Linking fatty liver diseases to hepatocellular carcinoma by hepatic stellate cells

Hepatic stellate cells(HSCs),a distinct category of non-parenchymal cells in the liver,are critical for liver homeostasis.In healthy livers,HSCs remain non-proliferative and quiescent.However,under conditions of acute or chronic liver damage,HSCs are activated and participate in the progression and regulation of liver diseases such as liver fibrosis,cirrhosis,and liver cancer.Fatty liver diseases(FLD),including nonalcoholic(NAFLD)and alcoholrelated(ALD),are common chronic inflammatory conditions of the liver.These diseases,often resulting from multiple metabolic disorders,can progress through a sequence of inflammation,fibrosis,and ultimately,cancer.In this review,we focused on the activation and regulatory mechanism of HSCs in the context of FLD.We summarized the molecular pathways of activated HSCs(aHSCs)in mediating FLD and their role in promoting liver tumor development from the perspectives of cell proliferation,invasion,metastasis,angiogenesis,immunosuppression,and chemo-resistance.We aimed to offer an in-depth discussion on the reciprocal regulatory interactions between FLD and HSC activation,providing new insights for researchers in this field.

Effects and Mechanism of Oxymatrine on Proliferation,Invasion and Migration of Hepatocellular Carcinoma Cells

[Objectives] To observe the effects of oxymatrine on the proliferation,invasion and migration of hepatocellular carcinoma cells,and to explore its possible molecular mechanism.[Methods]The hepatocellular carcinoma cell line Hep G2 was randomly divided into the negative control group and the low,medium and high concentration groups of oxymatrine.The negative control group was added to the cell culture medium,and the low,medium and high concentration groups of oxymatrine were added to the oxymatrine and cell culture medium with the final concentration of 1.0,2.0 and 4.0 mg/m L.The proliferation of each group was measured by MTT assay,and the inhibition rate of cell proliferation was calculated.The invasion and migration ability of each group was determined using Transwell chamber assay.The expression of E-cadherin and CD44 mRNA in each group was detected using Real-time PCR,while the expression of E-cadherin and CD44 protein was detected using Western blotting method.[Results] The inhibition rate of cell proliferation in the high concentration group of oxymatrine was higher than that in the medium and low concentration groups,and the inhibition rate of cell proliferation of medium concentration group was higher than that in the low concentration group.In the cell invasion and cell migration experiments,the number of transmembrane cells of low,medium and high concentration groups of oxymatrine was less than that in the negative control group(P < 0.05),the number of transmembrane cells of high concentration groups of oxymatrine was less than that in medium and low concentration groups,and the number of transmembrane cells of medium concentration group was less than in that of the low concentration group(P < 0.05).Compared with the negative control group,the expression of E-cadherin mRNA and protein increased in the low,medium and high concentration groups of oxymatrine,while the expression of CD44 mRNA and protein decreased(P < 0.05).[Conclusions] Oxymatrine has the effect of inhibiting the proliferation,invasion and migration of hepatocellular carcinoma cell line Hep G2 in vitro.The higher the concentration,the more significant the effect will be.The mechanism is possibly connected with the up-regulation of E-cadherin expression and down-regulation of CD44 expression.

Study on the mechanism of tRNA-ValAAC-5 in promoting the proliferation and migration of hepatocellular carcinoma cells

Objective:To demonstrate the role and mechanism of tRNA-ValAAC-5 expression in hepatocellular carcinoma(HCC)cells.Methods:The expression levels of tRNA-ValAAC-5 in HCC(Hep3B,HuH7,SNU398,Hep3G2)and human hepatocellular carcinoma(THLE2,THLE3)were detected by real-time PCR.HEP3B and Hep3G2 cells were respectively transfected with tRNA-ValAAC-5-inhibitor and tRNA-ValAAC-5-NC as the inhibitor group and the NC group.Then the ability of cell proliferation was detected by CCK-8 assay and the ability of invasion and metastasis was detected by Transwell assay.The protein expression levels of p21,Matrix metalloproteinase 2(MMP2)and Matrix metalloproteinase 9(MMP9)were determined by Western blot.Results:The relative expression of tRNA-ValAAC-5 in Hep3B,HuH7,SNU398 and Hep3G2 cells were significantly higher than THLE2 and THLE3 cells,the differences were statistically significant(P<0.05).After tRNA-ValAAC-5-inhibitor transfection,the expression of tRNA-ValAAC-5 in Hep3B and Hep3G2 cells were reduced than tRNA-ValAAC-NC group.Both of the differences were statistically significant(t=36.52,27.45,P<0.001),which indicated the transfection was successful.The proliferative ability of Hep3B and Hep3G2 cells transfected with tRNA-ValAAC-5-inhibitor after 24,48,72,96 h were inhibited effectively compared with tRNA-ValAAC-5-NC group.All of the differences were statistically significant in Hep3B(t=5.25,8.23,7.33,14.16,P<0.001)and Hep3G2(t=4.25,5.11,9.39,7.59,P<0.001)cells.The number of invasion and metastasis of Hep3B and Hep3G2 cells were reduced in tRNA-ValAAC-5-inhibitor group compared with tRNA-ValAAC-5-NC group,there was significant difference(t=14.01,21.85,P<0.001).The protein expression levels of P21 were lower,MMP2 and MMP9 were higher in tRNA-ValAAC-5-inhibitor group compared with tRNA-ValAAC-5-NC group,the differences were statistically significant in Hep3B(t=8.96,12.80,4.652,P<0.001)cells and Hep3G2(t=15.17,22.36,12.61,P<0.001)cells.Conclusion:tRNA-ValAAC-5 can effectively promote the proliferation,invasion and metastasis of HCC,and its possible mechanism is related to regulating the expression of p21,MMP2 and MMP9.

Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis

AIM To investigate the effect of adipose-derived mesenchymal stem cells(ADMSCs)and their conditioned media(CM) on hepatocellular carcinoma(HCC) cell tumorigenesis.METHODS The proliferation rate of HepG2 and PLC-PRF-5 HCC cancer cells was measured using the trypan blue exclusion method and confirmed using the cell-counting kit8(commonly known as CCK-8) assay. Apoptosis was detected by flow cytometry using annexin V-FITC. Protein and mRNA expression was quantified by ELISA and real time PCR, respectively. Migration and invasion rates were performed by Transwell migration and invasion assays. Wound healing was examined to confirm the data obtained from the migration assays.RESULTS Our data demonstrated that when co-culturing HCC cell lines with ADMSCs or treating them with ADMSC CM, the HCC cell proliferation rate was significantly inhibited and the apoptosis rate increased. The decreased proliferation rate was accompanied by an upregulation of P53 and Retinoblastoma mRNA and a downregulation of c-Myc and hTERT mRNA levels. More notably, ADMSCs and their CM suppressed the expression of the two important markers of HCC carcinogenicity, alpha-fetoprotein and Des-gamma-carboxyprothrombin. In addition, the migration and invasion levels of HepG2 and PLC-PRF-5 cells significantly decreased, potentially through increased expression of the tissue inhibitor metalloproteinases TIMP-1, TIMP-2 and TIMP-3.CONCLUSION These findings shed new light on a protective and therapeutic role for ADMSCs and their CM in controlling HCC invasiveness and carcinogenesis.

Potential roles of EZH2, Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma cell line Hep3B

AIM: To investigate the potential roles of enhancer of zeste homolog2(EZH2), Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma(HCC) cell line Hep3 B.METHODS: A total of 73 patients who underwent surgical resection at Fuzong Clinical Medical College of Fujian Medical University were enrolled in this study. Hep3 B cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37?℃. Vectors that containing c DNA of the EZH2 gene or mi R-203 targeted sh RNA plasmid were constructed, and then transfected into Hep3 B cells. The m RNA expression of mi R-203, EZH2, and Bmi-1 was analyzed using quantitative real-time polymerase chain reaction analysis, and the protein levels of EZH2 and Bmi-1 were detected by Western blot analysis. Effect of EZH2 or mi R-203 on cell proliferation was observed by methyl thiazolyl tetrazolium assay, and cell apoptosis was assessed using flow cytometry. Besides, effect of EZH2 or mi R-203 on tumor cell invasion was detected using Transwell assay.RESULTS: The m RNA levels of EZH2 and Bmi-1 in HCC tissues and in Hep3 B cells were significantly higher compared with those in normal samples(P < 0.01), while mi R-203 level was significantly lower in HCC tissues(P < 0.01). Hep3 B cells transfected with EZH2-sh RNA or mi R-203-sh RNA showed lower expression levels of EZH2 and Bmi-1(P < 0.05). Compared with controls, Hep3 B cells transfected with EZH2-sh RNA had relative slow cell proliferation, indicating that low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could inhibit Hep3 B cell proliferation(P < 0.05). The average apoptosis rate of Hep3 B cells transfected with EZH2-sh RNA vector was about 18.631%, while that of Hep3 B cells transfected with sh RNA vector was about 5.33%, suggesting that EZH2 was down-regulated by transfecting with EZH2-sh RNA, and the down-regulated EZH2 contributed to the cell apoptosis. Low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could reduce Hep3 B cell invasion(P < 0.05).CONCLUSION: Our study suggests that EZH2 and Bmi-1 are up-regulated while mi R-203 is downregulated in Hep3 B cells. Mi R-203 may contribute to the metastasis and enhance apoptosis of HCC cells by regulating EZH2 and Bmi-1. Our study may provide a theoretical basis for metastasis of HCC and targeted therapy of HCC.

Construction,identification and biological feature study of human hepatocellular carcinoma cells stably expressing HBeAg

Objective: To construct a HepG2 cell line which stably expressing Hepatitis B e antigen(HBeAg)and investigate the effects of HBeAg on the proliferation,migration and invasion of HepG2 cells. Methods: The lentivirus carrying HBeAg gene was constructed and packaged. HepG2 cells were infected with the lentivirus and screened with puromycin to obtain HepG2 cells which stably expressing HBeAg(HepG2-HBeAg cells).The expression levels of HBeAg mRNA and protein were detected by RT-qPCR and Western blot,respectively. The content of HBeAg secretion in cell supernatant in both HepG2-HBeAg cells and control cells(HepG2-NC cells and HepG2 cells)were detected by IFMA assay. Furthermore,CCK-8 proliferation assay,colony formation assay and transwell migration and invasion assays were conducted to compare the abilities of cell proliferation,migration and invasion,respectively. Results: The expression of HBeAg in the HepG2-HBeAg cell was significantly higher than those in HepG2 cells and HepG2-NC cells. Secreted HBeAg in the supernatant of HepG2-HBeAg cells was 26. 33±2. 13 PEIU/mL but was undetectable in supernatant of control cells. The proliferation,migration and invasion were all significantly lower in HepG2-HBeAg cells compared to control cells(P<0. 01). Conclusion: A HepG2 cell line which stably expressing HBeAg was constructed successfully,and the over-expression of HBeAg could attenuate the proliferation,migration and invasion of hepatocellular carcinoma cells.

Cancer-associated fibroblasts in hepatocellular carcinoma

The hepatic stellate cells in the liver are stimulated sustainably by chronic injury of the hepatocytes, activating myofibroblasts, which produce abundant collagen. Myofibroblasts are the major source of extracellular proteins during fibrogenesis, and may directly, or secreted products, contribute to carcinogenesis and tumor progression. Cancer-associated fibroblasts(CAFs) are one of the components of the tumor microenvironment that promote the proliferation and invasion of cancer cells by secreting various growth factors and cytokines. CAFs crosstalk with cancer cells stimulates tumor progression by creating a favorable microenvironment for progression, invasion, and metastasis through the epithelial-mesenchymal transition. Basic studies on CAFs have advanced, and the role of CAFs in tumors has been elucidated. In particular, for hepatocellular carcinoma, carcinogenesis from cirrhosis is a known fact, and participation of CAFs in carcinogenesis is supported. In this review, we discuss the current literature on the role of CAFs and CAF-related signaling in carcinogenesis, crosstalk with cancer cells, immunosuppressive effects, angiogenesis, therapeutic targets, and resistance to chemotherapy. The role of CAFs is important in cancer initiation and progression. CAF-targeted therapy may be effective for suppression not only of fibrosis but also cancer progression.

Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97

AIM: To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS: Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, and biological characteristics of the target clones selected by in vivo screening were studied. RESULTS: Two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potential were isolated from the parent cell line. Compared with MHCC97-L, MHCC97-H had smaller cell size (average cell diameter 43 microm vs 50 microm) and faster in vitro and in vivo growth rate (tumor cell doubling time was 34.2h vs 60.0h). The main ranges of chromosomes were 55-58 in MHCC97-H and 57-62 in MHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was (37.5 +/- 11.0) cells/field for MHCC97-H vs (17.7 +/- 6.3)/field for MHCC97-L. The proportions of cells in G0-G1 phase, S phase, and G2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65, 0.28/0.25 and 0.16/0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5wk after orthotopic implantation of tumor tissue were (246 +/- 66) microg.L(-1) for MHCC97-H and (91 +/- 66) microg.L(-1) for MHCC97-L. The pulmonary metastatic rate was 100% (10/10) vs 40% (4/10). CONCLUSION: Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.

Overexpression of DNA methyltransferase 1 and its biological significance in primary hepatocellular carcinoma

AIM： To explore the relationship between DNA methyltransferase 1 （DNMT1） and hepatitis B virus （HBV）-related hepatocellular carcinoma （HCC） and its biological significance in primary HCC. METHODS： We carried out an immunohistochemical examination of DNMT1 in both HCC and paired nonneoplastic liver tissues from Chinese subjects. DNMT1 mRNA was further examined in HCC cell lines by real-time PCR. We inhibited DNMT1 using siRNA and detected the effect of depletion of DNMT1 on cell proliferation ability and cell apoptosis in the HCC celt line SMMC-7721. RESULTS： DNMT1 protein expression was increased in HCCs compared to histologically normal nonneoplastic liver tissues and the incidence of DNMT1 immunoreactivity in HCCs correlated significantly with poor tumor differentiation （P = 0.014）. There were more cases with DNMT1 overexpression in HCC with HBV （42.85%） than in HCC without HBV （28.57%）. However, no significant difference in DNMT1 expression was found in HBV-positive and HBV-negative cases in the Chinese HCC group. There was a trend that DNMT1 RNA expression increased more in HCC cell lines than in pericarcinoma cell lines and normal liver cell lines. In addition, we inhibited DNMT1 using siRNA in the SMMC-7721 HCC cell line and found depletion of DNMT1 suppressed cells growth independent of expression of proliferating cell nuclear antigen （PCNA）, even in HCC cell lines where DNMT1 was stably decreased. CONCLUSION： The findings implied that DNMT1 plays a key role in HBV-retated hepatocellular tumorigenesis. Depletion of DNMT1 mediates growth suppression in SMMC-7721 cells.

人脐带间充质干细胞条件培养基及外泌体对肝癌细胞增殖、迁移、侵袭和凋亡的影响

背景:间充质干细胞可通过分泌含细胞因子、生长因子和外泌体的细胞外囊泡调节肿瘤微环境,对肿瘤细胞生物学行为进行精准调控。目的:研究人脐带间充质干细胞条件培养基及外泌体对肝癌细胞生物学特性的影响。方法:收集人脐带间充质干细胞培养上清,通过高速离心并过滤获得人脐带间充质干细胞条件培养基;收集人脐带间充质干细胞培养上清,通过超高速梯度离心法提取人脐带间充质干细胞外泌体。使用PKH26标记人脐带间充质干细胞外泌体,与肝癌细胞MHCC97-H共培养,荧光显微镜观察MHCC97-H细胞摄取外泌体情况。通过CCK-8增殖实验、Transwell迁移和侵袭实验以及流式凋亡实验评估人脐带间充质干细胞条件培养基、人脐带间充质干细胞外泌体对肝癌细胞生物学功能的影响。结果与结论:(1)人脐带间充质干细胞外泌体可被MHCC97-H细胞摄取且主要分布于细胞质中;(2)人脐带间充质干细胞条件培养基处理后,MHCC97-H细胞的增殖、迁移和侵袭能力显著增强(P<0.001,P<0.05,P<0.01),凋亡能力显著下降(P<0.001);而人脐带间充质干细胞外泌体处理后,MHCC97-H细胞的增殖能力下降(P<0.001),迁移、侵袭及凋亡能力显著增强(P<0.001);(3)结果表明,人脐带间充质干细胞条件培养基具有促进MHCC97-H细胞增殖、迁移、侵袭和抑制凋亡的能力;而人脐带间充质干细胞外泌体则具有促进MHCC97-H细胞迁移、侵袭及凋亡和抑制增殖的能力。

肝星状细胞通过胱硫醚γ-裂解酶/硫化氢(CSE/H2S)调控肝细胞癌细胞凋亡的作用及其机制

目的肝星状细胞(HSC)和硫化氢(H2S)信号分子作为肝细胞癌(HCC)微环境中重要的组分,参与调控HCC的发生发展等多种生物学进程。本研究通过HSC与肝癌细胞系共培养,探讨HSC通过分泌H2S参与调控肝癌细胞凋亡的作用及其机制。方法以HSC细胞系(LX-2)及肝癌细胞系(Hep G2、PLC/PRF/5)为研究对象。RT-q PCR和Western Blot(WB)法检测LX-2中H2S关键合成酶——胱硫醚γ-裂解酶(CSE)m RNA及表达水平;ELISA测定上清液LX-2产生的H2S浓度;二代测序、细胞免疫荧光、染色质免疫沉淀(Ch IP)及WB检测H2S(内源性和外源性)作用Hep G2和PLC/PRF/5细胞后,JNK/Jun B-TNFSF14信号通路基因、结合位点及相关蛋白。Transwell小室将LX-2分别与Hep G2和PLC/PRF/5共培养,CCK-8和流式细胞术检测肝癌细胞的细胞活力、凋亡,WB测定H2S-TNFSF14信号通路相关蛋白。所有细胞实验均重复3次。计量资料两组间比较采用成组t检验;多组间比较采用单因素方差分析或重复测量方差分析,进一步两两比较采用Dunnett-t检验。结果LX-2主要通过CSE合成H2S,LX-2培养上清液中H2S浓度随着时间延长逐渐增加[(22.89±0.08)pg/m L vs(28.29±0.15)pg/m L vs(36.19±1.90)pg/m L,F=79.63,P<0.05]。LX-2中CSE m RNA水平显著高于CBS m RNA和MPST m RNA(1.008±0.13 vs 0.320±0.014 vs 0.05±0.02,F=80.84,P<0.05)。当CSE被炔丙基甘氨酸(PPG)抑制之后,随着PPG浓度增加,H2S浓度下降(P<0.05)。LX-2分别与Hep G2、PLC/PRF/5共培养,随着培养时间延长,Hep G2(87.48%±0.82%vs70.48%±0.641%vs 52.89%±0.57%vs 45.20%±0.69%,F=1517.13,P<0.001)和PLC/PRF/5(92.41%±0.48%vs 74.10%±0.73%vs 53.70%±0.60%vs 44.00%±0.27%,F=2626.21,P<0.001)细胞活力降低;凋亡增加(Hep G2:12.88%±0.64%vs15.5%±0.16%vs 18.43%±0.37%vs 13.01%±0.58%,F=142.15,P<0.001;PLC/PRF/5:8.51±0.05 vs 12.80±0.33 vs 15.59±0.21 vs 10.72±0.30,F=676.40,P<0.001),第3天最显著。二代测序显示,内源性H2S(LX-2产生)和Na HS(外源性H2S供体)参与调控Hep G2中多种基因表达。Na HS和LX-2通过释放H2S,激活肝癌细胞中JNK/Jun B信号通路、上调凋亡基因TNFSF14表达,且p-Jun B与TNFSF14基因转录调控区域结合增加。结论在肝癌微环境中,HSC通过信号分子CSE/H2S,激活了肝癌细胞中JNK/Jun B信号通路,TNFSF14表达增加,从而促进了肝癌细胞凋亡。

Nlrp6过表达通过调控AMPK-Srebp1c轴抑制脂质合成抑制肝癌细胞的增殖

目的探讨Nlrp6通过调控脂质合成抑制肝细胞癌(HCC)进展的作用及其机制。方法通过分析癌症基因组图谱(TCGA)数据库中的RNA-seq数据和临床信息,评估Nlrp6在不同病理分级的HCC组织中的表达变化,并进行Kaplan-Meier生存分析及相关性分析。将腺病毒转染HepG2细胞过表达或敲低Nlrp6,使用棕榈酸(PA)构建肝脂肪变性模型。油红O染色评估Nlrp6对肝癌细胞HepG2脂质沉积的影响,CCK-8、Edu染色及克隆形成实验探讨Nlrp6对HepG2细胞增殖的影响,实时荧光定量PCR检测脂质合成相关基因的表达,Western blotting检测AMPK-Srebp1c轴相关蛋白的表达水平。使用肝脏特异性敲除Nlrp6小鼠,进行高脂饮食喂养24周,构建动物肝脂肪变性模型。取小鼠肝脏进行组织学染色,检测纤维化及肝癌标志物相关基因的表达,同时验证AMPK-Srebp1c信号通路的变化。结果生信分析结果显示在HCC患者肝脏组织中,Nlrp6的表达显著降低,且随着病理分级增加而进一步降低,同时Nlrp6的表达与患者生存期显著相关(P<0.0001)。细胞实验结果显示,Nlrp6过表达抑制了HepG2细胞的脂质沉积和细胞增殖,而Nlrp6敲低则产生相反效果(P<0.05)。q-PCR结果显示,Nlrp6可抑制脂质合成相关基因的表达(P<0.05),Western blotting结果表明过表达Nlrp6可促进AMPK的磷酸化,抑制调控脂质合成的转录因子Srebp1c的表达(P<0.05)。而Nlrp6敲低则抑制AMPK的磷酸化,促进Srebp1c的活化(P<0.05)。动物组织病理学染色结果表明,肝脏特异性敲除Nlrp6会促进肝脂肪变性及胶原沉积,q-PCR检测纤维化基因与染色结果一致(P<0.05),而肝癌标志物AFP的表达水平明显上升(P=0.06)。肝脏组织样本Western blotting结果证实Nlrp6的缺失会抑制AMPK的磷酸化,上调Srebp1c的表达(P<0.05)。结论Nlrp6通过AMPK-Srebp1c轴抑制脂质合成,其可能是抑制肝癌细胞增殖的重要途径之一,进而改善HCC的进展。

SRSF7对HepG2细胞增殖、迁移和侵袭的影响及其可能机制

目的:探讨富含丝氨酸/精氨酸剪接因子7(SRSF7)对肝细胞癌(HCC)细胞HepG2增殖、迁移和侵袭的影响及其可能机制。方法:通过癌症基因组图谱(TCGA)和Kaplan-Meier Plotter在线分析SRSF7在HCC和癌旁组织中的差异表达及其与患者预后的关系。常规培养HepG2细胞,用转染试剂将SRSF7 RNA敲减序列(siSRSF7#1和siSRSF7#2)、对照序列(NC)、SRSF7过表达载体(hSRSF7-oe)和对照载体(hSRSF7-nc)转染至HepG2细胞中,实验分为NC组、siSRSF7#1组、siSRSF7#2组、NC+hSRSF7-nc组、siSRSF7+hSRSF7-nc组和siSRSF7+hSRSF7-oe组。通过qPCR和WB法检测各组HepG2细胞中SRSF7 mRNA和蛋白的表达,MTS实验、平板克隆形成实验、划痕愈合实验、Transwell小室实验分别检测各组HepG2细胞的增殖、迁移和侵袭的能力。WB法检测各组HepG2细胞中JAK1/STAT3信号通路的相关蛋白的表达。结果:数据库数据分析显示SRSF7 mRNA在HCC组织中呈高表达(P<0.001),SRSF7 mRNA高表达与HCC患者不良预后有关联(P<0.05)。敲减SRSF7后,HepG2细胞的增殖、迁移和侵袭能力均显著下降(均P<0.01)。敲减SRSF7组细胞中JAK1和STAT3磷酸化水平显著降低(均P<0.05),同时过表达SRSF7后,JAK1和STAT3磷酸化水平又明显升高(均P<0.05)。结论:SRSF7在HCC组织中呈高表达,其可能通过调控JAK1/STAT3信号通路促进HepG2细胞的增殖、迁移和侵袭。

KIF2C对肝细胞癌细胞恶性生物学行为和人脐静脉内皮细胞血管生成的影响

目的:探究驱动蛋白-13家族2C(KIF2C)对肝细胞癌(HCC)细胞增殖、侵袭和迁移以及人脐静脉内皮细胞(HUVEC)血管生成的影响,为HCC治疗提供潜在靶点。方法:用数据库数据分析KIF2C mRNA和蛋白在HCC组织中的表达及其与血管生成相关因子(VEGFR2和HIF-1α)表达的相关性,常规培养人正常肝细胞QSG-7701、HUVEC和HCC细胞Huh-7、Hep3B2.1-7,用Lipofectamine 3000转染试剂将sh-NC和sh-KIF2C转染至Huh-7、Hep3B2.1-7细胞,qPCR检测各组QSG-7701、Huh-7和Hep3B2.1-7细胞中KIF2C mRNA的表达,WB法检测各组细胞中KIF2C蛋白的表达,细胞克隆形成实验检测敲低KIF2C对Hep3B2.1-7和Huh-7细胞克隆形成的影响,小管生成实验检测敲低KIF2C表达的Huh-7和Hep3B2.1-7细胞的条件培养液对HUVEC血管生成能力的影响。结果:数据库分析结果显示,KIF2C mRNA和蛋白在HCC组织中均呈高表达(均P<0.01),用qPCR和WB法检测人HCC中KIF2C mRNA和蛋白的表达水平,结果显示其mRNA和蛋白在各HCC细胞中也呈高表达(均P<0.01),与数据库数据分析结果相符。数据库数据分析还显示,KIF2C与HCC组织中VEGFR2、HIF-1α的表达水平呈正相关(P<0.05或P<0.01)。成功构建了稳定低表达KIF2C的Huh-7和Hep3B2.1-7细胞(均P<0.01),敲低KIF2C表达均可明显抑制Huh-7和Hep3B2.1-7细胞的增殖能力(均P<0.01)、侵袭和迁移能力(均P<0.01),敲低KIF2C表达的HCC细胞条件培养液均可显著抑制体外HUVEC的血管生成能力(P<0.05或P<0.01)。结论:KIF2C可促进Huh-7和Hep3B2.1-7细胞增殖、侵袭和迁移以及HUVEC血管生成的能力,提示KIF2C可能是治疗HCC的潜在靶点。

miR-542-5p通过靶向结合ENSA抑制肝细胞癌增殖、迁移和侵袭

目的探讨miR-542-5p靶向结合ENSA对肝细胞癌增殖、迁移和侵袭的影响。方法通过GSE36915数据矩阵分析miR-542-5p在肝癌组织和癌旁组织表达情况,构建miR-542-5p过表达肝癌细胞HepG2和Hep3B细胞系并使用RT-PCR验证,采用CCK-8、划痕、Transwell法检测细胞增殖、迁移和侵袭能力;使用miR-542-5p过表达Hep3B细胞系建立体内裸鼠移植瘤,观察移植瘤体积变化和重量变化,使用RT-PCR检测移植瘤miR-542-5p水平。通过GEO2R分析GSE101728、GSE7642的差异基因,使用miRWalk在线预测miR-542-5p的靶基因,筛选符合以上条件的基因得到ENSA;通过TCGA肝癌数据分析ENSA在肝癌组织和癌旁组织的表达情况,RT-PCR检测miR-542-5p过表达HepG2和Hep3B细胞ENSA水平,并在miR-542-5p过表达HepG2和Hep3B细胞系中转染ENSA过表达质粒,使用上述相同方法检测细胞增殖、迁移和侵袭能力。结果miR-542-5p在肝癌组织中表达水平显著低于癌旁组织(P<0.01),miR-542-5p过表达HepG2和Hep3B细胞系的光密度(A值)和相对细胞侵袭数均明显低于其阴性对照(miR-NC)(P<0.01),而划痕宽度明显高于其miR-NC(P<0.01);裸鼠移植瘤实验结果显示miR-542-5p过表达Hep3B细胞瘤的体积和重量明显低于miR-NC(P<0.01)。ENSA在肝癌组织中表达水平显著高于癌旁组织(P<0.01)且ENSA促进肝癌进展;miR-542-5p过表达HepG2和Hep3B细胞系中ENSA蛋白表达显著降低于其阴性对照(miR-NC)(P<0.01);miR-542-5p+ENSA双过表达HepG2和Hep3B细胞系的A值和细胞侵袭数均明显高于其miR-542-5p过表达细胞(P<0.01),而划痕宽度明显低于其miR-542-5p过表达细胞(P<0.01)。结论miR-542-5p可通过靶向抑制ENSA表达抑制肝细胞癌细胞增殖、迁移和侵袭。

Angiotensin-converting enzyme 2 and AMPK/mTOR pathway in the treatment of liver fibrosis:Should we consider further implications?

This editorial contains comments on the article by Zhao et al in print in the World Journal of Gastroenterology.The mechanisms responsible for hepatic fibrosis are also involved in cancerogenesis.Here,we recapitulated the complexity of the renin-angiotensin system,discussed the role of hepatic stellate cell(HSC)autophagy in liver fibrogenesis,and analyzed the possible implications in the development of hepatocarcinoma(HCC).Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers definitively contribute to reducing hepatic fibrogenesis,whereas their involvement in HCC is more evident in experimental conditions than in human studies.Angiotensin-converting enzyme 2(ACE2),and its product Angiotensin(Ang)1-7,not only regulate HSC autophagy and liver fibrosis,but they also represent potential targets for unexplored applications in the field of HCC.Finally,ACE2 overexpression inhibits HSC autophagy through the AMP-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)pathway.In this case,Ang 1-7 acts binding to the MasR,and its agonists could modulate this pathway.However,since AMPK utilizes different targets to suppress the mTOR downstream complex mTOR complex 1 effectively,we still need to unravel the entire pathway to identify other potential targets for the therapy of fibrosis and liver cancer.

miR-3612靶向SEMA4C调控肝细胞癌细胞的恶性生物学行为

目的:探讨miR-3612靶向信号素(SEMA)4C对肝细胞癌细胞增殖与侵袭能力的影响。方法:收集2020年5月至2021年9月间在皖南医学院第一附属医院弋矶山医院手术切除的肝细胞肝癌的40对癌组织和相应癌旁组织,常规培养肝细胞癌Hep3B和Huh7细胞,将其分为对照组、miR-3612 mimics-NC组、miR-3612 mimics组、miR-3612 inhibitor-NC组、miR-3612 inhibitor组、si-NC组、si-SEMA4C组、mimics-NC+pcDNA-NC组、miR-3612 mimics+pcDNA-NC组和miR-3612 mimics+pcDNA-SEMA4C组,用转染试剂将相应的核酸和质粒转染各组细胞。qPCR法检测miR-3612和SEMA4C mRNA在肝细胞癌组织和Hep3B和Huh7细胞中的表达,双荧光素酶报告基因实验和免疫共沉淀(RIP)实验验证miR-3612与SEMA4C的结合及调控关系,qPCR法和WB法检测转染后各组Hep3B和Huh7细胞中miR-3612、SEMA4C mRNA和蛋白的表达,CCK-8法、细胞划痕实验和Transwell小室实验分别检测各组Hep3B和Huh7细胞的增殖、迁移和侵袭能力。结果:miR-3612在肝细胞癌组织和Hep3B和Huh7细胞中呈低表达(P<0.001),而SEMA4C则呈高表达(P<0.001),过表达miR-3612可抑制Hep3B和Huh7细胞的增殖、迁移、侵袭和vimentin、SEMA4C蛋白的表达,促进E-cadherin蛋白的表达(P<0.05或P<0.01或P<0.001),敲低miR-3612则促进Hep3B和Huh7细胞的增殖、迁移、侵袭和SEMA4C蛋白的表达(P<0.05或P<0.01或P<0.001)。双荧光素酶报告基因实验和RIP实验证实miR-3612与SEMA4C可直接结合(P<0.001),miR-3612与SEMA4C的表达呈负相关也间接证明了这一点(P<0.001)。敲减SEMA4C能明显抑制Hep3B、Huh7细胞的增殖、侵袭和迁移能力(P<0.05或P<0.01或P<0.001),过表达SEMA4C可逆转过表达miR-3612对Hep3B和Huh7细胞增殖、迁移、侵袭和EMT的抑制作用(P<0.05或P<0.01或P<0.001)。结论:miR-3612通过调控SEMA4C表达影响Hep3B和Huh7细胞的恶性生物学行为,miR-3612有望成为临床肝细胞癌治疗的潜在靶点。

鼠李柠檬素下调NEK2抑制肝癌细胞恶性生物学行为

目的:分析鼠李柠檬素通过NIMA相关激酶2(NEK2)抑制肝癌细胞增殖、迁移和侵袭的机制。方法:网络药理学分析鼠李柠檬素可能的作用靶点为NEK2,TCGA_LIHC数据库分析肝癌组织中NEK2表达及预后的关系。检测不同浓度鼠李柠檬素对肝癌细胞增殖及NEK2蛋白表达的影响,设置对照组和鼠李柠檬素组,检测两组细胞增殖、迁移和侵袭。观察鼠李柠檬素对移植瘤体积、质量及瘤组织中Ki67蛋白表达。构建shNEK和稳定过表达NEK2细胞,观察鼠李柠檬素抑制NEK2对肝癌细胞生物学行为的影响。结果:与癌旁组织相比,癌组织NEK2的表达明显上调(P<0.05),且其表达量与预后有关(P<0.05)。随着鼠李柠檬素浓度越高,HepG2细胞和Hep3B细胞相对存活率降低(P<0.05)。鼠李柠檬素组增殖活性、侵袭、迁移细胞数均低于对照组(P<0.05),移植瘤体积和质量明显低于对照组(P<0.05),Ki67蛋白表达明显低于对照组(P<0.05),且随着鼠李柠檬素浓度越高、作用时间越久,NEK2蛋白表达下降更为明显(P<0.05)。NEK2基因沉默可以促进细胞增殖、侵袭和迁移,NEK2过表达可以逆转鼠李柠檬素抑制的细胞增殖、侵袭和迁移(P<0.05)。结论:鼠李柠檬素可以抑制NEK2蛋白表达,抑制肝癌细胞的增殖、迁移和侵袭。

骨形态发生蛋白9对肝细胞癌肿瘤干细胞干性、增殖和侵袭的影响及机制

目的探讨骨形态发生蛋白9(BMP9)对肝细胞癌(HCC)肿瘤干细胞(CSCs)干性、增殖和侵袭的影响及机制。方法取对数生长期的正常肝细胞、肝癌细胞、肝癌CSCs,采用RT-qPCR法检测BMP9 mRNA表达,采用Western blotting法检测BMP9蛋白表达。采用慢病毒干扰载体转染肝细胞癌肿瘤干细胞(HepG2-CSCs)以敲低BMP9表达,并分成HepG2-CSCs组、HepG2-CSCs-BMP9敲低组。加入MAPK/ERK信号激动剂(DIPQUO)后,将细胞分为三组:HepG2-CSCs组、HepG2-CSCs敲低组及HepG2-CSCs敲低+DIPQUO组。采用RT-qPCR实验检测HepG2-CSCs干性相关分子CD44、SOX2和OCT4表达,采用CCK-8实验检测细胞增殖能力,采用Transwell实验检测细胞侵袭能力,采用蛋白印迹法检测MAPK/ERK通路关键蛋白表达。结果与正常肝细胞比,肝癌细胞和肝癌肿瘤干细胞(HepG2-CSCs)中BMP9表达上调(P<0.05)。HepG2 CSCs细胞中BMP9 mRNA和蛋白表达高于HepG2细胞(P均<0.05)。BMP9敲低后,HepG2-CSCs-BMP9敲低组中HepG2-CSCs细胞中干细胞相关标志物CD44、SOX2、OCT4 mRNA表达较HepG2-CSCs组降低(P均<0.05)。与HepG2-CSCs组比较,HepG2-CSCs-BMP9敲低组中HepG2-CSCs在48、72和96 h的细胞增殖能力降低(P均<0.05),HepG2-CSCs细胞迁移和侵袭能力降低(P均<0.05)。较HepG2-CSCs组,HepG2-CSCs-BMP9敲低组中HepG2-CSCs中p-ERK1/2和p-MEK1/2蛋白表达显著降低(P均<0.05)。HepG2-CSCs敲低+DIPQUO组HepG2-CSCs中干细胞相关标志物CD44、SOX2、OCT4 mRNA表达增加(P均<0.05),HepG2-CSCs增殖和侵袭能力增强(P均<0.05)。结论BMP9敲低可抑制HepG2-CSCs干性维持并抑制细胞增殖、降低侵袭能力,机制可能与抑制MAPK/ERK信号通路有关。

RNF113A对肝细胞癌细胞恶性生物学行为的影响研究

目的:探究RNF113A对肝癌细胞增殖、迁移、侵袭、凋亡和自噬的影响。方法:肝癌细胞Hep3B分成control组和RNF113A过表达组,Hep G2分成control组和RNF113A敲低组,CCK-8实验检测细胞活力的变化,克隆形成实验检测细胞增殖能力的变化,Transwell实验检测细胞侵袭能力的变化,划痕实验检测细胞迁移能力的变化,流式细胞术检测细胞凋亡能力的变化,Western blot实验检测自噬相关基因和AMPK信号通路相关基因的蛋白表达变化。结果:与control组相比,RNF113A过表达组中Hep3B细胞的增殖、克隆、侵袭和迁移能力均提高,而凋亡和自噬能力均下降,AMPK信号通路受抑;而RNF113A敲低组中Hep G2细胞的增殖、克隆、侵袭和迁移能力显著下降,凋亡和自噬能力升高,促进AMPK信号通路的激活。结论:RNF113A通过抑制AMPK信号通路促进肝癌细胞的增殖、克隆、侵袭和迁移,并抑制凋亡和自噬。