TPX2 knockdown suppressed hepatocellular carcinoma cell invasion via inactivating AKT signaling and inhibiting MMP2 and MMP9 expression

Objective： Targeting protein for Xenopus kinesin-like protein 2 （TPX2） is a nuclear proliferation-related protein that plays a critical role in the formation of mitotic spindle. High expression of TPX2 has been observed in several types of tumors. However, the role of TPX2 in hepatocellular carcinoma （HCC） remains unclear. Our study aimed to investigate the effect of TPX2 on HCC cell invasion. Methods： The immortalized normal human liver cell line L02 and six HCC cell lines including SMMC- 7721, BEL-7402, Huh-7, HepG2, Hep3B and SKHepl were subjected to qRT-PCR and western blot for TPX2 mRNA and protein, respectively. Furthermore, TPX2 small interfering RNA （siRNA） was used to knock down TPX2 expression in SMMC-7721 and HepG2 cells. Cell proliferation and invasion were determined by MTT and transwell assays. Otherwise, expression of p-AKT, MMP2 and MMP9 were evaluated by western blot in SMMC-7721 cells. Results： The expression of TPX2 in HCC cell lines was markedly higher than that in normal human liver cell line. TPX2 knockdown using a specific TPX2-siP, NA reduced the number of invaded cells and inhibited cell proliferation in SMMC-7721 and HepG2 cells. Furthermore, TPX2 knockdown resulted in inactivation of AKT signaling and down-regulation of MMP2 and MMP9 expression in SMMC-7721 cells. Conclusions： Our study identified that TPX2 might contribute to tumor cell invasion through activating AKT signaling and subsequently increasing MMP2 and MMP9 in HCC.     

siRNA targeted against HAb18G/CD147 inhibits MMP-2 secretion, actin and FAK expression in hepatocellular carcinoma cell line via ERK1/2 pathway

HAb18G/CD147 has been identified as a factor that induces MMPs production. SiRNA targeted against HAb18G/CD147 was transfected into FHCC-98 cells (a HCC cell line) to knockdown its expression. The results showed that downregulating HAb18G/CD147 decreased ERK1/2, MMP-2 and FAK levels and inhibited cell motility and invasion, together with rearranged actin stress fiber formation, while had no effects on integrin alpha3beta1 expression. MEK1/2 inhibitor, U0126, inhibited MMP-2, FAK and actin expression in FHCC-98 cell line. The findings indicate that si-HAb18G inhibits gelatinase production, actin and FAK expression in FHCC-98 via an ERK1/2 signaling pathway.     

三氧化二砷对人肝癌SMMC-7721细胞侵袭转移能力的影响

目的：探讨三氧化二砷（As2O3）对人肝癌SMMC-7721细胞黏附、侵袭、转移能力的影响以及其机制。方法：采用MTT法观察人肝癌SMMC-7721细胞经As2O3作用前后对基底膜黏附能力的影响;Transwell膜侵袭系统观察SMMC-7721细胞经As2O3作用前后游走与穿透基底膜能力的改变。免疫细胞化学方法检测人肝癌细胞经As2O3作用前后CD44V6表达的改变。结果：As2O3能显著抑制人肝癌SMMC-7721细胞与m arigel的黏附、抑制细胞游走与穿透基底膜的作用（P〈0.05）,能抑制人肝癌细胞CD44v6的表达（P〈0.05）。结论：As2O3具有抑制人肝癌细胞的侵袭、转移能力,其抑制作用可能与CD44v6的表达下调有关。     

三氧化二砷对人肝癌SMMC-7721细胞侵袭转移能力的影响

目的:探讨三氧化二砷(As2O3)对人肝癌SMMC-7721细胞黏附、侵袭、转移能力的影响以及其机制。方法:采用MTT法观察人肝癌SMMC-7721细胞经As2O3作用前后对基底膜黏附能力的影响;Transwell膜侵袭系统观察SMMC-7721细胞经As2O3作用前后游走与穿透基底膜能力的改变。免疫细胞化学方法检测人肝癌细胞经As2O3作用前后CD44V6表达的改变。结果:As2O3能显著抑制人肝癌SMMC-7721细胞与m arigel的黏附、抑制细胞游走与穿透基底膜的作用(P<0.05),能抑制人肝癌细胞CD44v6的表达(P<0.05)。结论:As2O3具有抑制人肝癌细胞的侵袭、转移能力,其抑制作用可能与CD44v6的表达下调有关。     

Autophagy induced by baicalin involves downregulation of CD147 in SMMC-7721 cells in vitro

Baicalin has been demonstrated to exert anticancer effects mainly through induction of tumor cell apoptosis and cell cycle arrest. However, the precise mechanisms underlying its anticancer role remain to be elucidated. In the present study, we investigated whether autophagy was involved in the anticancer activity of baicalin in the human hepatocellular carcinoma (HCC) cell line SMMC-7721 and the possible molecular mechanisms. Our data showed that the viability of SMMC-7721 cells was significantly inhibited by baicalin in a dose- and time-dependent manner. Alongside apoptosis, autophagy was also induced by baicalin dose- and time-dependently with the involvement of the autophagy-associated protein Βeclin?1. Moreover, we demonstrated that cell death induced by baicalin was significantly inhibited by the apoptosis inhibitor z-DEVD-fmk or the autophagy inhibitor 3-MA, respectively. In addition, we found that CD147, a key molecule related both to apoptosis and autophagy, was markedly downregulated at the protein level in SMMC-7721 cells treated with baicalin. Collectively, this is the first study to suggest that baicalin induces autophagic cell death in SMMC-7721 cells, which involves the downregulation of CD147. Our study reveals a new mechanism for the anticancer effects of baicalin and puts forward a potential crucial role of CD147 in baicalin-induced cancer cell death.     

白眉蝮蛇去整合素rAdinbitor对人肝癌细胞系SMMC-7721细胞黏附与增殖的抑制作用

Objective:To investigate the effects of rAdinbitor on the adhesion and proliferation of human hepatoma cell strain SMMC-7721.Methods:Cell adhesion assay was used to observe the effect of rAdinbitor on the adhesion of SMMC-7721 cells to fibronectin (FN).Crystal violet staining was performed to detect the influence of rAdinbitor on the adhesion of SMMC7721 cells.MTT assay was employed to detect the inhibitory effects of different concentration of rAdinbitor on the proliferation of SMMC-7721 cells.The morphologic changes of the control SMMC-7721 cells and the apoptotic cells induced by 200 μg/mL rAdinbitor for 36 h were observed under light microscope after HE staining.Flow cytometry analysis was applied to determine the apoptosis rate of SMMC-7721 cells.Results:(1) FN promoted the adhesion of human hepatoma cell strain SMMC-7721 in a dose-dependent manner.(2) rAdinbitor could dose-dependently inhibit the adhesion of SMMC-7721 cells to FN.The higher the concentration was,the stronger the inhibition was.There was significant difference among the groups (P＜0.05).(3) rAdinbitor had a strong inhibition on the proliferation of SMMC-7721 cells and showed a dose-dependent manner (P＜0.05).After a 48 h exposure,the IC5o value of rAdinbitor was 177.83 μg/mL.(4) After exposure of SMMC-7721 cells to 200 μg/mL rAdinbitor for 36 h,the eady morphologic changes appeared and the apoptosis rate was 20.68%,significantly higher than that of the control group (2.38%,P＜0.05).Conclusion:rAdinbitor can dose-dependently inhibit the SMMC-7721 cells adhesion to FN,and can inhibit the proliferation in dose-dependent manner and promote their apoptosis.     

下调MLLT3/AF9基因对肝癌SMMC-7721细胞增殖与侵袭能力的影响

目的 本文旨在研究组蛋白甲基转移酶MLLT3/AF9在人肝癌组织中的表达,观察体外下调MLLT3/AF9的表达对肝癌SMMC-7721细胞增殖与侵袭能力的影响。方法 应用Real-time PCR技术检测100例肝癌组织、癌旁组织和肝癌细胞系SMMC-7721、HepG2、Hu7、BEL-7402中MLLT3/AF9基因的表达水平;应用RNA干扰下调MLLT3/AF9的表达,采用CCK-8、Transwell实验和划痕实验检测MLLT3/AF9下调组和对照组SMMC-7721细胞的增殖及侵袭能力。结果 Real-time PCR检测结果显示肝癌组织中的MLLT3/AF9基因的表达量显著高于癌旁组织; Real-time PCR和Western blot检测到LVMLLT3-RNAi组中MLLT3/AF9 mRNA和蛋白的表达水平都降低,CCK-8、Transwell和划痕实验结果显示LV-MLLT3-RNAi组细胞的增殖和侵袭能力降低。结论 MLLT3/AF9在肝癌组织中表达水平明显升高,体外下调MLLT3/AF9的表达可以有效抑制肝癌SMMC-7721细胞的增殖与侵袭。     

Suppression of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger 1 by RNA interference or amiloride inhibits human hepatoma cell line SMMC-7721 cell invasion

Na⁺/H⁺ exchanger 1 (NHE1), a primary regulator of intracellular pH (pHi) and extracellular pH (pHe), plays a significant role in acidifying the tumor microenvironment, possibly resulting in their malignant potential. However, currently, very little is known about the roles of NHE1 in invasion of hepatocellular carcinoma (HCC) cells. We have recently shown that NHE1 is over-expressed in HCC tissues and that this increased expression is associated with HCC invasiveness. In this study, we also found that NHE1 is over-expressed in HCC cell lines. Subsequently, we silenced NHE1 expression in the human HCC cell line SMMC-7721 using RNA interference (RNAi) and examined the invasiveness and proliferation of NHE1-silenced SMMC-7721 cells and the matrix metalloproteinase-2 (MMP-2) activity. The knockdown of NHE1 expression significantly inhibited the invasive ability of SMMC-7721 cells but had only a minor effect on the cellular proliferation rate. Moreover, NHE1 knockdown significantly reduced the secretion of MMP-2. Further experiment using amiloride (an inhibitor of NHE1) confirmed the above result. Together, these findings indicate that NHE1 has an important role in SMMC-7721 cell invasion and that NHE1 might be a new target of HCC treatment.     

缺氧诱导肝癌细胞获得干细胞特性的初步实验研究

目的:观察缺氧对肝癌干细胞特性的影响.方法:用肝癌细胞系HepG2、SMMC-7721建立细胞缺氧培养模型,利用倒置显微镜观察细胞形态学变化;克隆形成实验检测肿瘤细胞克隆形成能力;"肿瘤球"形成实验检测肿瘤细胞自我更新能力;实时定量RT-PCR和Western blot检测干细胞相关基因Oct-4、Sox-2、Nanog mRNA和蛋白表达;采用流式细胞术检测干细胞表面标志物CD133表达.结果:缺氧条件下HepG2、SMMC-7721细胞克隆形成率显著高于常氧组(P<0.05).缺氧条件下HepG2、SMMC-7721细胞肿瘤球形成率显著高于常氧组(P<0.05).常氧条件下HepG2、SMMC-7721细胞中干细胞相关基因Oct-4、Sox-2、Nanog mRNA及蛋白表达水平极低,缺氧处理48 h后,其mRNA及蛋白表达水平明显增高(P<0.05).缺氧条件下HepG2、SMMC-7721细胞CD133阳性表达率表达量显著增加(P<0.05).在缺氧36 h和48 h,CD133蛋白表达水平显著增强(P<0.05).结论:缺氧能诱导肝癌细胞干细胞特性的获得.     

CD147和CK19在肝细胞肝癌中的表达及临床意义

Objective

The aim of this study was to investigate the expressions of CD147 and CK19 in hepatocellular carcinoma (HCC) and their clinical significance.

Methods

The expressions of CD147 and CK19 were determined by tissue microarray and immunohistochemistry (IHC) in 272 cases of HCC and 81 cases of adjacent tumorous tissue.

Results

The positive expression of CD147 in HCC and adjacent tumorous tissue was 73.53% (200/272) and 13.58% (11/81) with significant difference (P < 0.05). The positive expression of CK19 in HCC and adjacent tumorous tissue was 14.34% (39/272) and 0 (0/81) with significant difference (P < 0.05). The positive expression of CD147 were closely correlated to the histological grade, clinical stage, tumor-free survival, diameter of tumor and embolus of cancer in aqueduct or portal vein; but not to the patients’ sex, age, liver cirrhosis, AFP level, infection of HBV, lymph node metastasis, number of tumor, invasion liver involucrum and the micro-satellites (P > 0.05). The expression of CK19 in HCC were closely correlate to the tumor-free survival, histological grade, diameter of tumor, liver cirrhosis, micro-satellites, lymph node metastasis and clinical stage; but not to patients’ sex, age, number of tumor, invasion liver involucrum, AFP level, infection of HBV and embolus of cancer in aqueduct or portal vein (P > 0.05). Among the patients of positive expression of CD147, the median replacing time and overall survival were 13 and 24 months, lower than 48 and 60 months in the patients of negative expression (P < 0.05). Among the patients of positive expression of CK19, the median replacing time and overall survival were 7 and 13 months, lower than 31 and 42 months in the patients of negative expression (P < 0.05). The expression of CD147 had no correlation with the expression of CK19 (r = 0.061, P = 0.317).

Conclusion

The positive of CD147 and CK19 closely correlate with the clinical prognosis of HCC, it may indicate poor prognosis of HCC.     

The effect of NET-1 on the proliferation, migration and endocytosis of the SMMC-7721 HCC cell line

To explore the effect of NET-1 on the proliferation, migration and endocytosis in the hepatocellular carcinoma (HCC) cell line SMMC-7721, we constructed the pU6H1-NET-1-siRNA (NET-1siRNA) and pcDNA3.1/myc-NET-1 (myc-NET-1) vectors and transfected them into SMMC-7721 cells. The expression levels of NET-1 mRNA and protein were detected using real-time quantitative RT-QPCR and western blotting. The proliferation rates of SMMC-7721 cells were determined by CCK-8 assays, flow cytometry (FCM) and immunohistochemistry staining. The migration in two or three dimensional space of SMMC-7721 cells were determined by wound-healing assay and in vitro invasion assay. The extent of endocytosis in SMMC-7721 cells was estimated by observing the amount of transferrin (Tfn) absorbed with capture ELISA assays, and Tfn endocytosis was observed under confocal immunofluorescence microscopy. The results show that: i) after transfecting NET-1 siRNA, the expression of NET-1 mRNA and protein in SMMC-7721 cells decreased significantly, the growth of cells was suppressed, which induced cell cycle arrest, the proliferation rates were dramatically reduced and the expression of Ki67 declined, and migration and endocytosis in cells were inhibited, compared with untreated cells (every P<0.01); ii) Following transfection with myc-NET-1, the expression of NET-1 mRNA and protein in SMMC-7721 cells increased, and both the proliferation of cells and the cell cycle were promoted (P<0.01, respectively). However, the abilities of cell migration and endocytosis were not affected compared with untreated cells. These data suggest that: i) the NET-1 gene may play an important role in proliferation, migration and endocytosis of cells; ii) siRNA technology may efficiently suppress the expression and function of NET-1 in HCC, suggesting that NET-1 may be a therapeutic target for HCC.     

miR-223-3p 通过靶向RAC1 调控肝细胞癌SMMC-7721 细胞的增殖和凋亡

目的：探讨miR-223-3p 通过调控Ras 相关C3 肉毒素底物1（Ras-related C3 botulinum toxin substrate 1，RAC1）对肝细胞癌（hepatocellular carcinoma，HCC）细胞增殖和凋亡的影响及其可能的作用机制。方法：选用2016 年8 月至2018 年8 月吉林市中心医院手术切除的30 例HCC 组织及其癌旁组织标本和人HCC 细胞系SMMC-7721、Bel-7402、HepG2 及人正常肝细胞QSG-7701，用qPCR检测HCC组织和细胞系中miR-223-3p的表达水平。分别将miR-223-3p mimics、miR-223-3p inhibitor 和siRAC1转染至SMMC-7721 细胞，通过CCK-8、克隆形成实验和Annexin V-FITC/PI 染色流式细胞术检测SMMC-7721 细胞的增殖、克隆形成和凋亡水平。用双荧光素酶报告基因实验检测miR-223-3p 与RAC1 的靶向关系，Western blotting 检测细胞中RAC1 蛋白的表达水平。结果：miR-223-3p 在HCC组织的表达水平显著低于癌旁组织（P<0.01），其表达水平与肿瘤大小、TNM分期及肿瘤分化病理特征相关（P<0.05 或P<0.01）；miR-223-3p 在HCC细胞系表达水平显著低于QSG-7701 细胞（均P<0.01），以在SMMC-7721细胞中表达水平最低。双荧光素酶报告基因实验证实RAC1 是miR-223-3p 靶基因，miR-223-3p 靶向负调控RAC1 的表达。转染miR-223-3p mimics 显著抑制SMMC-7721 细胞的增殖和克隆形成能力（P<0.05 或P<0.01），并促进细胞凋亡（P<0.01）；转染miR-223-3p inhibtor 则逆转miR-223-3p mimics 对细胞的抑制作用。结论：过表达miR-223-3p 抑制HCC细胞增殖和克隆形成能力并促进细胞凋亡，其机制可能与靶向下调RAC1表达有关。     

Adenovirus-mediated Il-24 expression suppresses hepatocellular carcinoma growth via induction of cell apoptosis and cycling arrest and reduction of angiogenesis

Previous studies have shown that interleukin (IL)-24 as a novel tumor suppressor gene has tumor-suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo. In this study, we explored the potential effect of adenovirus-mediated IL-24 gene therapy on human hepatocellular carcinoma (HCC) by using a HCC cell line, SMMC-7721. We constructed a recombinant adenovirus, AdVGFP/IL-24 expressing the marker green fluorescent protein (GFP) and the tumor-suppressor gene, IL-24. We demonstrated that AdVGFP/IL-24 treatment of SMMC-7721 cells in vitro significantly induced HCC cell cytotoxicity and apoptosis, and altered HCC cell cycling with an S-phase reduction and G2/M phase arrest, compared with AdVGFP, without IL-24 expresssion (p < 0.05). Furthermore, we also showed that the treatment of SMMC-7721 tumors by an intratumoral injection of AdVGFP/IL-24 significantly suppressed in vivo HCC growth in athymic nude mice, compared with AdVGFP treatment (p < 0.05). In addition, we also elucidated the molecular mechanism responsible for AdVGFP/IL-24-associated tumor suppression. These include: (1) upregulation of p53-independent apoptosis-associated caspase-3 and (2) downregulation of angiogenesis-associated vascular endothelial growth factor and CD34. Therefore, this study will provide a framework for future clinical applications of AdVGFP/IL-24 in HCC gene therapy.     

CD155/PVR plays a key role in cell motility during tumor cell invasion and migration

Background

Invasion is an important early step of cancer metastasis that is not well understood. Developing therapeutics to limit metastasis requires the identification and validation of candidate proteins necessary for invasion and migration.

Methods

We developed a functional proteomic screen to identify mediators of tumor cell invasion. This screen couples Fluorophore Assisted Light Inactivation (FALI) to a scFv antibody library to systematically inactivate surface proteins expressed by human fibrosarcoma cells followed by a high-throughput assessment of transwell invasion.

Results

Using this screen, we have identified CD155 (the poliovirus receptor) as a mediator of tumor cell invasion through its role in migration. Knockdown of CD155 by FALI or by RNAi resulted in a significant decrease in transwell migration of HT1080 fibrosarcoma cells towards a serum chemoattractant. CD155 was found to be highly expressed in multiple cancer cell lines and primary tumors including glioblastoma (GBM). Knockdown of CD155 also decreased migration of U87MG GBM cells. CD155 is recruited to the leading edge of migrating cells where it colocalizes with actin and αv-integrin, known mediators of motility and adhesion. Knockdown of CD155 also altered cellular morphology, resulting in cells that were larger and more elongated than controls when plated on a Matrigel substrate.

Conclusion

These results implicate a role for CD155 in mediating tumor cell invasion and migration and suggest that CD155 may contribute to tumorigenesis.     

RNAi-mediated silencing of CD147 inhibits tumor cell proliferation,invasion and increases chemosensitivity to cisplatin in SGC7901 cells in vitro

Background

CD147 is a widely distributed cell surface glycoprotein that belongs to the Ig superfamily. CD147 has been implicated in numerous physiological and pathological activities. Enriched on the surface of many tumor cells, CD147 promotes tumor growth, invasion, metastasis and angiogenesis and confers resistance to some chemotherapeutic drugs. In this study, we investigated the possible role of CD147 in the progression of gastric cancer.

Methods

Short hairpin RNA (shRNA) expressing vectors targeting CD147 were constructed and transfected into human gastric cancer cells SGC7901 and CD147 expression was monitored by quantitative realtime RT-PCR and Western blot. Cell proliferation, the activities of MMP-2 and MMP-9, the invasive potential and chemosensitivity to cisplatin of SGC7901 cells were determined by MTT, gelatin zymography, Transwell invasion assay and MTT, respectively.

Results

Down-regulation of CD147 by RNAi approach led to decreased cell proliferation, MMP-2 and MMP-9 activities and invasive potential of SGC7901 cells as well as increased chemosensitivity to cisplatin.

Conclusion

CD147 involves in proliferation, invasion and chemosensitivity of human gastric cancer cell line SGC7901, indicating that CD147 may be a promising therapeutic target for gastric cancer.     

FGFR3 promotes angiogenesis-dependent metastasis of hepatocellular carcinoma via facilitating MCP-1-mediated vascular formation

The biological role of fibroblast growth factor receptor 3 (FGFR3) in tumor angiogenesis of hepatocellular carcinoma (HCC) has not been discussed before. Our previous work had indicated FGFR3 was overexpressed in HCC, and silencing FGFR3 in Hu7 cells could regulate tumorigenesis via down-regulating the phosphorylation level of key members of classic signaling pathways including ERK and AKT. In the present work, we explored the role of FGFR3 in angiogenesis-dependent metastasis by using SMMC-7721 and QGY-7703 stable cell lines. Our results indicated FGFR3 could regulate in vitro cell migration ability and in vivo lung metastasis ability of HCC, which was in accordance with increased angiogenesis ability in vitro and in vivo. Using the supernatant from SMMC-7721/FGFR3 cells, we conducted a human angiogenesis protein microarray including 43 angiogenesis factors and found that FGFR3 modulated angiogenesis and metastasis of HCC mainly by promoting the protein level of monocyte chemotactic protein 1 (MCP-1). Silencing FGFR3 by short hairpin RNA (shRNA) could reduce MCP-1 level in lysates and supernatant of QGY-7703 cells and SMMC-7721 cells. Silencing MCP-1 in QGY-7703 or SMMC-7721 cells could induce similar phenotypes compared with silencing FGFR3. Our results suggested FGFR3 promoted metastasis potential of HCC, at least partially if not all, via facilitating MCP-1-mediated angiogenesis, in addition to previously found cell growth and metastasis. MCP-1, a key medium between HCC cells and HUVECs, might be a novel anti-vascular target in HCC.     

肝癌细胞 67kDa层粘连蛋白受体的表达

背景与目的:本实验室最近发现人肝癌细胞 SMMC-7721表达的 67kDa层粘连蛋白受体( 67kDa Laminin receptor,67LR)与层粘连蛋白的结合能力明显高于正常肝细胞 L-02表达的 67LR,而 67LR在肝癌细胞和正常肝细胞中的表达水平尚不清楚.本研究旨在探讨 67LR在肝癌细胞中的表达,及其与层粘连蛋白结合能力的关系.方法:以人肝癌细胞 SMMC-7721、 HepG2和正常肝细胞 L-02为材料,采用 131I标记的层粘连蛋白测定其与细胞的结合能力;采用流式细胞术和 RT-PCR分析 67LR蛋白和 mRNA的表达水平. 结果: (1)相同条件下 SMMC-7721、 HepG2 细胞与层粘连蛋白特异结合量分别为 (17.54± 0.49) ng/105 cell、 (11.18± 0.53) ng/105 cell,而 L-02细胞的特异结合量为 (8.36± 0.48) ng/105 cell,表明肝癌细胞与层粘连蛋白的结合能力明显高于正常肝细胞( P< 0.01) ;(2)流式细胞术分析, SMMC-7721细胞的 67LR阳性表达率为 34.7%, L-02细胞的 67LR阳性表达率为 55.3%,而 HepG2 细胞则几乎不表达 67LR; (3)RT-PCR产物进行半定量分析发现,两株肝癌细胞的 67LR mRNA表达水平明显高于 L-02细胞.结论:肝癌细胞与层粘连蛋白的结合能力明显高于正常肝细胞 L-02,而细胞膜表面的 67LR水平却低于 L-02细胞,提示 SMMC-7721细胞 67LR的亲和力可能高于 L-02细胞,而且还涉及其它层粘连蛋白受体.     

三氧化二砷对人肝癌SMMC-7721细胞中P27kip1、JAB1表达的影响及其作用机制

背景与目的:三氧化二砷(arsenic trioxide,As2O3)作为治疗实体瘤的新药已应用于临床,但其作用机理尚不清楚.本研究拟探讨As2O3对肝癌SMMC-7721细胞增殖的影响,及其对细胞周期素依赖性激酶抑制剂(cyclin-dependent kinase inhibitors,CDKIs)P27kip1和P27kip1相关蛋白c-Jun结合蛋白-1(c-Jun activation domain-binding protein 1,JAB1)表达的调控作用.方法:体外培养人肝癌细胞株SMMC-7721,用0～8 μmol/L As2O3处理96 h,用WST-8法检测细胞的存活率.用2 μmol/L As2O3作用72 h,在指点时间点收集细胞,用Western blot技术检测P27kip1、JAB1在SMMC-7721细胞中的表达,同时采用核浆分离方法及细胞免疫荧光技术检测P27kip1、JAB1表达和亚细胞定位的改变.结果:与对照组比较,As2O3可明显抑制SMMC-7721细胞的增殖,96 h时IC50为(1.81±0.41)μmol/L.2μmol/LAs2O3处理12 h后,SMMC-7721细胞中P27kip1蛋白表达增加,而JAB1蛋白表达减少.在As2O3处理后12 h、24 h,P27kip1与JAB1均发生从胞浆向胞核的易位.免疫荧光检测P27kip1和JAB1的亚细胞定位情况,结果也显示2 μmol/L As2O3可诱导两者在胞核的积聚.结论:As2O3可下调JAB1的表达,从而影响P27kip1的核内外分布及表达,并影响P27kip1的功能状态,进而参与调控肝癌细胞的增殖.     

Hypomethylation of the hsa-miR-191 Locus Causes High Expression of hsa-miR-191 and Promotes the Epithelial-to-Mesenchymal Transition in Hepatocellular Carcinoma

hsa-miR-191 is highly expressed in hepatocellular carcinoma (HCC), but the factors regulating this elevated expression are unknown. This study aimed to investigate the epigenetic mechanisms of increased hsa-miR-191 expression by analyzing the relationship between the DNA methylation status of hsa-miR-191 and miR-191 expression. Methylation-specific polymerase chain reaction (PCR), bisulfite sequencing PCR, Northern blot, and quantitative real-time PCR were performed to examine hsa-miR-191 methylation and expression levels. Western blot, transwell, and scratch assays were performed to examine the function and molecular mechanisms of hsa-miR-191. Approximately 58.9% of hsa-miR-191 expression was higher in HCC tissues than in adjacent noncancerous tissues; this high expression was associated with poor prognosis. The hypomethylation observed in some HCC cell lines and HCC tissues was correlated with the hsa-miR-191 expression level. This correlation was validated by treatment with the 5-aza-DAC demethylation agent. The level of hypomethylation was 63.0% in 73 clinical HCC tissue samples and was associated with increased (2.1-fold) hsa-miR-191 expression. The elevated expression of hsa-miR-191 in the SMMC-771 HCC cell line induced the cells to transition into mesenchymal-like cells; they exhibited characteristics such as loss of adhesion, down-regulation of epithelial cell markers, up-regulation of mesenchymal cell markers, and increased cell migration and invasion. Inhibiting hsa-miR-191 expression in the SMMC-7721 cell line reversed this process (as assessed by cell morphology and cell markers). Furthermore, hsa-miR-191 probably exerted its function by directly targeting TIMP metallopeptidase inhibitor 3 and inhibiting TIMP3 protein expression. Our results suggest that hsa-miR-191 locus hypomethylation causes an increase in hsa-miR-191 expression in HCC clinical tissues and that this expression induces HCC cells to transition into mesenchymal-like cells.