天冬酰胺合成酶在肝细胞癌中的表达与预后分析及功能研究
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摘要
肝细胞癌（HCC）是世界范围内最常见恶性肿瘤，其发病率在所有恶性肿瘤中排第五位，死亡率位于第三位。HCC也是我国常见高发肿瘤之一，每年新增患者占全世界新增患者的一半，且发病率逐年上升。HCC高复发、高转移、对化疗敏感性低、预后差，是全球最常见的致死性恶性肿瘤之一。HCC同乙型肝炎病毒（HBV）感染、丙型肝炎病毒（HCV）感染、酗酒以及黄曲霉毒素污染等因素相关，其发生与发展是由体内多种基因参与的，多种步骤协同作用的一个复杂过程。
手术切除、肝脏移植、局部消融以及经皮肝动脉化疗栓塞术（TACE）是目前HCC的常用治疗方法，其中，外科根治性切除是目前HCC首选治疗方法之一，但术后极易复发，并成为死亡的主要原因。手术切除及肝移植是目前治疗HCC最为有效的方法，但是HCC患者在患病早期症状并不明显，并且缺少特异性高的肿瘤标志物以及供肝缺乏等原因，只有少数病人能够获得手术切除或者肝移植的机会，加之HCC具有高度恶性的生物学行为，手术后容易出现肿瘤的复发以及转移，从而影响患者生存率，在全世界范围内，HCC的5年生存率仅有3%-5%，HCC术后5年生存率也仅有30%-40%。此外，对化疗药物的不敏感，也是HCC患者生存率低的一个原因。目前临床上常用的TNM分期和BCLC分期，是HCC预后的重要参考指标，但传统的临床分期来预测肝癌的预后是有缺陷的，HCC的预后，与其自身分子生物学行为密切相关。此外，传统的HCC病理学诊断、分型以及分类方法也存在局限性，无法从分子水平上体现肝癌的生物学行为，无法充分反映HCC的真实本质，更无法准确预测HCC的分子生物学行为。因此，寻找能够反映HCC分子生物学特征的标志，并对其功能进行研究，是目前肝癌研究的重要方向。
天冬酰胺合成酶（ASNS）主要定位于胞浆中，依赖ATP催化天冬氨酸与谷氨酰胺生成天冬酰胺与谷氨酸。ASNS在仓鼠的ts11细胞中发挥促进细胞通过G1期从而完成细胞周期的作用；ASNS过表达可以减轻胰腺癌细胞因为葡萄糖剥夺或者顺铂诱导的肿瘤细胞的凋亡，提示其在胰腺癌细胞中可能起到抗凋亡的作用；ASNS在急性淋巴细胞白血病以及部分急性髓性细胞白血病对天冬酰胺酶治疗耐药的病人中的表达升高，故ASNS的表达强弱被认为同天冬酰胺酶是否耐药相关；ASNS也可以作为在卵巢癌及胰腺癌的细胞系中天冬酰胺酶治疗敏感性的生物学标志，ASNS低表达的肿瘤细胞对天冬酰胺酶更敏感；此外，还有研究认为ASNS在去势手术耐受的前列腺癌（CRPC）中高表达，利用ASNS抑制剂来耗竭天冬酰胺可能是治疗CRPC病人的一个新的方法策略。
我们前期通过蛋白组学技术筛选出ASNS在肝细胞癌肿瘤组织中特异性高表达，提示ASNS可能在肝细胞癌的发生发展中发挥作用。目前对ASNS在肝细胞癌中的表达情况、预后分析以及功能研究尚无文献报道。因此，本研究的目的一方面研究ASNS在肝癌中的表达情况并结合临床资料探讨其表达与肝癌发生发展的关系；另一方面研究ASNS在肝癌中的生物学作用及机制；为深入了解肝细胞癌的生物学行为以及为可能的靶向治疗提供理论依据。
实验方法：
1.运用real-timePCR、westernblot以及IHC等方法检测肝细胞癌、癌旁及正常肝组织中ASNS的表达情况；
2.IHC方法检测大鼠DEN诱癌过程中ASNS在肝脏中表达的动态变化过程；
3.结合临床资料统计分析ASNS与相关临床指标及预后之间的关系；
4.资料收集及统计方法：
(1)随机选取2002-2007年在我院行肝癌根治术的原发性肝细胞癌患者标本269例，制作组织芯片，并收集完善病史及随访资料；以免疫组化方法检测组织芯片标本中ASNS的表达，并统计分析ASNS的表达与肝癌患者各项临床特征（肿瘤大小、血管侵犯、肝内播散和远处转移等）及远期生存（无瘤生存率、总生存率和生存时间等）的关系。
(2)用SPSS17.0软件进行统计分析。结果以median(Q1-Q3)表示。χ2检验或Fisher确切概率法用来进行定性资料的比较。组间定量分析用wilcoxon配对秩检验。相关性分析采用spearman秩相关。累计生存和复发率的计算采用Kaplan-Meier，生存曲线用Log-rank绘制并进行生存分析。P<0.05视为统计显著。
5.构建ASNS过表达及RNA干扰质粒并转染肝癌细胞系，westernblot鉴定效果；构建ASNSRNA干扰慢病毒并感染肝癌细胞系，获得ASNS稳定敲减的MHCC97H及MHCCLM3细胞株。
6.CCK-8方法检测ASNS高低表达对多种肝癌细胞系增殖的影响；
7.平板克隆形式实验检测ASNS高低表达细胞株的克隆形成能力；
8.Transwell方法检测ASNS高低表达细胞株的迁移及侵袭能力；
9.spheroid克隆形成实验检测ASNS高低表达对细胞株的干细胞自我更新能力的影响；
10.用westernblot检测在TNFα与CHX刺激下ASNS高低表达对细胞凋亡的影响；
11.扩增ASNS高低表达稳转细胞系行裸鼠皮下荷瘤，观察肿瘤生长情况；
12.ASNS高低表达稳转细胞株行裸鼠尾静脉注射，观察肺转移情况及裸鼠生存情况；
13.CCK-8方法检测ASNS高低表达细胞株在天冬酰胺酶处理后的增殖能力；
14.裸鼠皮下荷瘤后腹腔内注射天冬酰胺酶，观察ASNS高低表达细胞株肿瘤生长情况；
15.观察高低表达ASNS肝癌细胞对抗外界刺激能力（H2O2）的影响；
实验结果:
1.ASNS在肝癌组织中高表达：
(1)ASNS在58例原发性肝细胞癌组织中的mRNA表达水平显著高于及癌旁组织样本中，两者差别具有统计学意义(P=0.0008)。与18例正常肝组织相比，58对原发性肝细胞癌及癌旁组织样本中ASNS的mRNA表达水平显著增高，差别均有统计学意义(P=0.0036，P=0.0258)。58对原发性肝癌与癌旁组织中，ASNS的mRNA表达水平，癌组织中高于癌旁组织的有44对（44/58，76%）。
(2)ASNS在10对肝癌及其癌旁组织样本中的蛋白表达，7对（70%）在癌组织中表达高于癌旁组织。
(3)在269例肝癌病人组织芯片标本中，ASNS在肝癌组织中蛋白水平的表达高于癌旁组织及正常肝脏组织，差别具有统计学意义（P<0.01，P<0.01）；
2.在大鼠DEN诱癌过程中，ASNS在肝脏中的表达逐渐升高：IHC显示随着DEN诱癌时间延长，ASNS表达逐渐升高，在晚期肝硬化期及早期肝癌期ASNS的表达水平达到最高，随着时间的延长与肿瘤的进展，ASNS的表达水平下降，而对照组大鼠肝脏ASNS的表达无明显变化，始终低于DEN组。
3.临床资料统计分析：
(1)卡方检验显示ASNS在肝细胞癌中的表达水平与血清AFP水平、肿瘤直径、镜下血管侵犯、肿瘤包膜完整性、BCLC分期及TNM分期负相关，与术前白蛋白水平正相关；
(2)ASNS表达与1，3，5年生存时间呈正相关并具有统计学意义，ASNS是影响肝癌患者术后总体生存率的独立预后保护因素（Hazardration0.744，95%CI0.565-0.979）。
4.构建了ASNS过表达及RNA干扰质粒，构建了ASNS的RNA干扰慢病毒并分别构建和鉴定了MHCCLM3及MHCC97H高低表达的稳定细胞系；
5.ASNS的差异表达对肝癌生物学功能的影响：
(1)瞬时转染过表达ASNS抑制肝癌细胞增殖，ASNS敲减促进肝癌细胞增殖；
(2)ASNS敲减增强肝癌细胞克隆隆形成能力；
(3)ASNS过表达下调CyclinD1表达并引起细胞周期阻滞于G1期；ASNS敲减促进细胞周期的进展并上调CyclinD1表达；
(4)ASNS敲减促进皮下荷瘤裸鼠的肿瘤增殖能力；
(5)ASNS敲减增强肝癌细胞迁移能力及侵袭能力（P<0.01），过表达ASNS抑制肝癌细胞的迁移能力（P<0.01）；
(6)ASNS敲减增强尾静脉注射裸鼠的肝癌细胞的肺转移能力（P<0.01），增加其死亡率（P<0.05）；
(7)ASNS敲减抑制肝癌细胞的凋亡；
(8)ASNS敲减可减轻肝癌细胞中的氧化应激；
(9)ASNS敲减可增强肝癌细胞的自我更新能力（P<0.01）。
6.ASNS敲减增强肝癌细胞对天冬酰胺酶的敏感性：
(1)天冬酰胺酶对ASNS低表达的SMMC7721与PLC细胞株增殖的抑制明显强于ASNS高表达的MHCCLM3与MHCC97H细胞株；在天冬酰胺酶处理下，ASNS敲减的肝癌细胞增殖能力显著低于对照细胞株（P<0.01）；
(2)皮下注射ASNS敲减肝癌细胞的裸鼠经腹腔注射天冬酰胺酶，其肿瘤体积较皮下注射对照细胞组裸鼠显著减小（P<0.01）。
结论:
本研究发现ASNS在肝癌组织中高表达，与血清AFP水平、术前白蛋白水平、肿瘤直径、镜下血管侵犯、肿瘤包膜完整性、BCLC分期及TNM分期密切相关。ASNS是肝癌患者术后生存的独立预后因素，有望成为肝癌预后评估的一项有前景的指标。进一步体内和体外实验研究表明ASNS在肝癌发生过程中起抑制肿瘤细胞增殖、迁移、侵袭及自我更新，促进凋亡与增强氧化应激的作用，其抑制增殖可能通过抑制细胞周期素CyclinD1的表达而发挥其生物学作用。另外，ASNS的表达与天冬酰胺酶处理的敏感性有关，提示ASNS有可能成为肝细胞癌用天冬酰胺酶治疗的一个潜在的标志性分子。
Hepatocellular carcinoma (HCC) is one of the most common malignant neoplasms and the third leading cause of death from cancer worldwide. Despite many kinds of treatments, such as surgical resection, local ablation, transplantation, transcatheter arterial chemoembolization (TACE) and so on, the prognosis still remains dismal. Across all countries,5-year overall survival of HCC is only3-5%. The poor prognosis of patients with HCC is largely due to the high frequency of recurrence and metastasis after surgical resection, in addition to resistance to systemic chemotherapy. Therefore, the limited therapeutic options and poor prognosis have triggered the search for molecular markers related to clinical outcomes, which will provide new targets for intervention of progression of HCC.
The asparagine synthetase (ASNS) gene, encoding the enzyme that catalyzes the biosynthesis of asparagine from aspartate in an ATP-dependent reaction for which glutamine is the nitrogen source. The transcription of ASNS gene is highly regulated by the nutritional status of the cell. Early studies showed that elevated expression of ASNS was correlated with drug resistance of leukemic cells to L-asparaginase, which is a universally used component of childhood acute lymphoblastic leukemia (ALL) and some forms of acute myeloblastic leukemia (AML) treatments. Similarly, ASNS was considered as a causal, predictive biomarker for L-asparaginase activity in ovarian cancer cell. Furthermore, it has also been showed that enhanced expression of ASNS protects pancreatic cancer cells from apoptosis induced by glucose deprivation and cisplatin. Recent studies suggested that ASNS was up-regulated in castration-resistant prostate cancer (CRPC) and depletion of asparagine using ASNS inhibitors might be a novel strategy for targeting CRPC cells. However, the expression and the functional roles of ASNS in solid tumours still remain uncertain, especially in HCC. The significance of ASNS expression level in the prognostic evaluation of HCC patients who undergo hepatectomy has not been reported yet. By proteomics approach, we identified ASNS higher expressed in HCC tumour tissues compared with normal liver specimens. Hence, it is meaningful to investigate the clinical significance and biological function of ASNS in the development of HCC.
This study aimed to investigate the ASNS expression pattern and determine its contribution to HCC progression and clinical prognostic values. In addition, the functional role of ASNS in HCC development and its therapeutic potential were also addressed. The results indicated that expression of ASNS was significantly correlated with clinical characteristics, predicting HCC patients'outcome after surgical resection and chemotherapy target for HCC.
In the present study, we investigated ASNS is overexpressed in HCC tumour specimens.By proteomics approach, we identified that ASNS protein level was much higher in HCC tumour tissues than normal liver specimens. Real-time RT-PCR assay was performed for transcripts of ASNS from frozen paired samples derived from58patients with HCC and18normal live tissues. The expression of ASNS mRNA varied greatly between tumour and their adjacent tissues, with76%(44of58) tumour tissues expression higher than normal like tissues. The upregulation of ASNS was further confirmed by Immunoblotting and immuneohistochemistry assay.
ASNS protein was stained in liver tissues of the DEN carcinogenesis rat by immuneohistochemistry to observe the dynamic expression of ASNS in the process from hepatitis, cirrhosis to HCC. ASNS expression was significantly higher in DEN treated liver than control group after injecting DEN for8weeks, and up to highest expression in12-14weeks, which period is advanced cirrhosis and early HCC stage. While the ASNS expression was moderately decreased in16-20weeks, which period is advanced HCC stage. In accordance with the findings in animal model experiments, in clinical samples, high expression of ASNS was associated with smaller tumour size and early tumour stage and low ASNS expression was correlated with larger tumour size and advanced tumour stage.
The relationship between ASNS expression levels in tumour tissues and the clinicopathological characteristics of269patients was examed in the TMA analysis. Pearson χ2test indicated that expression of ASNS was significantly correlated with serum AFP level (P=0.040), tumour size (P=0.002), microscopic vascular invasion (P=0.003), tumour encapsulation (P=0.007), TNM stage (P=0.039) and BCLC stage (P=0.039). Other clinical characteristics were not closely related to the expression of ASNS, including age, gender, tumour number, intrahepatic metastasis, distant metastasis, portal venous invasion, cirrhosis, and hepatitis background. Kaplan-Meier survival curves with comparisons of high ASNS expression versus low expression in269HCC patients are shown. ASNS expression levels were negatively correlated with1-year,3-year and5-year survival rates (52.6%,29.2%and25.3% for high ASNS expression versus37.4%,20.0%and13.9%for low expression; P=0.002,0.005and0.002). Additionally, the1-year,3-year and5-year disease-free survival rates in high ASNS expression patients were lower than those in low ASNS expression patients, however, no significant difference was observed.Univariate analysis of recurrence-related and survival-related clinicopathological variables revealed that ASNS and serum AFP levels were predictors for OS. Gender, HBV, tumour size, intrahepatic metastasis, portal venous invasion, distant metastasis, microscopic vascular invasion, encapsulation, TNM stage and BCLC stage were statistically correlated with both recurrence and survival. These individual parameters were further subjected to multivariate Cox proportional hazards model, which demonstrated that ASNS expression level, together with HBV, intrahepatic metastasis, microscopic vascular invasion and BCLC stage was strongly associated with OS. ASNS was an independent prognostic indicator for the survival of HCC patients (HR0.744,95%CI0.565-0.979, P=0.035).
We used lentivirus delivered shRNA targeting ASNS to infect HCC cells, and constructed stably transfected cells. Also, HCC cells were transient transfected with GV142/ASNS vector. We found that overexpression of ASNS in HCC cells suppressed cell proliferation by CCK-8assay. Furthermore, ASNS knockdown revealed increased proliferation and colony formation in ASNS knockdown cells compared with that of control cells. The Cell cycle distribution demonstrated ASNS knockdown could hasten cell cycle progression In addition, we found Cyclin Dl protein decreased in overexpressed ASNS cells, while increased in knockdown cells. Stably transfected HCC cells were inoculated into flanks of nude mice and the effect of ASNS on xenograft tumour growth was also observed. Compared with control cells, ASNS knockdown cells showed a significant increase in tumour size. These results indicated that ASNS knockdown promoted HCC growth both in vitro and in vivo.
Transwell experiments showed that depletion of ASNS markedly increased cell migration and invasion capacity. Restoration of ASNS expression in SMMC7721cells repressed cell migration. For further examining the effect of ASNS knockdown, stably transfected MHCC97H cells were injected into tail vein of nude mice. Six weeks later, ASNS knockdown resulted in significantly increased number and sizes of pulmonary metastatic lesions. Furthermore, ASNS knockdown group had a shorter survival period in comparison with control group. These results demonstrate an enhanced metastatic potential in low ASNS expression HCC cells. HCC cells were treated with L-asparaginase and cell proliferation was detected by CCK-8. Compared with cells with high ASNS (MHCCLM3and MHCC97H), L-asparaginase exerted a much higher effect on cells with low ASNS expression (SMMC7721and PLC). Moreover, in the detection of stably transfected HCC cells, it showed decreased proliferation in ASNS knockdown cells in the presence of L-asparaginase compared with control. Futhermore, the effect of L-asparaginase was also observed in vivo. Nude mice bearing MHCCLM3tumour xenografts were treated with L-asparaginase every other day by intraperitoneal injection. ASNS knockdown group revealed a delayed tumour formation and a significant reduction in tumour size compared with that of control group. These results revealed an increased sensitivity of low ASNS expression cells to L-asparaginase.
In this study, we presented the upregulation of ASNS at both mRNA and protein levels in HCC. The association of ASNS expression with clinicopathological features was further investigated in HCC patients by TMA, which showed a significant correlation of ASNS expression with many clinical features, indicating low expression of ASNS was associated with malignant clinicopathological characteristics. In DEN carcinogenesis rat, ASNS was high expressed from inflammation stage to advanced HCC stage and reached a peak at advanced cirrhosis and early HCC stage. Moreover, moderated decreased ASNS expression was observed in advanced cancer stage in comparison with early cancer stage, which was validated by human HCC clinical specimens. The Kaplan-Meier analysis showed that patients with HCC, who had high ASNS expression in general, had better prognosis than those with low expression. Multivariate analysis revealed that ASNS expression level was an independent and significant prognostic indicator affecting survival after surgical resection.
In further confirming the role of ASNS in HCC, the result showed increased proliferation and colony formation ability in vitro and elevated tumourigenicity of xenografts in nude mice in low ASNS cells. In accordance with correlation analysis, these results imply that ASNS is a tumour growth suppressor in HCC. Overexpression of ASNS severely inhibited the migration capacity and ASNS knockdown significantly increased migration and invasion abilities in vitro. The result was further confirmed by pulmonary metastatic model in vivo.
In later investigations, we also observed that ASNS overexpression increased apoptosis sensitivities of cells upon various stimuli. HCC cells with low ASNS expression were more sensitive to L-asparaginase than that exhibit high ASNS expression both in vitro and in vivo.
In conclusion, this study explored the effects of ASNS in tumour progression and prognosis significant in HCC patients. ASNS expression level was an independent and significant prognostic indicator affecting survival after surgical resection. Low expression of ASNS might be an indicator for the patients treated with L-asparaginase.
手术切除、肝脏移植、局部消融以及经皮肝动脉化疗栓塞术（TACE）是目前HCC的常用治疗方法，其中，外科根治性切除是目前HCC首选治疗方法之一，但术后极易复发，并成为死亡的主要原因。手术切除及肝移植是目前治疗HCC最为有效的方法，但是HCC患者在患病早期症状并不明显，并且缺少特异性高的肿瘤标志物以及供肝缺乏等原因，只有少数病人能够获得手术切除或者肝移植的机会，加之HCC具有高度恶性的生物学行为，手术后容易出现肿瘤的复发以及转移，从而影响患者生存率，在全世界范围内，HCC的5年生存率仅有3%-5%，HCC术后5年生存率也仅有30%-40%。此外，对化疗药物的不敏感，也是HCC患者生存率低的一个原因。目前临床上常用的TNM分期和BCLC分期，是HCC预后的重要参考指标，但传统的临床分期来预测肝癌的预后是有缺陷的，HCC的预后，与其自身分子生物学行为密切相关。此外，传统的HCC病理学诊断、分型以及分类方法也存在局限性，无法从分子水平上体现肝癌的生物学行为，无法充分反映HCC的真实本质，更无法准确预测HCC的分子生物学行为。因此，寻找能够反映HCC分子生物学特征的标志，并对其功能进行研究，是目前肝癌研究的重要方向。
天冬酰胺合成酶（ASNS）主要定位于胞浆中，依赖ATP催化天冬氨酸与谷氨酰胺生成天冬酰胺与谷氨酸。ASNS在仓鼠的ts11细胞中发挥促进细胞通过G1期从而完成细胞周期的作用；ASNS过表达可以减轻胰腺癌细胞因为葡萄糖剥夺或者顺铂诱导的肿瘤细胞的凋亡，提示其在胰腺癌细胞中可能起到抗凋亡的作用；ASNS在急性淋巴细胞白血病以及部分急性髓性细胞白血病对天冬酰胺酶治疗耐药的病人中的表达升高，故ASNS的表达强弱被认为同天冬酰胺酶是否耐药相关；ASNS也可以作为在卵巢癌及胰腺癌的细胞系中天冬酰胺酶治疗敏感性的生物学标志，ASNS低表达的肿瘤细胞对天冬酰胺酶更敏感；此外，还有研究认为ASNS在去势手术耐受的前列腺癌（CRPC）中高表达，利用ASNS抑制剂来耗竭天冬酰胺可能是治疗CRPC病人的一个新的方法策略。
我们前期通过蛋白组学技术筛选出ASNS在肝细胞癌肿瘤组织中特异性高表达，提示ASNS可能在肝细胞癌的发生发展中发挥作用。目前对ASNS在肝细胞癌中的表达情况、预后分析以及功能研究尚无文献报道。因此，本研究的目的一方面研究ASNS在肝癌中的表达情况并结合临床资料探讨其表达与肝癌发生发展的关系；另一方面研究ASNS在肝癌中的生物学作用及机制；为深入了解肝细胞癌的生物学行为以及为可能的靶向治疗提供理论依据。
实验方法：
1.运用real-timePCR、westernblot以及IHC等方法检测肝细胞癌、癌旁及正常肝组织中ASNS的表达情况；
2.IHC方法检测大鼠DEN诱癌过程中ASNS在肝脏中表达的动态变化过程；
3.结合临床资料统计分析ASNS与相关临床指标及预后之间的关系；
4.资料收集及统计方法：
(1)随机选取2002-2007年在我院行肝癌根治术的原发性肝细胞癌患者标本269例，制作组织芯片，并收集完善病史及随访资料；以免疫组化方法检测组织芯片标本中ASNS的表达，并统计分析ASNS的表达与肝癌患者各项临床特征（肿瘤大小、血管侵犯、肝内播散和远处转移等）及远期生存（无瘤生存率、总生存率和生存时间等）的关系。
(2)用SPSS17.0软件进行统计分析。结果以median(Q1-Q3)表示。χ2检验或Fisher确切概率法用来进行定性资料的比较。组间定量分析用wilcoxon配对秩检验。相关性分析采用spearman秩相关。累计生存和复发率的计算采用Kaplan-Meier，生存曲线用Log-rank绘制并进行生存分析。P<0.05视为统计显著。
5.构建ASNS过表达及RNA干扰质粒并转染肝癌细胞系，westernblot鉴定效果；构建ASNSRNA干扰慢病毒并感染肝癌细胞系，获得ASNS稳定敲减的MHCC97H及MHCCLM3细胞株。
6.CCK-8方法检测ASNS高低表达对多种肝癌细胞系增殖的影响；
7.平板克隆形式实验检测ASNS高低表达细胞株的克隆形成能力；
8.Transwell方法检测ASNS高低表达细胞株的迁移及侵袭能力；
9.spheroid克隆形成实验检测ASNS高低表达对细胞株的干细胞自我更新能力的影响；
10.用westernblot检测在TNFα与CHX刺激下ASNS高低表达对细胞凋亡的影响；
11.扩增ASNS高低表达稳转细胞系行裸鼠皮下荷瘤，观察肿瘤生长情况；
12.ASNS高低表达稳转细胞株行裸鼠尾静脉注射，观察肺转移情况及裸鼠生存情况；
13.CCK-8方法检测ASNS高低表达细胞株在天冬酰胺酶处理后的增殖能力；
14.裸鼠皮下荷瘤后腹腔内注射天冬酰胺酶，观察ASNS高低表达细胞株肿瘤生长情况；
15.观察高低表达ASNS肝癌细胞对抗外界刺激能力（H2O2）的影响；
实验结果:
1.ASNS在肝癌组织中高表达：
(1)ASNS在58例原发性肝细胞癌组织中的mRNA表达水平显著高于及癌旁组织样本中，两者差别具有统计学意义(P=0.0008)。与18例正常肝组织相比，58对原发性肝细胞癌及癌旁组织样本中ASNS的mRNA表达水平显著增高，差别均有统计学意义(P=0.0036，P=0.0258)。58对原发性肝癌与癌旁组织中，ASNS的mRNA表达水平，癌组织中高于癌旁组织的有44对（44/58，76%）。
(2)ASNS在10对肝癌及其癌旁组织样本中的蛋白表达，7对（70%）在癌组织中表达高于癌旁组织。
(3)在269例肝癌病人组织芯片标本中，ASNS在肝癌组织中蛋白水平的表达高于癌旁组织及正常肝脏组织，差别具有统计学意义（P<0.01，P<0.01）；
2.在大鼠DEN诱癌过程中，ASNS在肝脏中的表达逐渐升高：IHC显示随着DEN诱癌时间延长，ASNS表达逐渐升高，在晚期肝硬化期及早期肝癌期ASNS的表达水平达到最高，随着时间的延长与肿瘤的进展，ASNS的表达水平下降，而对照组大鼠肝脏ASNS的表达无明显变化，始终低于DEN组。
3.临床资料统计分析：
(1)卡方检验显示ASNS在肝细胞癌中的表达水平与血清AFP水平、肿瘤直径、镜下血管侵犯、肿瘤包膜完整性、BCLC分期及TNM分期负相关，与术前白蛋白水平正相关；
(2)ASNS表达与1，3，5年生存时间呈正相关并具有统计学意义，ASNS是影响肝癌患者术后总体生存率的独立预后保护因素（Hazardration0.744，95%CI0.565-0.979）。
4.构建了ASNS过表达及RNA干扰质粒，构建了ASNS的RNA干扰慢病毒并分别构建和鉴定了MHCCLM3及MHCC97H高低表达的稳定细胞系；
5.ASNS的差异表达对肝癌生物学功能的影响：
(1)瞬时转染过表达ASNS抑制肝癌细胞增殖，ASNS敲减促进肝癌细胞增殖；
(2)ASNS敲减增强肝癌细胞克隆隆形成能力；
(3)ASNS过表达下调CyclinD1表达并引起细胞周期阻滞于G1期；ASNS敲减促进细胞周期的进展并上调CyclinD1表达；
(4)ASNS敲减促进皮下荷瘤裸鼠的肿瘤增殖能力；
(5)ASNS敲减增强肝癌细胞迁移能力及侵袭能力（P<0.01），过表达ASNS抑制肝癌细胞的迁移能力（P<0.01）；
(6)ASNS敲减增强尾静脉注射裸鼠的肝癌细胞的肺转移能力（P<0.01），增加其死亡率（P<0.05）；
(7)ASNS敲减抑制肝癌细胞的凋亡；
(8)ASNS敲减可减轻肝癌细胞中的氧化应激；
(9)ASNS敲减可增强肝癌细胞的自我更新能力（P<0.01）。
6.ASNS敲减增强肝癌细胞对天冬酰胺酶的敏感性：
(1)天冬酰胺酶对ASNS低表达的SMMC7721与PLC细胞株增殖的抑制明显强于ASNS高表达的MHCCLM3与MHCC97H细胞株；在天冬酰胺酶处理下，ASNS敲减的肝癌细胞增殖能力显著低于对照细胞株（P<0.01）；
(2)皮下注射ASNS敲减肝癌细胞的裸鼠经腹腔注射天冬酰胺酶，其肿瘤体积较皮下注射对照细胞组裸鼠显著减小（P<0.01）。
结论:
本研究发现ASNS在肝癌组织中高表达，与血清AFP水平、术前白蛋白水平、肿瘤直径、镜下血管侵犯、肿瘤包膜完整性、BCLC分期及TNM分期密切相关。ASNS是肝癌患者术后生存的独立预后因素，有望成为肝癌预后评估的一项有前景的指标。进一步体内和体外实验研究表明ASNS在肝癌发生过程中起抑制肿瘤细胞增殖、迁移、侵袭及自我更新，促进凋亡与增强氧化应激的作用，其抑制增殖可能通过抑制细胞周期素CyclinD1的表达而发挥其生物学作用。另外，ASNS的表达与天冬酰胺酶处理的敏感性有关，提示ASNS有可能成为肝细胞癌用天冬酰胺酶治疗的一个潜在的标志性分子。
Hepatocellular carcinoma (HCC) is one of the most common malignant neoplasms and the third leading cause of death from cancer worldwide. Despite many kinds of treatments, such as surgical resection, local ablation, transplantation, transcatheter arterial chemoembolization (TACE) and so on, the prognosis still remains dismal. Across all countries,5-year overall survival of HCC is only3-5%. The poor prognosis of patients with HCC is largely due to the high frequency of recurrence and metastasis after surgical resection, in addition to resistance to systemic chemotherapy. Therefore, the limited therapeutic options and poor prognosis have triggered the search for molecular markers related to clinical outcomes, which will provide new targets for intervention of progression of HCC.
The asparagine synthetase (ASNS) gene, encoding the enzyme that catalyzes the biosynthesis of asparagine from aspartate in an ATP-dependent reaction for which glutamine is the nitrogen source. The transcription of ASNS gene is highly regulated by the nutritional status of the cell. Early studies showed that elevated expression of ASNS was correlated with drug resistance of leukemic cells to L-asparaginase, which is a universally used component of childhood acute lymphoblastic leukemia (ALL) and some forms of acute myeloblastic leukemia (AML) treatments. Similarly, ASNS was considered as a causal, predictive biomarker for L-asparaginase activity in ovarian cancer cell. Furthermore, it has also been showed that enhanced expression of ASNS protects pancreatic cancer cells from apoptosis induced by glucose deprivation and cisplatin. Recent studies suggested that ASNS was up-regulated in castration-resistant prostate cancer (CRPC) and depletion of asparagine using ASNS inhibitors might be a novel strategy for targeting CRPC cells. However, the expression and the functional roles of ASNS in solid tumours still remain uncertain, especially in HCC. The significance of ASNS expression level in the prognostic evaluation of HCC patients who undergo hepatectomy has not been reported yet. By proteomics approach, we identified ASNS higher expressed in HCC tumour tissues compared with normal liver specimens. Hence, it is meaningful to investigate the clinical significance and biological function of ASNS in the development of HCC.
This study aimed to investigate the ASNS expression pattern and determine its contribution to HCC progression and clinical prognostic values. In addition, the functional role of ASNS in HCC development and its therapeutic potential were also addressed. The results indicated that expression of ASNS was significantly correlated with clinical characteristics, predicting HCC patients'outcome after surgical resection and chemotherapy target for HCC.
In the present study, we investigated ASNS is overexpressed in HCC tumour specimens.By proteomics approach, we identified that ASNS protein level was much higher in HCC tumour tissues than normal liver specimens. Real-time RT-PCR assay was performed for transcripts of ASNS from frozen paired samples derived from58patients with HCC and18normal live tissues. The expression of ASNS mRNA varied greatly between tumour and their adjacent tissues, with76%(44of58) tumour tissues expression higher than normal like tissues. The upregulation of ASNS was further confirmed by Immunoblotting and immuneohistochemistry assay.
ASNS protein was stained in liver tissues of the DEN carcinogenesis rat by immuneohistochemistry to observe the dynamic expression of ASNS in the process from hepatitis, cirrhosis to HCC. ASNS expression was significantly higher in DEN treated liver than control group after injecting DEN for8weeks, and up to highest expression in12-14weeks, which period is advanced cirrhosis and early HCC stage. While the ASNS expression was moderately decreased in16-20weeks, which period is advanced HCC stage. In accordance with the findings in animal model experiments, in clinical samples, high expression of ASNS was associated with smaller tumour size and early tumour stage and low ASNS expression was correlated with larger tumour size and advanced tumour stage.
The relationship between ASNS expression levels in tumour tissues and the clinicopathological characteristics of269patients was examed in the TMA analysis. Pearson χ2test indicated that expression of ASNS was significantly correlated with serum AFP level (P=0.040), tumour size (P=0.002), microscopic vascular invasion (P=0.003), tumour encapsulation (P=0.007), TNM stage (P=0.039) and BCLC stage (P=0.039). Other clinical characteristics were not closely related to the expression of ASNS, including age, gender, tumour number, intrahepatic metastasis, distant metastasis, portal venous invasion, cirrhosis, and hepatitis background. Kaplan-Meier survival curves with comparisons of high ASNS expression versus low expression in269HCC patients are shown. ASNS expression levels were negatively correlated with1-year,3-year and5-year survival rates (52.6%,29.2%and25.3% for high ASNS expression versus37.4%,20.0%and13.9%for low expression; P=0.002,0.005and0.002). Additionally, the1-year,3-year and5-year disease-free survival rates in high ASNS expression patients were lower than those in low ASNS expression patients, however, no significant difference was observed.Univariate analysis of recurrence-related and survival-related clinicopathological variables revealed that ASNS and serum AFP levels were predictors for OS. Gender, HBV, tumour size, intrahepatic metastasis, portal venous invasion, distant metastasis, microscopic vascular invasion, encapsulation, TNM stage and BCLC stage were statistically correlated with both recurrence and survival. These individual parameters were further subjected to multivariate Cox proportional hazards model, which demonstrated that ASNS expression level, together with HBV, intrahepatic metastasis, microscopic vascular invasion and BCLC stage was strongly associated with OS. ASNS was an independent prognostic indicator for the survival of HCC patients (HR0.744,95%CI0.565-0.979, P=0.035).
We used lentivirus delivered shRNA targeting ASNS to infect HCC cells, and constructed stably transfected cells. Also, HCC cells were transient transfected with GV142/ASNS vector. We found that overexpression of ASNS in HCC cells suppressed cell proliferation by CCK-8assay. Furthermore, ASNS knockdown revealed increased proliferation and colony formation in ASNS knockdown cells compared with that of control cells. The Cell cycle distribution demonstrated ASNS knockdown could hasten cell cycle progression In addition, we found Cyclin Dl protein decreased in overexpressed ASNS cells, while increased in knockdown cells. Stably transfected HCC cells were inoculated into flanks of nude mice and the effect of ASNS on xenograft tumour growth was also observed. Compared with control cells, ASNS knockdown cells showed a significant increase in tumour size. These results indicated that ASNS knockdown promoted HCC growth both in vitro and in vivo.
Transwell experiments showed that depletion of ASNS markedly increased cell migration and invasion capacity. Restoration of ASNS expression in SMMC7721cells repressed cell migration. For further examining the effect of ASNS knockdown, stably transfected MHCC97H cells were injected into tail vein of nude mice. Six weeks later, ASNS knockdown resulted in significantly increased number and sizes of pulmonary metastatic lesions. Furthermore, ASNS knockdown group had a shorter survival period in comparison with control group. These results demonstrate an enhanced metastatic potential in low ASNS expression HCC cells. HCC cells were treated with L-asparaginase and cell proliferation was detected by CCK-8. Compared with cells with high ASNS (MHCCLM3and MHCC97H), L-asparaginase exerted a much higher effect on cells with low ASNS expression (SMMC7721and PLC). Moreover, in the detection of stably transfected HCC cells, it showed decreased proliferation in ASNS knockdown cells in the presence of L-asparaginase compared with control. Futhermore, the effect of L-asparaginase was also observed in vivo. Nude mice bearing MHCCLM3tumour xenografts were treated with L-asparaginase every other day by intraperitoneal injection. ASNS knockdown group revealed a delayed tumour formation and a significant reduction in tumour size compared with that of control group. These results revealed an increased sensitivity of low ASNS expression cells to L-asparaginase.
In this study, we presented the upregulation of ASNS at both mRNA and protein levels in HCC. The association of ASNS expression with clinicopathological features was further investigated in HCC patients by TMA, which showed a significant correlation of ASNS expression with many clinical features, indicating low expression of ASNS was associated with malignant clinicopathological characteristics. In DEN carcinogenesis rat, ASNS was high expressed from inflammation stage to advanced HCC stage and reached a peak at advanced cirrhosis and early HCC stage. Moreover, moderated decreased ASNS expression was observed in advanced cancer stage in comparison with early cancer stage, which was validated by human HCC clinical specimens. The Kaplan-Meier analysis showed that patients with HCC, who had high ASNS expression in general, had better prognosis than those with low expression. Multivariate analysis revealed that ASNS expression level was an independent and significant prognostic indicator affecting survival after surgical resection.
In further confirming the role of ASNS in HCC, the result showed increased proliferation and colony formation ability in vitro and elevated tumourigenicity of xenografts in nude mice in low ASNS cells. In accordance with correlation analysis, these results imply that ASNS is a tumour growth suppressor in HCC. Overexpression of ASNS severely inhibited the migration capacity and ASNS knockdown significantly increased migration and invasion abilities in vitro. The result was further confirmed by pulmonary metastatic model in vivo.
In later investigations, we also observed that ASNS overexpression increased apoptosis sensitivities of cells upon various stimuli. HCC cells with low ASNS expression were more sensitive to L-asparaginase than that exhibit high ASNS expression both in vitro and in vivo.
In conclusion, this study explored the effects of ASNS in tumour progression and prognosis significant in HCC patients. ASNS expression level was an independent and significant prognostic indicator affecting survival after surgical resection. Low expression of ASNS might be an indicator for the patients treated with L-asparaginase.
引文
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