锚蛋白重复序列22(ANKRD22)对人肝癌细胞的影响及其机制
DOI: 10.12449/JCH240519
Effect of ankyrin-repeat domain-containing protein 22 on human hepatoma cells and its mechanism
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摘要:
目的 探讨锚蛋白重复序列22(ANKRD22)对人肝癌细胞增殖、侵袭和迁移的影响及其分子机制。 方法 通过TCGA数据库分析正常肝组织及肝细胞癌组织中ANKRD22的表达水平及其与预后的关系。通过qRT-PCR和Western Blot检测人正常肝细胞(L-02)和人肝癌细胞系(Huh7、Hep G2、MHCC-97H、SK-HEP-1、SMMC-7721)中ANKRD22的表达情况。通过CCK-8、EdU、划痕实验及Transwell检测ANKRD22对肝癌细胞增殖、侵袭和迁移能力的影响。通过Western Blot检测ANKRD22与细胞周期蛋白、EMT相关蛋白之间的关系。通过KEGG、ssGSEA分析进一步探究ANKRD22在肝癌细胞中的作用机制,并进行实验验证。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,进一步两两比较采用LSD-t检验。 结果 TCGA数据库中ANKRD22在肝细胞癌组织中较正常肝组织高表达(t=5.083,P<0.05),且ANKRD22高表达患者的总生存期及疾病相关生存期均显著低于ANKRD22低表达的患者(P值均<0.05)。肝癌细胞系中ANKRD22的表达量均高于正常肝细胞(P值均<0.05)。增殖实验结果提示,ANKRD22过表达组的EdU阳性率、增殖速度均高于空载对照(Vector)组(t值分别为19.60、6.72,P值均<0.001);si-ANKRD22#2组及si-ANKRD22#3组的EdU阳性率、增殖速度较si-NC组均降低(P值均<0.001)。Cyclin E1、Cyclin D1、CDK7、CDK4在过表达组中表达高于Vector组(t值分别为3.54、4.95、6.34、5.19,P值均<0.01);在si-ANKRD22#2组及si-ANKRD22#3组中表达均低于si-NC组(P值均<0.001)。P27在过表达组中表达低于Vector组(t=6.12,P<0.001),在si-ANKRD22#2组及si-ANKRD22#3组中表达均高于si-NC组(P值均<0.001)。侵袭、迁移实验结果提示,ANKRD22过表达组的迁移速度、穿膜数量(迁移组和侵袭组)均高于Vector组(t值分别为5.01、25.60、3.67,P值均<0.05);si-ANKRD22#2组及si-ANKRD22#3组的迁移速度、穿膜数量(迁移组和侵袭组)较si-NC组均降低(P值均<0.01)。N-cadherin、Vimentin、Snail在过表达组中表达高于Vector组(t值分别为12.13、8.85、13.97,P值均<0.001),在si-ANKRD22#2组及si-ANKRD22#3组中表达均低于si-NC组(P值均<0.001);E-cadherin在过表达组中表达低于Vector组(t=4.98,P<0.01),在si-ANKRD22#2组及si-ANKRD22#3组中表达均高于si-NC组(P值均<0.001)。KEGG富集分析及ssGSEA分析提示,ANKRD22在肝细胞癌中与PI3K/AKT/mTOR信号通路相关,在过表达组中,p-AKT/AKT、p-PI3K/PI3K、p-mTOR/mTOR均较Vector组升高(t值分别为12.21、3.43、9.75,P值均<0.01);在si-ANKRD22#2组及si-ANKRD22#3组中表达均低于si-NC组(P值均<0.001)。 结论 ANKRD22在肝癌细胞中高表达,能促进肝癌细胞的增殖、侵袭和迁移能力,并且能促进PI3K/AKT/mTOR信号通路的激活。 Abstract:Objective To investigate the effect of ankyrin-repeat domain-containing protein 22 (ANKRD22) on the proliferation, invasion, and migration of human hepatoma cells and its molecular mechanism. Methods The TCGA database was used to analyze the expression level of ANKRD22 in normal liver tissue and hepatocellular carcinoma tissue and its association with prognosis. Western Blot and qRT-PCR were used to measure the expression of ANKRD22 in human normal liver cells (L-02) and human hepatoma cells (Huh7, HepG2, MHCC-97H, SK-HEP-1, and SMMC-7721); CCK-8 assay, EdU, wound healing assay, and Transwell assay were used to observe the effect of ANKRD22 on the proliferation, invasion, and migration of hepatoma cells; Western Blot was used to investigate the association of ANKRD22 with cyclins and EMT-related proteins; KEGG and ssGSEA analyses were performed to investigate the mechanism of action of ANKRD22 in hepatoma cells, and related experiments were conducted for validation. The independent-samples t-test was used for comparison of continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results In the TCGA database, the expression level of ANKRD22 in hepatoma tissue was significantly higher than that in normal liver tissue (t=5.083, P<0.05), and the patients with a high expression level of ANKRD22 had longer overall survival and disease-related survival than those with a low expression level of ANKRD22 (P<0.05). The expression level of ANKRD22 in various human hepatoma cell lines was higher than that in human normal liver cells (all P<0.05). Cell proliferation assay showed that the ANKRD22 overexpression group had significantly higher EdU positive rate and proliferation rate than the Vector group (t=19.60 and 6.72, both P<0.001), and compared with the si-NC group, the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower EdU positive rate and proliferation rate (all P<0.001). Compared with the Vector group, the overexpression group had significantly higher expression levels of Cyclin E1, Cyclin D1, CDK7, and CDK4 (t=3.54, 4.95, 6.34, and 5.19, all P<0.01), and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group (all P<0.001). The overexpression group had a significantly lower expression level of P27 than the Vector group (t=6.12, P<0.001), and the si-ANKRD22#2 group and the si-ANKRD22#3 group had a significantly higher expression level than the si-NC group (both P<0.001). Invasion and migration experiments showed that compared with the Vector group, the ANKRD22 overexpression group had significantly higher migration rate and number of crossings through the membrane (migration group and invasion group) (t=5.01, 25.60, and 3.67, all P<0.05), and compared with the si-NC group, thesi-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower migration rate and number of crossings through the membrane (migration group and invasion group) (all P<0.01). The overexpression group had significantly higher expression levels of N-cadherin, Vimentin, and Snail than the Vector group (t=12.13, 8.85, and 13.97, all P<0.001), and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group (all P<0.001); the overexpression group had a significantly lower expression level of E-cadherin than the Vector group (t=4.98, P<0.01), and the si-ANKRD22#2 group and the si-ANKRD22#3 group had a significantly higher expression level than the si-NC group (both P<0.001). The KEGG enrichment analysis and the ssGSEA analysis showed that ANKRD22 was associated with the PI3K/AKT/mTOR signaling pathway in hepatocellular carcinoma, and the overexpression group had significantly higher expression levels of p-AKT/AKT, p-PI3K/PI3K, and p-mTOR/mTOR than the Vector group (t=12.21, 3.43, and 9.75, all P<0.01), and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group (all P<0.001). Conclusion ANKRD22 is highly expressed in hepatoma cells and can promote the proliferation, invasion, and migration of hepatoma cells and the activation of the PI3K/AKT/mTOR signaling pathway. -
Key words:
- Carcinoma, Hepatocellular /
- Ankyrin Repeat /
- Cell Proliferation /
- Cell Movement
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全球每年死于肝病(肝硬化、病毒性肝炎和肝癌)的人数超过200万,占全球总死亡人数的4%(每25例死亡中就有1例死于肝病)[1],早期诊断和及时干预治疗是预防肝脏疾病发病率和死亡率的关键。随着科技和内镜技术的进步,内镜已逐渐在诊断和治疗肝脏疾病中崭露头角。本综述讨论了内镜在各种肝脏疾病检测、诊断和治疗中发挥的作用。
1. 超声内镜引导下肝穿刺活检(EUS-LB)
迄今为止,大部分肝脏疾病如非酒精性脂肪性肝病(NAFLD)、肝硬化、肝癌等的诊断金标准都依赖于肝组织活检来评估肝脏的受损程度及病变。主流的肝穿刺活检的方式大致分为以下3种:超声引导下经皮肝穿刺活检、经颈静脉肝穿刺活检,以及EUS-LB。其中经皮穿刺是应用最为广泛的一种检测方法,但部分在患者有腹水,或是出现凝血功能障碍的情况下,其安全性难以保障[2],而EUS-LB技术相较传统穿刺适应证范围扩大,具有不良反应少、恢复时间较短[3]等优点,3种方法的比较详见表1。目前对于EUS-LB存在争议的部分在于标本欠完整性,导致诊断率不佳,但技术的改进和器械的不断发展也大大改善了该问题。研究[7]表明,在技术规范的操作下,EUS所导致的穿刺组织破碎的概率将大幅度减低,其核心标本的形态结构将得到最大程度的保留。在一项对比经皮肝穿刺活检与EUS-LB的研究[8]中,所有行EUS-LB的患者都得到了诊断,且81%的患者有≥10个完整的肝门管,与经皮方法相当(P=0.607)。此外,研究[9-11]表明将探针从22G更换为19G可提高标本的完整性。值得一提的是,一项涵盖1 489例患者的Meta分析[12]表明,仅通过内镜医师进行的宏观现场评估也足以获取充分的样本,达到病理诊断需求(准确率91.3%,敏感度91.5%,特异度98.9%,阳性预测值98.8%)。
综上所述,EUS-LB目前在有腹水、凝血功能障碍、肥胖等合并症,或是病灶在左叶或尾状叶的患者中是一种安全、可信的诊断方式,其不良反应小、恢复快等优势有望在将来成为主流的临床肝穿刺方式。
2. EUS引导下肝囊肿/肝脓肿穿刺引流
通常情况下肝囊肿不需要特殊治疗,然而当巨大肝囊肿引起疼痛、胆管受压或破裂等临床症状时则需行治疗。目前,经皮穿刺引流是肝囊肿引流的一线方法,而EUS引导下穿刺引流有其独特的优点:(1)实现一步到位的内引流,消除了患者自行拔管的风险;(2)当病灶位于尾状叶时,EUS引导下内引流比经皮引流更容易进行干预[6];(3)清晰观察和定位脓肿,划分介入结构,避免意外并发症;(4)避免皮肤感染和瘘管形成[13]。总结研究[14-15]报告的14例使用EUS引导下行腔内支架引流的单纯性肝囊肿老年患者(平均年龄71.9岁),13例患者支架保持在原位,其中仅1例患者出现不良反应(出血),且在10.5个月的随访中单纯性肝囊肿均未复发,提示EUS引导下的内引流对于治疗高龄患者的单纯性肝囊肿是一项安全、有效的干预措施。
传统情况下,位于尾状叶的肝脓肿难以通过经皮穿刺进行干预,需要外科开腹或腹腔镜引流以免脓肿破裂流入腹腔,而EUS内引流术为这类患者提供了新的、创伤更小且安全的选择。目前,对于成年患者的肝脓肿EUS下内引流已有多篇文献[16-18]报道其有效性及安全性。此外,在容易出现自行拔除体外引流管情况的特殊人群中(如儿童[19]或意识不清的老人[20]),EUS内引流的安全性及有效性也有相应报道验证。
综上所述,对于囊肿或脓肿位于难以经皮穿刺引流的部位(如尾状叶)的或是存在意识障碍的各个年龄段患者,EUS引导下的内引流是一种安全、有效和微创的替代方法。
3. EUS引导下肝肿瘤治疗
虽然EUS引导下肝肿瘤治疗不是一线疗法,但该技术的出现为经腹或经皮难以解决的疑难或难治病例提供了更多治疗选择,而且可以最大限度地减少全身不良反应,从而提高生活质量[6]。目前常见的技术有以下几种。
3.1 EUS引导下的肝脏热疗
de Nucci等[21]报道1例使用EUS引导下射频消融术治疗的Ⅱ-Ⅲ-Ⅳb段囊下巨大肝细胞癌,有效破坏约70%的肿瘤组织,提示EUS引导射频消融术可以有效治疗传统经皮方法无法处理的左肝病变。EUS引导下冷冻消融术[22]以及高强度聚焦超声[23]目前仅在活体猪实验中得到可行性、有效性及安全性的评估,尚无临床实验可供参考。
3.2 EUS引导下的无水乙醇注射治疗
已有研究[24-25]证实EUS引导下无水乙醇注射对于难以经皮介入治疗的肝细胞癌及肝脏转移癌患者治疗的安全性及有效性,具备一定应用前景。
3.3 EUS引导下的放射治疗
Jiang等[26]对26例难以经腹介入的左侧肝恶性肿瘤患者行EUS引导下I125近距离放射治疗或乙醇注射,结果显示,接受I125治疗的患者肿瘤完全应答率达92.3%,提示EUS下近距离放射治疗是一种有效、安全的根治性手术或姑息性控制的治疗方式。
4. 内镜下血管介入治疗
传统内镜的胃底食管静脉曲张治疗是目前应用最为广泛的内镜下血管介入技术,当前主流术式包括:内镜下注射硬化剂、内镜下套扎、内镜下注胶术。其中硬化剂注射治疗是控制食管静脉(EV)曲张的高效方法,但在血流速度较快的胃底静脉(GV)中硬化剂难以停滞起效。与硬化剂治疗相比,套扎术具有止血快、治疗次数少、GV再出血率低、减少活动性喷出性EV出血等有效性和安全性方面的优势[27]。而内镜下静脉曲张注胶术已被证明是GV的最佳治疗方案[28]。
EUS引导下血管介入治疗最早于2000年报道[29],目前技术发展趋于成熟,广泛应用于胃静脉曲张治疗和门静脉压力梯度(PPG)测定。EUS引导下的胃静脉曲张治疗与传统的内镜相比具有以下优势:在急性出血时直接观察受到阻碍时,提高注射治疗的精确度[30];止血弹簧圈使得在低再出血率(0~16%)、低副作用(0~7%)的情况下,保证高止血率(约99%)[31-33];可以提供止血情况的实时反馈[34]。
常规的经颈静脉肝静脉压力梯度测定是由介入科进行,但存在辐射、需要造影剂等缺点。EUS引导下PPG测定(EUS-PPG)是一种可直接测量肝静脉PPG的新型技术。研究[35]表明,EUS-PPG成功率高(91.7%),且EUS-PPG与经颈静脉肝静脉压力梯度测定显著相关(Pearson相关系数为0.923,P<0.001),提示EUS-PPG是评估门静脉高压的一种安全而准确的方法。一项包含83例接受EUS-PPG患者的单中心回顾性研究[36]中,EUS-PPG成功率为100%,而且在同时接受EUS-LB的71例患者中无不良事件发生,其中98.6%的组织学标本足以得出病理学诊断。因此,该技术为肝硬化患者提供了一种内镜下同时进行肝穿刺以及PPG测定的安全、可靠方法,并可减少侵入性操作的次数。
5. 内镜下的减重技术对脂肪肝的疗效
目前,NAFLD患者在全球的患病比例约为25%,而随着生活水平及膳食结构的改变,该比例日益增高[37],通常建议将减重在内的生活方式干预作为一线治疗手段[38]。对于肥胖的NAFLD患者而言,不仅缺乏有效的治疗药物,而且外科代谢手术因其创伤大难以被患者接受。因此,内镜下减重手术现已成为研究新热点(表2)。
内镜下IGB是内镜辅助下将一个充入液体的气球临时引入胃部,以达到饱腹感,多项研究[40-41]证实了其对NAFLD/非酒精脂肪性肝炎的改善作用。Bazerbachi等[42]研究报道了21例早期纤维化的肥胖患者在接受IGB 6个月后,80%的患者获得了2分及以上的改善,其安全性也相对有保障(仅5%患者报告了术后疼痛,此外无严重不良事件)。Jirapinyo等[44]报道了肝纤维化≥F2的45例肥胖患者在接受内镜下胃折叠术6~12个月后,肝损伤相关生化指标以及肝瞬时弹性值显著下降(P=0.000 1~0.03)。
小肠相关内镜下减重手术是较为新颖的技术手段。十二指肠空肠旁路套管模仿外科减重的胃旁路术,通过在近端小肠置入可拆卸的套管,以减少食物在小肠的吸收面积从而实现减重效果的一种技术。多项研究[45-48]表明十二指肠空肠旁路套管可以在3~6个月内迅速改善NAFLD相关的血清学指标(P<0.05)及肝弹性成像情况。小肠黏膜消融术(DMR)是对乳头后十二指肠黏膜进行水热消融,从而改变黏膜和吸收特性。Mingrone等[49]的多中心随机对照研究中显示,欧洲患者DMR术后12周的磁共振质子密度脂肪分数较基线下降30%以上的比例显著高于假手术组(P=0.008),而在巴西人群中未见显著差异,提示了不同人群中NAFLD对于DMR的应答不同。van Baar等[50]的一项多中心前瞻性研究提示DMR术后6个月,转氨酶显著下降(P<0.001)。
目前,内镜下减肥技术仅有IGB通过FDA批准的同时被ASMBS认证,适用于BMI 30~40 kg/m2且无法通过节食和运动减肥的肥胖成年患者。小肠相关内镜下减重技术尚在研发阶段,但由于其在减重的同时具有对代谢途径的干预,目前研究表明其发展前景可期,有望为肥胖合并NAFLD的患者带来快速、小创伤的有效治疗手段。
6. 结语
内镜技术的发展为肝脏疾病的诊疗带来了许多新的选择:EUS-LB随着技术的成熟以及器械的更新逐渐成为诊断肝脏疾病的可靠、快速、安全的方式;EUS引导下的肝脓肿/囊肿穿刺引流为使得患者无需体外带管,显著提高了患者生活质量,同时对位于尾状叶或肝左叶等不便于经皮穿刺的位置提供了新的微创选择;内镜下的血管介入技术,从传统的静脉曲张硬化剂注射治疗发展到如见EUS下止血弹簧圈置入等技术,适应了临床多样化的需求,此外甚至可以在一次侵入性操作下同时安全、快速地完成门静脉压力测量与肝活检;内镜下减重技术的出现使得肥胖的NAFLD/非酒精脂肪性肝炎患者在小创伤、无体表切口的情况下实现减重以及肝脏非侵入性指标的快速显著改善。然而部分临床研究也显示地域或人种对于技术的不同应答。我国人群的内镜下肝脏疾病治疗及改善有待进一步研究及探索,从而对临床实践提供参考与依据。
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