【作者】 张黎；

【导师】 孙国平；

【作者基本信息】 安徽医科大学 ， 肿瘤学， 2021， 硕士

【摘要】 研究背景肝癌是临床上最常见的恶性肿瘤之一,其发病率及死亡率分别位居世界第六和第三,肝细胞癌（HCC）是肝癌中最常见的类型,占所有肝癌病例的75%～85%。多数肝癌患者肿瘤发现时已处于较晚期,肝癌复发率高,且总体预后较差,这些更增加了肝癌的治疗难度。目前,系统性化疗是肝癌患者的重要治疗手段,但近年来肝癌细胞对化疗产生耐药抵抗,因此探索肝癌发生分子机制,寻找肝癌治疗新靶点,增加肝癌细胞对抗癌药物的敏感性,对肝癌患者的治疗及生存有着极其重要的临床意义。细胞内外各种因素如缺氧、营养缺乏、氧化应激、Ca²⁺浓度改变等刺激,可使未折叠或错误折叠蛋白质在内质网中累积,这进一步加剧内质网的功能障碍,导致内质网应激（ERS）的激活。大量研究发现,持续活化的ERS可促进肿瘤生长转移、诱导肿瘤耐药反应及抑制抗肿瘤免疫。细胞为了减少错误折叠的蛋白质带来的损害、缓解应激状态,启动未折叠蛋白反应（UPR）。UPR的三条通路由3种跨膜传感器激活,包括活化转录因子-6（ATF6）、肌醇必需酶1α（IRE1α）、蛋白激酶R样内质网激酶（PERK）,UPR通过减少蛋白质翻译和增加蛋白折叠酶和分子伴侣的表达来缓解内质网应激。转录激活因子4（ATF4）是一种应激诱导的转录因子,是参与UPR的PERK途径的重要组成部分,ATF4及其靶基因与肿瘤血管生成和转移有关,是治疗癌症的潜在靶点。己糖激酶结构域成分1（HKDC1）是最近发现的第五种己糖激酶,在细胞葡萄糖代谢中起着至关重要的作用,现已有研究发现HKDC1可能在乳腺癌、肺癌、结直肠癌、肝癌及淋巴瘤中发挥致癌作用,但目前HKDC1在肝癌中的研究机制甚少。本篇研究旨在探究HKDC1在肝癌中的表达与肿瘤进展及临床预后的关系,进一步阐明其对肝癌细胞增殖、侵袭和迁移的影响,以及增强肝癌细胞对药物敏感性的作用,并探讨其可能的分子机制。目的（1）探究HKDC1在肝癌中的表达情况及其表达与肝癌进展及不良预后的关系。（2）阐明HKDC1对肝癌细胞侵袭、迁移、增殖以及药物敏感性的影响。（3）进一步探讨HKDC1发挥促癌作用可能的分子机制。方法（1）将肝细胞癌患者的石蜡包埋、福尔马林固定的癌及癌旁组织样本制备成组织芯片,用免疫组织化学法进行染色分析,检测HKDC1在癌组织和癌旁组织中的表达情况。同时,用Western blot法检测来自不同HCC患者的3对新鲜癌旁组织及肝癌组织的HKDC1蛋白表达情况。（2）使用Kaplan-Meier生存函数和TCGA肝癌基因组图谱中配对的肝癌/癌旁组织数据集,描述并分析HKDC1过表达与肝细胞癌患者病理分级、临床分期及总生存期的关系。（3）Western blot法检测正常肝细胞系LO2和五种肝癌细胞系Huh7、LM3、Hep G2、Hep3B及PLC/PRF/5中HKDC1的表达情况,后续实验选用HKDC1表达相对最高的Hep G2和HKDC1表达相对最低的Huh7细胞进行,并在Hep G2细胞中采用瞬时转染技术转染小发夹RNA敲除HKDC1,构建HKDC1低表达肝癌细胞模型;用相同方法使HKDC1在Huh7细胞中过表达,构建HKDC1高表达肝癌细胞模型。通过克隆形成实验、划痕实验、Transwell实验、流式细胞凋亡术等对细胞增殖、迁移、侵袭及药敏性进行检测。（4）在Hep G2、Huh7细胞系中采用瞬时转染技术敲除ATF4,Western blot法检测ATF4敲低后HKDC1蛋白表达变化。同时,用CCK-8法、克隆形成实验、划痕实验、Transwell实验、流式细胞术检测肝癌细胞ATF4敲低后增殖、迁移能力及凋亡的变化。结果（1）免疫组化结果显示HKDC1在肝癌组织中高表达,但在癌旁组织中明显低表达,HKDC1在肝癌组织中的表达明显高于癌旁正常组织。同样,Western blot结果显示,与相对应的癌旁组织相比,肝癌组织标本中HKDC1明显过表达。进一步的生存分析提示HKDC1高表达的肝癌患者总生存期较短。（2）TCGA肝癌基因组图谱中配对的肝癌/癌旁组织数据集分析表明,HKDC1在肝癌组织中的表达明显高于正常肝组织,且与患者的病理分级及临床分期呈正相关。基于TCGA数据的Kaplan-Meier生存分析也显示HKDC1高表达的肝癌患者总生存时间较短。（3）克隆形成实验结果表明,Huh7细胞内的HKDC1过表达促进了细胞的增殖,Hep G2细胞中的HKDC1沉默则会导致细胞增殖受到抑制。Transwell侵袭实验与划痕实验表明,Huh7细胞中的HKDC1过表达增强了细胞的迁移及侵袭能力;而降低Hep G2细胞中的HKDC1表达可显著抑制细胞迁移及侵袭。另外,流式细胞凋亡实验结果也显示,敲低HKDC1能降低Hep G2细胞对索拉菲尼的凋亡抵抗,HKDC1过表达能降低Huh7细胞对索拉菲尼的药物敏感性。（4）Western blot结果显示,敲低ATF4可下调肝癌细胞中HKDC1的蛋白表达。CCK-8法、克隆形成实验、划痕实验、流式细胞术结果显示,敲低ATF4可降低Hep G2及Huh7两种肝癌细胞系的增殖及迁移能力,并促进其凋亡。结论（1）HKDC1在肝癌中过表达,且其表达与肝癌进展及不良预后相关。（2）HKDC1能促进肝癌细胞增殖、侵袭及迁移,并降低肝癌细胞对索拉非尼的药物敏感性。（3）HKDC1可能受ATF4的调控以增强其表达,从而发挥促癌作用。更多还原

【Abstract】 BackgroundLiver cancer is one of the most common malignant tumor type in clinical practice.Its morbidity and mortality rank respectively seventh and second in the world.Hepatocellular carcinoma（HCC）is the most common type of liver cancer,accounting for 90%of all liver cancer cases.Most patients with liver cancer are already at an advanced stage when their tumors are discovered.The recurrence rate of liver cancer is high,and the overall prognosis is poor,which makes the treatment of liver cancer more difficult.At present,systemic chemotherapy is an important treatment for patients with liver cancer,but in recent years,liver cancer cells have developed resistance to chemotherapy.Therefore,exploring the molecular mechanism of liver cancer,finding new targets for the treatment of liver cancer,and increasing the sensitivity of liver cancer cells to anti-cancer drugs have extremely important clinical significance for the treatment and survival of liver cancer patients.Various factors inside and outside the cell,such as hypoxia,nutrient deficiency,oxidative stress,and Ca²⁺concentration changes,can cause unfolded or misfolded proteins to accumulate in the endoplasmic reticulum,which further aggravates the dysfunction of the endoplasmic reticulum,and lead to activation of ERS.A large number of studies have found that continuously activated ERS can promote tumor growth and metastasis,induce tumor drug resistance and inhibit anti-tumor immunity.In order to reduce the damage caused by misfolded proteins and relieve stress,the cell initiates the unfolded protein response（UPR）.The three pathways of UPR are activated by three transmembrane sensors:activated transcription factor-6（ATF6）,inositol essential enzyme 1α（IRE1α）,and protein kinase RNA-like endoplasmic reticulum kinase（PERK）.UPR relieves ERS by reducing protein translation and increasing the expression of protein folding enzymes and molecular chaperones.Activator of transcription 4（ATF4）is a stress-induced transcription factor and an important part of the PERK pathway involved in UPR.ATF4 and its target genes are related to tumor angiogenesis and metastasis,and are potential targets for cancer treatment.Hexokinase domain component 1（HKDC1）is the fifth type of hexokinase recently discovered,which plays a vital role in cellular glucose metabolism.Studies have found that HKDC1 may play a carcinogenic effect in breast cancer,lung cancer,colorectal cancer,liver cancer and lymphoma,but the current research mechanism of HKDC1 in liver cancer is very limited.This study aims to explore the relationship between the expression of HKDC1 in liver cancer and clinical staging and prognosis,further clarify its effect on the proliferation,invasion and migration of liver cancer cells,and enhance the effect of liver cancer cells on drug sensitivity,and explore its possible molecular mechanism.Objective（1）To explore the expression of HKDC1 in HCC and its relationship with liver cancer progression and poor prognosis.（2）To clarify the effects of HKDC1 on the proliferation,invasion,migration and drug sensitivity of HCC cell lines.（3）To further explore the possible molecular mechanism of cancer-promoting effect of HKDC1.Methods（1）Preparing Tissue microarray from the paraffin-embedded and formalin-fixed cancer and para-cancerous tissue samples of HCC patients.Staining analysis was performed by immunohistochemistry to detect the expression of HKDC1 in cancer tissues and adjacent tissues.At the same time,the Western Blot method was used to detect the expression levels of HKDC1 protein in 3 pairs of fresh adjacent tissues and liver cancer tissues from different HCC patients.（2）Kaplan-Meier survival function and the paired liver cancer/paracancerous tissue data set in the TCGA Liver Cancer Genome Atlas was used to describe and analyze the relationship between HKDC1 overexpression and the pathological grade,clinical stage and prognosis of HCC patients.（3）Western Blot method was used to detect the expression of HKDC1 in normal liver cell line LO2 and five liver cancer cell lines Huh7,LM3,Hep G2,Hep3B and PLC/PRF/5.We selected Hep G2 with the highest expression of HKDC1 and Huh7 cell line with the lowest expression for follow-up experiments.In Hep G2 cells,the small hairpin RNA was transfected with transient transfection technology to knock out HKDC1,and in Huh7 cells,HKDC1 was overexpressed by transfection.Through clone formation experiment,scratch experiment,Transwell experiment,flow cytometry,etc.,the proliferation,migration,invasion and drug sensitivity of liver cancer cells were detected.（4）Use transient transfection technology to knock out ATF4 with small interfering RNA.Western Blot method was used to detect the change of HKDC1 protein expression after ATF4 interference.At the same time,CCK-8 method,clone formation experiment,scratch experiment,Transwell experiment,and flow cytometry were used to detect changes in the proliferation,invasion,migration and apoptosis of liver cancer cells.Results（1）The results of immunohistochemistry showed that HKDC1 was highly expressed in liver cancer tissues,but was significantly lower in adjacent tissues.Western Blot results showed that compared with the corresponding adjacent tissues,HKDC1 was significantly overexpressed in liver cancer tissue samples.（2）Survival analysis suggests that HCC patients with high HKDC1 expression have a poor prognosis.The analysis of the TCGA data showed that the expression of HKDC1in liver cancer tissues was significantly higher than that in normal liver tissues,and it was positively correlated with the pathological grade and clinical stage of patients.Kaplan-Meier survival analysis based on TCGA data also showed that the overall survival time of liver cancer patients with high HKDC1 expression was shorter.（3）The results of the clone formation experiment showed that the silencing of HKDC1in Hep G2 inhibited cell proliferation,and overexpression in Huh7 promoted cell proliferation.Scratch experiments and Transwell invasion experiments show that reducing the expression of HKDC1 in Hep G2 can significantly inhibit the migration and invasion of HCC,and overexpression in Huh7 cells promotes the migration and invasion of HCC.In addition,the results of flow cytometry apoptosis experiments also show that knockdown of HKDC1 can reduce the apoptosis resistance of Hep G2 to sorafenib,and overexpression of HKDC1 can reduce the drug sensitivity of Huh7 to sorafenib.（4）Western Blot results showed that the knockdown of ATF4 can down-regulate the expression of HKDC1 in HCC.The results of CCK-8 method,clone formation experiment,scratch experiment,and flow cytometry showed that ATF4 silencing can inhibit the proliferation and migration of Hep G2 and huh7,and promote their apoptosis.Conclusion（1）HKDC1 is overexpressed in HCC and its expression is related to the progression and poor prognosis of HCC.（2）HKDC1 knockdown can inhibit the proliferation,invasion and migration of HCC cells,and enhance the drug sensitivity of HCC cells.（3）HKDC1 may be regulated by ATF4 to enhance its expression,thereby playing a role in promoting cancer.更多还原