色法(IF),分析肝癌细胞系MHCC-97H中FBXW7与MCM7的互作关系;通过慢病毒感染过表达FBXW7和(或) MCM7,小干扰RNA技术下调FBXW7和(或) MCM7的表达;采用Western blot检测FBXW7对MCM7蛋白表达的影响,CCK-8实验检测肝癌细胞的增殖活性,EdU细胞增殖检测分析肝癌细胞的增殖能力,平板克隆实验分析肝癌细胞的集落形成能力。结果 在MHCC-97H细胞中,FBXW7与MCM7存在互作关系:过表达FBXW7抑制了MCM7的蛋白表达水平、并抑制了肝癌MHCC-97H细胞的增殖(P <0.05),在过表达FBXW7的基础上增加MCM7的表达后,细胞增殖能力提高(P <0.05);下调FBXW7后细胞中MCM7表达增加、细胞增殖能力增强(P <0.05),而下调FBXW7的同时干扰MCM7表达后,细胞增殖能力减弱(P <0.05)。结论 FBXW7能抑制肝癌细胞增殖,其机制与负向调节肝癌细胞中促癌蛋白MCM7的表达水平有关。

超声多模态检查联合血清Egfl7、VEGF、OPN对肝癌微血管侵犯的诊断价值及预后价值分析

目的探讨与分析超声多模态检查联合血清表皮生长因子样结构域蛋白7(epidermal growth factor-like domain protein 7,EGFL7)、血管内皮生长因子(vascular endothelial growth factor,VEGF)、骨桥蛋白(osteopontin,OPN)对肝癌微血管侵犯的诊断价值及预后价值。方法2018年2月到2021年5月选择在本院诊治的肝癌患者78例作为研究对象,所有患者都给予超声多模态检查联合血清Egfl7、VEGF、OPN检测,同时给予手术病理检查,判断微血管侵犯状况,随访患者的预后并进行预测价值分析。结果78例患者中检出微血管侵犯阳性28例,占比35.9%。阳性组的肝癌血管分级、血管分布与阴性组对比有明显差异(P<0.05)。阳性组的血清Egfl7、VEGF、OPN含量都明显高于阴性组(P<0.05)。所有患者随访到2022年10月1日,平均随访时间28.47±2.10个月,死亡22例,死亡率为78.6%。多因素Logistic回归分析显示微血管侵犯、血管分级、血管分布与血清Egfl7、VEGF、OPN含量都为影响患者随访死亡的重要因素(P<0.05)。结论肝癌微血管侵犯患者多表现为超声多模态血管分级程度高与混合血流状况,伴随有血清Egfl7、VEGF、OPN的高表达,超声与联合血清Egfl7、VEGF、OPN对肝癌微血管侵犯的诊断预后预测都有很好的价值。

靶向CD24分子3E10增强肝癌HuH-7细胞的化疗敏感度

目的探讨靶向分子CD24单克隆抗体3E10调节肝癌HuH-7细胞对化疗药顺铂敏感度的作用及其分子机制。方法 Western blot和流式细胞术检测抗CD24抗体3E10的特异性;Annexin V-FITC/PI双染色检测细胞凋亡;Real-time PCR和Western blot检测耐药基因和肿瘤干细胞特性基因的表达水平;Western blot检测JAK/STAT3和PI3K/AKT信号通路的活性水平。结果 3E10可有效识别CD24蛋白及HuH-7细胞。3E10显著增强HuH-7细胞对顺铂的敏感度(P<0.05),抑制率由(10.3±3.0)%提高至(34.4±10.8)%。3E10显著降低HuH-7细胞耐药基因ABCB1、ABCB5、ABCC1和肿瘤干性基因NANOG、CD24的表达水平及干性基因β-catenin的磷酸化水平(P<0.05)。3E10显著降低STAT3和AKT的磷酸化水平(P<0.05)。结论靶向CD24分子3E10通过降低HuH-7细胞的耐药性、肿瘤干性和JAK/STAT3、PI3K/AKT信号通路活性来增强HuH-7细胞对化疗药顺铂的敏感度。

右美托咪定对人肝癌Huh-7细胞增殖和迁移的影响

目的探讨右美托咪定对人肝癌Huh-7细胞增殖和迁移的影响及其潜在机制。方法实验分成细胞和动物两部分。首先,将人肝癌Huh-7细胞随机分为6组:对照组(CON组)、0.1 nmol/L右美托咪定组(D1组)、1 nmol/L右美托咪定组(D2组)、10 nmol/L右美托咪定组(D3组)、氧糖剥夺/复氧复糖组(OGD/R组)、右美托咪定+氧糖剥夺/复氧复糖组(D+OGD/R组)。采用MTT比色法检测细胞活性,Western blot测定SIRT1的蛋白表达量;采用Transwell法检测细胞迁移能力。其次,将SPF级BALB/c-nu雄鼠6只,随机分为对照组(CON组)和右美托咪定组(DEX组)。皮下种植Huh-7细胞,DEX组裸鼠注射右美托咪定(5 mg/kg),CON组注射相应体积的生理盐水,连续6 d,种瘤30 d后处死裸鼠,取出瘤体,计算肿瘤体积。结果右美托咪定可以促进Huh-7细胞的细胞活力及迁移能力,减轻因OGD/R造成的损伤,同时增加SIRT1的表达。而右美托咪定对裸鼠肿瘤的体积无明显影响。结论右美托咪定可以增强人肝癌Huh-7细胞株的增殖和迁移能力,这种作用可能是通过上调SIRT1表达来完成。

延龄草总皂苷通过促进人β防御素2(HBD-2)的表达抑制HuH-7细胞侵袭和迁移

目的探讨延龄草总皂苷调控人β防御素2(HBD-2)对HuH-7细胞侵袭和迁移的影响和机制。方法采用(0.5、1.0、2.0、4.0)mg/L延龄草总皂苷处理HuH-7细胞,噻唑蓝(MTT)法检测细胞增殖,TranswellTM小室测定细胞侵袭和迁移能力,Western blot法检测细胞中HBD-2、基质金属蛋白酶2(MMP2)、MMP9的蛋白水平。将HBD-2小干扰RNA(siRNA)转染至HuH-7细胞中,用延龄草总皂苷处理,实时定量PCR和Western blot法验证干扰效果,TranswellTM法分别检测细胞侵袭和迁移能力。结果除0.5 mg/L延龄草总皂苷对HuH-7细胞增殖无影响外,其他各剂量延龄草总皂苷均可抑制HuH-7细胞增殖。(0.5、1.0)mg/L的延龄草总皂苷处理均可下调细胞MMP2、MMP9蛋白水平、增加HBD-2蛋白水平并抑制细胞侵袭和迁移。HBD-2 siRNA转染可明显降低HuH-7细胞HBD-2水平。下调HBD-2提高延龄草总皂苷处理的HuH-7细胞侵袭和迁移能力并增加细胞MMP2、MMP9蛋白水平。结论延龄草总皂苷通过促进HBD-2表达抑制HuH-7细胞侵袭和迁移。

miR-493-5p、miR-7、miR-24在肝癌组织中的表达及与预后的相关性分析

目的 探讨微RNA(microRNA,miRNA)-493-5p、miR-7、miR-24在肝癌组织中的表达情况及其与预后的相关性。方法 选取2017年5月至2018年5月在石家庄市中医院接受手术治疗的54例肝癌患者,采用RT-PCR检测肝癌组织及癌旁组织中miR-493-5p、miR-7、miR-24的表达水平,并分析其与肝癌患者预后的相关性。结果 与癌旁组织相比,肝癌组织中miR-493-5p、miR-7的表达水平明显降低,miR-24的表达水平明显升高,差异有显著性(P<0.05);与Ⅰ/Ⅱ期、中/高分化、无淋巴结转移的肝癌患者相比,Ⅲ/Ⅳ期、低分化、有淋巴结转移的肝癌患者癌组织中miR-493-5p、miR-7表达水平明显降低,miR-24表达水平明显升高,差异均有显著性(P<0.05)。相关分析结果显示,miR-493-5p、miR-7与肝癌患者的TNM分期、淋巴结转移呈负相关,与分化程度呈正相关,而miR-24与肝癌患者的TNM分期、淋巴结转移呈正相关,与分化程度呈负相关。死亡组患者miR-493-5p、miR-7表达水平低于生存组,miR-24表达水平高于生存组(P<0.05)。多因素回归分析显示,miR-24为肝癌患者3年内死亡的独立危险因素,而miR-493-5p、miR-7为其独立保护因素(P<0.05)。结论 miR-493-5p、miR-7、miR-24在肝癌组织中呈异常表达,并与肝癌的临床病理特征相关,有望用于肝癌患者的预后评估。