【作者】 毛海燕；

【导师】 卜平；

【作者基本信息】 扬州大学 ， 中西医结合临床， 2020， 博士

【摘要】 结直肠癌是消化系统常见的恶性肿瘤之一,在中国乃至全球其发病率居于第三,死亡率居于第二,具有“高发病率、高死亡率”的特点。结直肠癌在早期的一些轻微症状,易被诊断为其他消化道疾病而忽视,当临床症状明显时,病情已广泛转移而失去手术机会。尽管随着诊断方法和手段的提高,早期发现的病例增多,但是总生存期仍不是很理想,随着研究的进展,晚期患者尽管采用放化疗、靶向治疗及免疫治疗等治疗手段,但疗效也未尽人意,因此,寻找行之有效的分子靶点指导临床个体化精准治疗具有十分重要的意义。胆绿素还原酶A(Biliverdin reductase A,BLVRA)是一种进化上高度保守的可溶性NADH依赖酶,广泛分布于动植物细胞浆内,是胆绿素代谢的限速酶,能够促进胆绿素向胆红素转化,也是一种抗氧化剂,能够维持细胞内氧化还原反应的动态平衡,其调节细胞的生长,参与细胞信号转导,能促进肿瘤细胞的增殖、侵袭和迁移,抑制肿瘤细胞的凋亡。BLVRA在多种恶性肿瘤中呈高表达水平,但其在结直肠癌中的表达水平及生物学作用尚不明确。汉黄芩素是从黄芩及半枝莲等中药中提取的的一种黄酮类化合物,具有清热解毒之功效,现代研究表明其具有较强的抗肿瘤活性,包括阻滞细胞周期、抑制细胞增殖、促进细胞凋亡、减少肿瘤新生血管形成等方面,同时还可以对化疗药物有增敏作用,且其抗肿瘤作用与多个信号通路有关,而汉黄芩素能否通过调控BLVRA发挥抗肿瘤作用尚未见相关报道。为了探讨BLVRA在结直肠癌中发生发展中的生物学行为,以及汉黄芩素能否通过抑制BLVRA达到治疗结直肠癌的作用。本文通过检测结直肠癌患者外周血及肿瘤组织中BLVRA表达水平发现其与结直肠癌的发生、发展密切相关。体外实验发现敲低BLVRA的表达,结肠癌细胞增殖能力降低、凋亡率增加、迁移和侵袭能力降低、减少上皮-间充质转化(EMT)进程,而过表达BLVRA的结肠癌细胞生物学活性恰恰相反,提示BLVRA与结肠癌细胞的恶化相关,且BLVRA的相关作用是通过调控Wnt/β-catenin信号通路发生的。汉黄芩素能够通过抑制BLVRA的表达,抑制结肠癌细胞的增殖能力、迁移和侵袭能力,诱导CRC细胞的凋亡,抑制EMT进程。具体内容包括以下四个部分:第一部分BLVRA在结直肠癌患者中的表达水平及临床意义目的:分析结直肠癌患者外周血及肿瘤组织中BLVRA的表达情况,探讨其与临床特征的相关性及生存期的关系。方法:收集2015年至2017年扬州大学附属医院经病理确诊的110例结直肠癌患者外周血、活检肿瘤组织及癌旁组织,采用ELISA、qRT-PCR及免疫组化等方法检测BLVRA表达情况,采用卡方检验分析BLVRA的不同表达水平与患者的年龄、性别、发病部位、分期、分化程度、淋巴结及远处转移情况等临床病理特征的相关性,采用Kapla n-Meier法绘制BLVRA不同表达水平的PFS及OS生存曲线,采用对数秩检验比较不同BLVRA表达的累积无进展生存情况及累积总生存差异,分别以性别、年龄、发病部位、分期、分化程度、淋巴结转移、远处转移、BLVRA表达为自变量,进行单因素及多因素COX回归分析。结果:结直肠癌患者外周血中BLVRA的表达高于健康体检者,其在肿瘤组织及癌旁组织均有表达,但肿瘤组织中表达水平明显高于癌旁组织(P<0.001),且BLVRA高表达的患者PFS及OS更短(P<0.001)。BLVRA的表达水平与患者分期、分化程度、淋巴结转移及远处转移相关(P<0.001),与患者年龄(P=0.881)、性别(P=0.143)及发病部位(P=0.387)无关。COX单因素分析显示患者分期、分化程度、淋巴结转移、远处转移及BLVRA表达水平均与患者预后相关(P<0.001),而年龄(P=0.594)、性别(P=0.903)及发病部位(P=0.649)与患者预后无关。COX多因素分析表明患者分期、分化程度、淋巴结转移、远处转移及BLVRA表达水平均与患者预后相关(P<0.001),而年龄(P=0.220)、性别(P=0.057)及发病部位(P=0.202)与患者预后无关。结论:结直肠癌患者外周血及肿瘤组织中的BLVRA表达水平明显高于健康体检者及癌旁组织;BLVRA高表达的患者PFS及OS明显缩短,提示预后不良;肿瘤分期、分化程度、淋巴结和远处转移及BLVRA表达水平是影响结直肠癌患者不良预后的独立因素。第二部分BLVRA对结肠癌细胞生物学行为的影响目的:构建BLVRA敲低及过表达载体,体外实验探讨其对结肠癌HT-29及SW620细胞增殖能力、细胞凋亡、迁移和侵袭及上皮-间质转化的影响。方法:首先采用qRT-PCR及Western blot法检测结肠癌细胞F株HHT-29、SW620 SW480、HCT116、LOVO及正常肠上皮细胞FHC中BLVRA mRNA及蛋白表达水平;构建BLVRA敲低及过表达载体和各自的阴性对照载体,进行细胞转染,qRT-PCR及Western blot检测转染后BLVRA mRNA及蛋白水平,证实BLVRA敲低及过表达细胞株成功建立;采用 MTT、流式细胞术、Transwell、免疫荧光、Western blot等方法检测细胞增殖能力、细胞凋亡率及细胞凋亡因子蛋白水平、细胞迁移和侵袭能力及上皮-间质转化(EMT)相关蛋白的变化。结果:qRT-PCR及Western blot结果均显示五株结肠癌细胞株中HT-29细胞的BLVRA mRNA及蛋白表达水平最高(P<0.01),SW620细胞的BLVRA mRNA及蛋白表达水平最低(P<0.05),选择HT-29及SW620细胞进行后续实验;细胞转染后qRT-PCR及Western blot结果显示HT-29细胞中BLVRA mRNA及蛋白表达降低(P<0.001),SW620细胞中 BLVRA mRNA 及蛋白表达升高(P<0.001);敲低 BLVRA后MTT结果显示HT-29细胞增殖能力降低,且随着时间延长,作用明显(P<0.001),流式细胞术提示细胞凋亡率增加(P<0.001),Western blot提示抗凋亡因子Bcl-2及survivin的蛋白水平降低(P<0.05),促凋亡因子BAX及Caspase-7的蛋白水平增加(P<0.05),Transwell结果显示细胞迁移和侵袭数目明显下降(P<0.001),免疫荧光及West6rnblot均显示E-Cadhern表达升高,Vimentin表达下降(P<0.05);过表达BLVRA后MTT结果显示SW620细胞增殖能力提高,且随着时间延长增殖明显(P<0.001),流式细胞术提示细胞凋亡率降低(P<0.001),Western blot提示抗凋亡因子Bcl-2及survivin的蛋白水平升高(P<0.05),而促凋亡因子BAX及Caspase-7的蛋白水平降低(P<0.05),Transwell结果显示细胞迁移和侵袭数目明显增加(P<0.001),免疫荧光结果显示E-Cadherin表达下降,Vimentin表达上升,Weste blot显示出一致的结果(P<0.05)。结论:结肠癌细胞株中BLVRA表达水平均高于正常肠上皮细胞;siRNA-BLVRA能够抑制HT-29细胞的增殖能力、促进细胞凋亡、抑制细胞的迁移和侵袭能力,并通过抑制上皮-间充质转化(EMT)进程来进一步弱化CRC细胞的迁移和侵袭能力,而BLVRA过表达在SW620细胞中则起着相反的生物学作用。第三部分BLVRA影响结肠癌细胞生物学行为的初步机制探讨目的:探讨BLVRA是否通过调控Wnt/β-catenin信号通路发生作用。方法:采用Western blot检测转染前后结肠癌细胞中Wnt/β-catenin通路及其靶蛋白的表达变化;加予该通路抑制剂(IWR-1)及激动剂(CHIR98014)干预后Western blot检测Wnt/β-catenin通路及其靶蛋白的表达变化。结果:BLRA敲低后Wnt 5a及β-catenin蛋白表达水平下降(P<0.01),p-β-catenin蛋白表达水平上升(P<0.01),靶蛋白cyclin D1、C-myc、p-GSK-3β、COX-2表达下降(P<0.05),p-PTEN表达水平增加(P<0.05),激动剂干预后起着相反的作用(P<0.05);BLVRA过表达后上调Wnt 5a、β-catenin(P<0.01)及靶蛋白cyclin D1、C-myc、p-GSK-3β、COX-2的表达(P<0.05),降低p-β-catenin(P<0.01)及靶蛋白p-PTEN(P<0.05)的表达,抑制剂干预后则起着相反的作用(P<0.05)。结论:siRNA-BLVRA能够抑制Wnt/β-catenin通路,激动剂干预后再次激活Wnt/β-catenin通路;BLVRA过表达则能激活Wnt/β-catenin通路,抑制剂干预后再次抑制Wnt/β-catenin通路,提示BLVRA是通过调控Wnt/β-catenin通路发生作用的。第四部分汉黄芩素调控BLVRA抑制结肠癌细胞增殖、迁移和侵袭及诱导凋亡的机制研究。目的:探讨汉黄芩素对结肠癌HT-29及SW620细胞增殖、迁移、侵袭和凋亡及EMT进程的影响,并探讨其是否通过调控BLVRA发生作用。方法:取对数生长期的HT-29和SW620细胞,加入不同浓度的汉黄芩素(0,20,40,80,160μg/mL),并设立 5-FU(12.5μg/mL)为阳性对照组,采用 MTT 法检测汉黄芩素及5-FU对结肠癌细胞增殖能力的影响;流式细胞术检测汉黄芩素及5-FU对结肠癌细胞凋亡的影响;通过Transwell实验检测汉黄芩素及5-FU对结肠癌细胞迁移和侵袭的影响;Western blot检测细胞凋亡因子Bcl-2、BAX及EMT相关蛋白 E-Cadherin、Vimentin 及 snail 的变化。结果:汉黄芩素抑制结肠癌细胞的增殖能力,且呈现时间及浓度依赖性(P<0.01);汉黄芩素能够提高结肠癌细胞凋亡率,抑制抗凋亡因子Bcl-2蛋白的表达,增加促凋亡因子BAX蛋白的表达水平,且随着汉黄芩素浓度的增加作用明显(P<0.01);汉黄芩素能够抑制结肠癌细胞的迁移和侵袭能力,且呈现浓度依赖性(P<0.01);Weste blot结果显示汉黄芩素能够增加E-Cadherin的表达,降低Vimentin及snail蛋白的表达水平,且呈现浓度依赖性(P<0.05);汉黄芩素素能降低两种CRC细胞中BLVRA的蛋白表达水平,并且与浓度相关(P<0.05)。结论:汉黄芩素能够抑制结肠癌HT-29和SW620细胞的增殖、迁移和侵袭能力,诱导细胞凋亡,抑制上皮-间充质转化(EMT)进程,且呈浓度依赖性,其作用可能是通过调控BLVRA的表达产生的。更多还原

【Abstract】 Colorectal cancer(CRC)is one of the common malignancy of the digestive tract.Among all types of malignant tumors,it has the third-highest incidence and the second-highest death rates,both worldwide and in China.It has the characteristics of high morbidity and high mortality.Colorectal cancer has no obvious positive signs or only slight symptoms in the early stage,which is easy to be ignored.When the clinical symptoms are obvious,the lesions have developed into the advanced stage,and the opportunity of operation was lost.Although with the improvement of diagnostic methods and means,the number of cases found in the early stage are increasing,the overall survival is still not statisfied.With the progress of research,although the advanced patients have the chance to be treated with radiotherapy and chemotherapy,targeted treatment and immunotherapy,the efficiency is still low.It is important to find the exact targets to guide the individualized treatment.Biliverdin reductase A(biliverdin reductase A,BLVRA)is a conserved soluble NADH dependent enzyme,which is widely exsisted in the cytoplasm of animals and plants.It converts biliverdin to bilirubin in the heme metabolism pathway,which is also an antioxidant.It maintains the dynamic balance of redox reaction in cells,regulates cell growth,participates in cell signal transduction,and promotes the proliferation of tumor cells,invasion and migration.It also inhibits the apoptosis of cells.BLVRA is highly expressed in a varietyf of tumors,but whether it acts as "oncogene" in colorectal cancer remains unclear.Wogonin is a kind of flavonoid extracted from Scutellaria baicalensis Georgi.It has wide antitumor activities that can induce cell cycle arrest,promote cell apoptosis,reduce tumor neovascularization,and can also enhance the sensitivity of tumor cells to chemotherapy drugs.Its antitumor effect is related to multiple signal pathways.Whether the effects were regulated by BLVRA has not been studied yet.In this study,first,the expressions of BLVRA in peripheral blood and tumor tissues of patients with colorectal cancer were detected by ELISA,qRT-PCR and immunohis-tochemistry respectively.Second,the effects of BLVRA knockdown and overexpression on proliferation,migration,invasion,epithelial-mesenchymal transition(EMT)and apoptosis of colorectal cancer cells were observed after cell transfection.Third,whether the effects of BLVRA was regulated by Wnt/p-catenin signaling pathway was also been investigated.Fourth,the effects of Wogonin on the proliferation,migration,invasion,epithelial-mesenchymal transition(EMT)and apoptosis of colon cancer cells were also been researched.Part Ⅰ:The expression of BLVRA in colorectal cancer patients and its clinical significanceObjective:To investigate the expression of BLVRA in peripheral blood and tumor and adjacent tissues of colorectal cancer patients,evaluated the relationship with clinicopath-ological parameters and survival.Methods:The study was carried out using samples from 110 patients who were histopathologically diagnosed as colorectal adenocarcinoma cases at the Affiliated Hospital of Yangzhou University from 2015 to 2017.As controls,110 healthy volunteers joined in this study.The tissues and the plasma of peripheral blood were acquired before the first treatment.ELISA,Real-time PCR and Immunohistochemistry(IHC)were applied to test the level of BLVRA in patients and volunteers.Kaplan-Meier was used to draw PFS and OS survival curves of different expression levels of BLVRA,and log rank test was used to compare the cumulative progression free survival and cumulative total survival differences.Cox regression analysis was performed according to age,location,stage,differentiation,lymph node metastasis,distant metastasis and BLVRA expression.Results:The expression of BLVRA in the peripheral blood of patients with colorectal cancer was higher than that of healthy people.It was expressed both in tumor tissue and adjacent tissue,which is significantly higher in tumor tissue(P<0.001).The PFS and OS of patients with high expression of BLVRA were shorter than low expression of BLVRA(P<0.001).The expression level of BLVRA has no connection with age(P=0.881),gender(P=01.43)and the location of the disease(P=0.387).COX univariate analysis showed that stage,differentiation,lymph node metastasis,distan t metastasis and the level of BLVRA were all related to poor prognosis(P<0.001),but age(P=0.594),gender(P=0.903)and location(P=0.649)were not connected with prognosis.COX multivariate analysis showed that stage,degree of differentiation,lymph node metastasis,distant metastasis and BLVRA expression were all related to poor prognosis(P<0.001),but age(P=0.220),gender(P=0.057)and location(P=0.202)are without relationship to prognosis.Conclusions:The expression of BLVRA in the peripheral blood and tumor tissues of colorectal cancer patients was significantly higher than that of the healthy people and the adjacent tissues;the PFS and OS of patients with high expression of BLVRA were significantly shortened,indicating poor prognosis;tumor stage,differentiation,metastasis and expression level of BLVRA were independent factors affecting the poor prognosis of colorectal cancer patients.Part Ⅱ:The biological effects of BLVRA in colorectal cancer cellsObjective:To investigate the effects of BLVRA on the proliferation,apoptosis,migration,invasion and epithelial stromal transformation of colon cancer HT-29 and SW620 cells after transfected with BLVRA interference and overex-pressionvectors.Methods:First,the expression of BLVRA mRNA and protein in colon cancer cell lines HT-29,SW620,SW480,HCT116,LoVo and normal intestinal epithelial cells FHC were detected by real-time PCR and Western blot.BLVRA knockdown and overexpression vectors and their negative control vectors were constructed for cell transfection,real-time PCR and Western blot were used to test BLVRA mRNA and protein levels after transfection to confirm that BLVRA knockdown and overexpression cell lines were successfully established.MTT,flow cytometry,Transwell,immunofluorescence and Western blot were performed to gauge cell proliferation,apoptosis rate,expression of apoptotic factors,cell migration and invasion ability,and the changes of epithelial mesenchymal transformation(EMT).Results:The results of Real-time PCR and Western blot showed that the expression level of BLVRA mRNA and protein in HT-29 cells was the highest in the five colon cancer cell lines(P<0.01),and that in SW620 cells was the lowest(P<0.05).HT-29 and SW620 cells were selected for the follow-up experiments.The expression of BLVRA mRNA and protein was decreased(P<0.001),and the expression of BLVRA mRNA and protein was increased in SW620 cells(P<0.001)after transected;MTT results showed that after BLVRA knockdown,the proliferation ability of HT-29 cells was inhibited,and with the time dependent(P<0.001).Flow cytometry showed that the apoptosis rate was increased(P<0.001),western blot revealed that the protein level of Bcl-2 and Survivin were reduced(P<0.05),while Bax and caspase-7 were enhanced(P<0.05).Transwell implied that the number of cell migration and invasion were significantly decreased(P<0.001).The expression of E-cadherin was increased,and vimentin was decreased(P<0.05)with immunofluorescence and western blot(P<0.05).On the other hand,MTT showed that after overexpression of BLVRA,the proliferation ability of SW620 cells was increased,which also with the time dependent(P<0.001).Flow cytometry exhibited that the apoptosis rate was decreased(P<0.001),western blot indicated that the protein levels of Bcl-2 and Survivin were upregulated(P<0.05),while the level of Bax and caspase-7 were downregulated(P<0.05).Transwell displayed that the number of cell migration and invasion were increased significantly(P<0.001).The expression of E-cadherin was reduced,and vimentin was increased(P<0.05)with immunofluorescence,which is consistent with the results of western blot(P<0.05).Conclusions:The expression of BLVRA in colon cancer cell lines was higher than that in normal intestinal epithelial cells;siRNA-BLVRA could inhibit the proliferation,apoptosis,migration and invasion of HT-29 cells,and restrain the process of epithelial mesenchymal transition(EMT),while the overexpression of BLVRA played an opposite role in SW620 cells.Part Ⅲ:The potential mechanisms of BLVRA in colon cancer cellsObjective:To discuss whether the effects of BLVRA was regulated by Wnt/β-catenin signal pathway.Methods:Western blot were applied to test the expression of Wnt/β-catenin and its targeted proteins before and after been treated with inhibitor(IWR-1)and activator(CHIR98014).Results:Knockdown of BLVRA downregulated the expression of Wnt,β-catenin and its targeted proteins,such as cyclin D1、C-myc、p-GSK-30、COX-2,while the expression of p-β-catenin and p-PTEN were upregulated.O n the contrary,overexpression of BLVRA promoted the expression of Wnt,β-catenin and its targeted proteins,such as cyclin D1、C-myc、p-GSK-3β、COX-2,the expression of p-β-catenin and p-PTEN were restrained.After been treated with activator and inhibitor,the opposite expression of the Wnt/β-catenin targeted protein were observed.Conclusions:siRNA-BLVRA can inhibit Wnt/β-Catenin signal pathway,and it can be stimulated again after treating with activator.Overexpression of BLVRA can activate Wnt/β-Catenin pathway,and it can be inhibited again after treating with inhibitor.All the results indicated that the effects of BLVRA were regulated by Wnt/β-Catenin signal pathway.Part Ⅳ The mechanisms of Wogonin inhibiting the colon cancer cells proliferation,migration,invasion and inducing apoptosis via BLVRAObjective:To investigate the effects of Wogonin on the proliferation,migration,invasion,apoptosis and EMT in conlon cancer cells HT-29 and SW620,and to certify whether its effect is regulated by BLVRA.Methods:CRC cell lines HT-29 and SW620 in logarithmic growth phase were used.Wogonin with different concentrations(0,20,40,80,160μg/mL)and 5-FU(12.5μg/mL)were added in the cells.MTT assay was used to test the proliferation of HT-29 and SW620 treated with Wogonin.Flow cytometry was adopted to examine the apoptosis rate of HT-29 and SW620 treated with Wogonin for 24 h.Transwell was applied to detect the migration and invasion of HT-29 and SW620 after Wogonin treatment.The apoptosis related proteins of Bcl-2,BAX and EMT related proteins E-Cadherin,Vimentin,snail were detected with western blot.Results:Wogonin could inhibite the proliferation of HT-29 and SW620 with time and concentration dependence(P<0.01).Wogonin could significantly enhance the apoptosis rate of HT-29 and SW620 cells with concentration dependence(P<0.01).It also decrease the expression of anti-apoptosis protein Bcl-2,increase the expression of BAX(P<0.01).Wogonin restrained the migration and invasion of HT-29 and SW620 in a dose-dependent manner(P<0.01).Western blot showed Wogonin promoted the expression of E-Cadherin,reduced the expressions of Vimentin and snail with concentration dependence(P<0.05).In both HT-29 and SW620 cells,Wogonin inbibited the expression of BLVRA in a dose-dependent manner(P<0.05).Conclusions:Wogonin could obviously inhibit the proliferation,migration and invasion,induce apoptosis in HT-29 and SW620 cells,and the mechanism may be related to the expression of BLVRA.更多还原