【作者】 李红；

【导师】 沈玉先；

【作者基本信息】 安徽医科大学 ， 药理学， 2022， 博士

【摘要】 肝内胆管癌（Intrahepatic cholangiocarcinoma,ICC）是第二大肝脏常见恶性肿瘤,发病率仅次于肝细胞癌（Hepatocellular carcinoma,HCC）,占消化道肿瘤的10%,其恶性程度高,预后较差,占全世界癌症死亡率的13%。尽管HCC和ICC都是原发性肝癌,但两者在发病机制以及治疗方式等方面具有很大的差异。ICC预后差的主要原因之一是其发病机制不清楚,早期诊断困难并且缺乏有效的治疗手段。中脑星形胶质细胞源性神经营养因子（Mesencephalic Astrocyte-derived Neurotrophic Factor,MANF）是新型神经营养因子家族的成员之一,内质网应激能上调其表达和分泌。本课题组前期研究发现在HCC癌组织中MANF表达降低,且能通过调节NF-κB/Snail信号通路抑制HCC的发生发展;在ICC中研究发现,MANF的表达与在HCC中相反,提示它在ICC中可能发挥不同的作用。有研究报道MANF表达水平与胆管癌的预后成负相关,敲低MANF能增加化疗药物索拉非尼引起的胆管癌细胞凋亡,提示MANF可能为胆管癌的治疗提供新的策略,但MANF在ICC中的具体作用机制不明。本课题组研究发现MANF与过氧化物酶6（Peroxiredoxins6,PRDX6）有相互作用,且MANF有可能影响PRDX6的表达。PRDX6是过氧化物酶PRDXs蛋白家族成员之一,同时拥有谷胱甘肽过氧化物酶（Glutathione peroxidase,GPx）功能和磷脂酶A2（Phospholipase A2,PLA2）活性。PRDX6被发现在肿瘤的发生过程中具有促癌和抑癌的双重作用,MANF与PRDX6在HCC中均表达降低,且发挥抑癌的作用,但两者在ICC中的作用、相互关系及其作用机制尚不清楚。目的研究MANF与PRDX6在ICC中的作用以及作用机制。方法运用免疫组织化学检测74例ICC癌组织以及癌旁组织中MANF的表达情况,免疫荧光检测ICC中表达MANF的细胞种类。分析MANF的表达与其临床病理特征的相关性。运用慢病毒感染、筛选构建稳定敲低MANF的ICC细胞株,Western blot验证敲低效果。然后用MTT法、平板克隆形成、划痕法、Transwell迁移和侵袭实验分别观察敲低MANF对HCCC9810和Hu CCT1细胞增殖、克隆形成、迁移以及侵袭性能力的影响。裸鼠成瘤实验观察Hu CCT1细胞敲低MANF基因后成瘤能力的变化,探讨MANF在ICC发生中的作用。免疫组织化学检测74例ICC癌组织以及癌旁组织中PRDX6的表达情况,免疫荧光检测ICC中表达PRDX6的细胞种类。分析PRDX6与临床病理特征的相关性,以及与MANF表达的关系。硫代乙酰胺（thioacetamide,TAA）诱导ICC大鼠模型观察全敲PRDX6后成瘤情况的变化。敲除组和对照组ICC瘤组织RNA测序,寻找差异基因,研究PRDX6在ICC发生中的作用以及作用机制。基于前期MANF与PRDX6酵母双杂交的结果,在ICC组织中运用免疫荧光检测MANF与PRDX6有无共定位,免疫共沉淀实验了解MANF与PRDX6是否存在相互作用。q PCR和Western blot检测HCCC9810和Hu CCT1细胞敲低MANF对PRDX6表达的影响。进一步检测裸鼠成瘤实验的实验组和对照组ICC组织中MANF和PRDX6的表达情况,研究敲低MANF对PRDX6表达的影响。结果1 MANF在ICC中的表达以及临床意义74例ICC患者肝组织进行免疫组化染色,结果显示在癌组织中MANF蛋白阳性率为93.2%,以中、强阳性为主,癌旁组织MANF蛋白表达阳性率为31.1%,以弱阳性为主,癌组织MANF蛋白表达明显高于癌旁组。免疫荧光发现ICC组织中癌细胞、巨噬细胞表达MANF,肝脏星状细胞则不表达该蛋白。MANF的表达与肿瘤分化程度相关,分化程度低的肿瘤组织MANF表达更高。与肿瘤数量也相关,肿瘤数量≥2的肿瘤组织MANF表达更高。同时MANF在晚期（Ⅲ/Ⅳ）ICC组织中表达明显高于早期（Ⅰ/Ⅱ）。但是MANF表达与年龄、性别及其它因素无关。2 MANF对ICC细胞株恶性生物学行为的影响构建稳定敲低MANF及载体对照组的ICC细胞株。MTT实验结果发现敲低MANF的HCCC9810和Hu CCT1细胞增殖率低于对照组。平板克隆形成实验显示敲低MANF的HCCC9810和Hu CCT1细胞相对于对照组,形成克隆数目明显减少。这些结果提示,敲低MANF后这两种ICC细胞增殖能力降低。划痕实验显示,敲低MANF的两种细胞迁移能力均降低。Transwell迁移实验同样表明HCCC9810和Hu CCT1细胞敲低MANF后迁移能力下降。Transwell侵袭实验结果提示两种细胞敲低MANF后侵袭能力降低。这些结果表明敲低MANF,抑制HCCC9810和Hu CCT1细胞迁移和侵袭能力。裸鼠成瘤实验结果发现,注射敲低MANF的Hu CCT1细胞与载体对照组相比,皮下肿瘤体积小,重量低。免疫组织化学实验发现敲低MANF组的瘤组织Ki67阳性细胞比对照组少。提示敲低MANF能抑制裸鼠体内Hu CCT1细胞增殖,从而抑制肿瘤的生长。3 PRDX6在ICC中的表达以及临床意义免疫组织化学染色发现74例ICC患者癌组织中PRDX6蛋白阳性率为94.6%,以中、强阳性为主,而癌旁组织PRDX6蛋白表达阳性率为38%,以弱阳性为主。PRDX6蛋白表达,癌组织组明显高于癌旁组。免疫荧光发现ICC组织中癌细胞、巨噬细胞表达PRDX6,肝脏星状细胞则不表达。PRDX6的表达与肿瘤分化程度相关,分化程度低的肿瘤组织PRDX6表达更高。与肿瘤数量也相关,肿瘤数量≥2的肿瘤组织PRDX6表达更高。同时PRDX6在晚期（Ⅲ/Ⅳ）ICC组织中表达明显高于早期（Ⅰ/Ⅱ）。而PRDX6表达与年龄、性别以及淋巴转移等因素无关。在ICC组织内,PRDX6与MANF的表达存在相关性,两者的临床意义一致,且表达细胞相同,均在癌细胞与巨噬细胞内有表达,肝星状细胞不表达。4大鼠PRDX6敲除对TAA诱导的ICC形成的影响7-8周野生型（WT）和PRDX6基因敲除(PRDX6^-/-)雌性大鼠用300mg/L硫代乙酰胺（thioacetamide,TAA）饮水造模,27周后处死取肝脏组织。统计发现,WT大鼠（n=8）平均肿瘤数（157.143±121.136）,PRDX6^-/-大鼠（n=8）平均肿瘤数（30.375±13.773）,PRDX6敲除后,肿瘤数量明显减少。PRDX6^-/-大鼠的肝/总体体重比降低,直径≥1cm的肿瘤WT组也多于PRDX6^-/-组。两组肝组织取材、切片,免疫组织化学实验表明基因敲除组PRDX6表达较低,两组肿瘤组织胆管型细胞角蛋白CK19阳性。PRDX6^-/-组肿瘤组织中Ki67阳性细胞明显减少。HE染色100倍视野下测量切片总面积和肿瘤面积,每个样本取10个视野,取平均值,计算肿瘤面积占肝总面积的百分比。与WT组的（46.169±17.873）相比,PRDX6^-/-组的平均肿瘤面积/总面积只有（10.651±8.223）,明显减少。q PCR检测PRDX6^-/-组肿瘤组织中Ki67的表达低于正常对照组。以上结果表明,PRDX6敲除抑制肿瘤细胞增殖,ICC肿瘤形成减少。取两组大鼠TAA诱导ICC模型的肝脏肿瘤组织做RNA测序分析,分析PRDX6敲除组和对照组瘤组织差异基因表达。发现敲除PRDX6后有127个基因上调,321个基因下调（n?=3）。两组之间DEGs的KEGG通路分析发现细胞信号转导通路基因变化最大,有47个基因表达发生了改变。WNT信号通路是基因表达变化最多的通路,其中WNT7A、WNT7B、FZD2、Ccnd2和MMP7在敲除PRDX6后表达明显下调。q PCR和免疫组织化学实验验证了这一结果。提示PRDX6可能通过调控WNT7A/B信号通路促进ICC的发生。5 MANF通过与PRDX6相互作用调节其蛋白水平前期课题组酵母双杂交实验发现MANF与PRDX6有相互作用。在ICC组织内,免疫荧光实验表明MANF与PRDX6有共定位,免疫共沉淀实验也证明MANF与PRDX6确实有相互作用。Western blot发现HCCC9810和Hu CCT1细胞敲低MANF后PRDX6蛋白表达降低;q PCR检测发现,敲低MANF不影响PRDX6的m RNA水平的变化。同样检测裸鼠成瘤实验的ICC组织中MANF与PRDX6的表达发现,MANF敲低后PRDX6蛋白表达也降低,m RNA水平没有明显变化。以上结果表明在ICC中,MANF与PRDX6存在相互作用,且敲低MANF,PRDX6的蛋白表达降低。结论1 MANF在ICC癌组织中高表达,并且与肿瘤分化程度、肿瘤数量以及肿瘤分期有关,敲低MANF抑制ICC细胞的增殖、迁移、侵袭和裸鼠移植瘤的生长。2 PRDX6在ICC癌组织中高表达,并且与肿瘤分化程度、肿瘤数量以及肿瘤分期有关,与MANF表达在ICC癌组织中高度相关。敲除PRDX6抑制ICC大鼠模型中肿瘤的形成,可能与调控WNT7A/7B信号通路有关。3 MANF与PRDX6有相互作用,敲低MANF后PRDX6的蛋白表达水平下调,m RNA水平没有明显变化,机制不清。更多还原

【Abstract】 Intrahepatic cholangiocarcinoma（ICC）is the second most common hepatic malignancy after hepatocellular carcinoma（HCC）and has high malignancy and poor prognosis.It accounts for 10%of the digestive tract tumors and 13%of cancer mortality worldwide.HCC and ICC are both primary liver cancers;however,there are significant differences in their pathogenesis and treatment.One reason for poor prognosis of ICC is unclear pathogenesis,difficult early diagnosis and lack of effective treatment.Mesencephalic astrocyte derived neurotrophic factor（MANF）is a member of a novel neurotrophic factor family.Our previous study found that the expression of MANF is decreased in HCC.Additionally,it inhibits the development of HCC by regulating the NF-κB signaling pathway.In ICC,the expression of MANF is increased,suggesting that it may play different roles in ICC.Studies have reported that the expression level of MANF is negatively correlated with the prognosis of cholangiocarcinoma.It can promote the apoptosis of cholangiocarcinoma cells induced bysorafenib,suggesting that this may be a new strategy for cholangiocarcinoma treatment;however,its mechanism in ICC remains unclear.MANF interacts with peroxiredoxin6（PRDX6）,and may affect its expression.PRDX6 is a member of the prdx protein family,which is associated with GSH peroxidase function.It is found that PRDX6 plays a dual role in promoting and inhibiting cancer in the tumorigenesis.The expression of MANF and PRDX6 is decreased in HCC,and they play an anti-tumor role.However,the role,relationship,and mechanism of these two genes in ICC remains unclear.ObjectiveTo study the role and mechanism of MANF and PRDX6 in ICC.MethodsImmunohistochemistry was used to detect the expression of MANF in 74 cases of ICC cancer and adjacent tissues,and immunofluorescence was used to detect the types of cells expressing MANF in ICC.The correlation between the expression of MANF and its clinicopathological features was analyzed.Lentivirus infection and screening were used to construct stable knockdown ICC cell lines.Western blot was used to verify the knockdown effect.Then the effects of MANF knockdown on the proliferation,colony formation,migration and invasion of ICC cells were detected by MTT assay,plate clone formation,scratch assay,Transwell migration and invasion assay.The tumorigenicity of Hu CCT1 cells after knockdown of MANF gene was detected in nude mice.Thus exploring the role of MANF in the occurrence of ICC.Immunohistochemistry was used to detect the expression of PRDX6 in 74 cases of ICC cancer and adjacent tissues,and immunofluorescence was used to detect the types of cells expressing PRDX6 in ICC.Correlation analysis of PRDX6 expression with its clinicopathological features and MANF expression.TAA induced ICC rat model was used to observe the changes of tumor formation after PRDX6 knockout.RNA sequencing of ICC tumor tissues in knockout group and control group was used to search for differential genes and explore the mechanism of PRDX6 in the occurrence of ICC.Based on the results of yeast two hybrid between MANF and PRDX6,immunofluorescence was used to detect the colocalization of MANF and PRDX6 in ICC tissues,and immunoprecipitation was used to detect the interaction between MANF and PRDX6.q PCR and Western blot detected the effect of knockdown MANF in HCCC9810 and Hu CCT1 cells on PRDX6 expression.The expression of MANF and PRDX6 in the nude mouse tumorigenesis experimental group and the control group was detected,and the effect of knockdown MANF on PRDX6expression was studied.Results1 Expression and clinical significance of MANF in ICCThe results of immunohistochemical staining in 74 cases of ICC liver tissue showed a 93.2%mainly moderate to strong positive rate of MANF protein expression in cancer tissue.Additionally,the positive protein expression of MANF in paracancerous tissue was 31.1%,and typically weak positive.The protein expression of MANF in the cancer tissue group was significantly higher than in the paracancerous group.Immunofluorescence showed that MANF was expressed in cancer cells and macrophages,but not in hepatic stellate cells.Furthermore,the expression of MANF was correlated with the degree of tumor differentiation;poorly differentiated tumor tissue had significantly increased expression of MANF.The expression of MANF was also correlated with the number of tumors present;ICC with?2 tumors had higher expression of MANF.Correspondingly,the expression of MANF was significantly higher in advanced ICC than in early ICC.However,the expression of MANF was not related to age,gender,or other factors.2 Effect of MANF on the malignant behavior of ICC cell linesWe constructed HCCC9810 and Hu CCT1 stably knocked down MANF and vector control groups.The MTT results showed a lower proliferation rate in cells with MANF knockdown when compared with the control group.Compared with the control group,additionally,the numbers of colonies formed in cells with MANF knockdown were significantly reduced.These results suggest that knockdown of MANF can decrease the proliferation of HCCC9810 and Hu CCT1.The scratch test and transwell migration assay showed that migration ability was decreased after knockdown on MANF.Further,the transwell invasion assay showed decreased invasion ability of the two kinds of cells decreased after knockdown of MANF.These results suggested that knockdown of MANF inhibits the migration and invasion of cells.Furthermore,the subcutaneous tumor volume and weight of Hu CCT1 cells transfected with MANF knockdown in nude mice were smaller than those in the control group.Immunohistochemistry showed that there were fewer Ki67 positive cells in the tumor tissue of the knockdown MANF group than that of the control group.This indicated that knockdown of MANF inhibits cell proliferation in nude mice,which,in turn,inhibi ts tumor growth.3 Expression and clinical significance of PRDX6 in ICCImmunohistochemical staining showed a 94.6%moderate and strong positivity rate of PRDX6 protein expression in 74 cases of ICC.By contrast,the positivity rate of PRDX6 protein expression in paracancerous tissues was 38%,and predominantly weakly positive.PRDX6 protein expression was significantly higher in cancer tissue group than in paracancerous group.Immunofluorescence revealed PRDX6 expression in cancer cells and macrophages,but not in hepatic stellate cells.This expression was correlated with the degree of tumor differentiation;poorly differentiated tumor tissue had significantly increased PRDX6 expression.Additionally,this expression was correlated with the number of tumors;cases of ICC with?2 tumors had higher PRDX6 expression.PRDX6expression was not associated with age,gender,lymphatic metastasis and so on.The expression of PRDX6 was correlated with the expression of MANF in cases of ICC;however,the clinical significance and cell localization of MANF and PRDX6 was the same.4 Effects of rat PRDX6 knockout on TAA-induced ICC formationWild type（WT）and PRDX6 knockout(PRDX6^-/-)rats were induced by drinking300 mg/L thioacetamide（TAA）for 27 weeks.The mean number of tumors in WT and PRDX6^-/-rats（n=8）was 157.143±121.136 and 30.375±13.773,respectively.Interestingly,the number of tumors decreased significantly after PRDX6 knockout.Furthermore,the liver:total body weight ratio was decreased in PRDX6^-/-rats.Immunohistochemistry showed low expression of PRDX6 in the knockout group,and positive expression of the ICC marker,CK19,in the two groups.The number of Ki67positive cells in PRDX6^-/-group was significantly decreased.The total section and tumor area was measured under 100?field of vision of HE staining.Ten fields of vision were taken for each sample,and the mean value was calculated to measure the percentage of tumors in the total tumor area.The mean tumor area was significantly decreased in the PRDX6^-/-group（10.651±8.223）,when compared with the WT group（46.169±17.873）.Furthermore,the expression of Ki67 in PRDX6^-/-group was lower than that in WT group by q PCR.These results suggested that PRDX6 knockout inhibited tumor cell proliferation and reduced ICC tumor formation.RNA sequencing analysis revealed 127 and 321 genes that were up-and down-regulated,respectively,after PRDX6 knockout（n=3）.KEGG pathway analysis of the differentially expressed genes between two groups showed that signal transduction pathway genes were most changed,with 47 changed genes.The WNT signaling pathway is the most variable pathway in gene expression.WNT7A,WNT7B,FZD2,Ccnd2 and MMP7 were down-regulated after PRDX6 knockout.The results were confirmed by q PCR and immunohistochemistry.These results suggested that PRDX6promotes the development of ICC by regulating the WNT7A/B signaling pathway.5 MANF regulates its protein level by interacting with PRDX6The interaction between MANF and PRDX6 was revealed via yeast two-hybrid screening.Immunofluorescence showed MANF and PRDX6 colocalization in ICC tissue.Furthermore,immunoprecipitation assay proved the interaction between MANF and PRDX6.Western blot showed decreased protein expression of PRDX6 after the knockdown of MANF in HCCC9810 and Hu CCT1.q PCR revealed that the m RNA expression of PRDX6 was not affected after MANF knockdown.Similarly,the protein expression of MANF and PRDX6 was analyzed in nude mice ICC tissue,which found decreased PRDX6 protein expression after MANF knockdown and the level of m RNA did not change significantly.These results indicated that there is an interaction between MANF and PRDX6 in ICC tissue,where knockdown of MANF reduced the protein expression of PRDX6.Conclusion1 MANF is highly expressed in ICC,and is related to tumor differentiation,tumor number and tumor stage.Knockdown of MANF inhibits the proliferation,migration,invasion of ICC cells and the growth of transplanted tumor in nude mice.2 PRDX6 is highly expressed in ICC cancer tissues,and is related to tumor differentiation,tumor number and tumor stage,and is highly correlated with the expression of MANF in ICC.PRDX6 knockout can reduce the tumor formation in ICC rat model.PRDX6 may promote the development of ICC by regulating WNT7A/B signaling pathway.3 MANF interacts with PRDX6.After knockdown of MANF,the protein expression of PRDX6 was down regulated,and the m RNA did not change significantly,and the mechanism is unclear.更多还原