转录因子KLF8在肝细胞肝癌生长与侵袭转移中的作用研究
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摘要
肝细胞肝癌(hepatocellular carcinoma,HCC)是世界上的第三大癌症杀手,在中国处在第二位,占全部恶性肿瘤死亡人数的18.8%。虽然手术切除仍是可能获得治愈的主要方法,但根治性切除术后复发率仍然相当高,还有更多的肝癌患者因肿瘤转移而失去根治的机会。复发特别是转移性复发是延长肝癌病人生存的一个主要障碍。不幸的是当前可获得的病理学及免疫学指标还不足以敏感到对转移性肿瘤进行精确预测,辅助治疗也不能有效预防转移复发,因而肿瘤转移仍是研究中的一个极重要的课题。探讨肝癌发生发展和转移复发的相关因素及分子机理,寻找肝癌早期诊断、预测转移的生物标记和干预治疗的靶分子,对其诊断和治疗具有重要的理论指导和临床应用意义。
Kr(u|¨)ppel-like factor 8(KLF8)最初是作为Kr(u|¨)ppel样Cys2/His2锌指转录因子蛋白质家族的一个转录抑制蛋白而被识别的,通过与辅抑制子碳端结合蛋白(CtBP)相联系抑制基因表达。有研究表明KLF8的表达在卵巢癌、肾癌以及乳腺癌的细胞系以及卵巢癌的原发肿瘤组织中是升高的,KLF8是v-Src诱导的致癌转换所必需的,在人类肿瘤的进展中发挥作用。最近研究显示KLF8是一个很强的上皮间质转换(EMT)和侵袭的诱导蛋白。而其在肝(癌)细胞中的表达及其与HCC发生、侵袭转移及临床预后等的关系尚无相关研究。
本课题旨在通过对比和分析不同转移潜能人HCC细胞系的KLF8表达及由314例有完整的临床和随访资料的HCC组织构建的组织芯片、有和无转移HCC组织标本中KLF8的表达,研究KLF8在HCC中的表达情况及其过表达与HCC临床预后的关系;通过靶向KLF8的小干扰RNA和构建表达shRNA-KLF8的慢病毒载体pGCSIL-KLF8并将其包装成慢病毒颗粒来(转染)感染HCCLM3细胞,在体外和体内探索下调KLF8基因表达对HCC生长及侵袭转移的作用,并通过比较下调和未下调KLF8的HCCLM3之间基因转录谱的差异初步探讨KLF8促进HCC生长与侵袭转移的作用机制,为HCC转移的基因治疗提供新的靶点。
第一部分转录因子KLF8在不同肝癌细胞系及组织中的表达
目的:观察正常肝细胞系L-02、不同转移潜能的肝癌细胞系及不同肝(癌)组织中KLF8的表达,初步探讨KLF8表达在肝细胞肝癌中的意义。方法:Real-timePCR、western blot、免疫细胞化学检测不同肝(癌)细胞系中KLF8的mRNA和蛋白质水平;RT-PCR检测不同肝(癌)组织中的KLF8 mRNA水平;免疫组织化学检测不同肝(癌)组织中的KLF8蛋白表达。结果:在不同的肝癌细胞系中KLF8的mRNA及蛋白质水平随着肝癌细胞系侵袭转移潜能的增强而显著升高(P＜0.05),KLF8主要表达于癌细胞的核内;KLF8的mRNA水平在正常肝组织中低,而在肝细胞肝癌组织中较高,在有明确转移的肝细胞肝癌组织中更高;KLF8蛋白表达的阳性率在正常肝组织和肝硬化组织中低而在肝细胞肝癌组织中很高,在肝癌转移灶中最高(强阳性率达83.3%,p＜0.001)。结论:KLF8在不同转移潜能的肝癌细胞系及不同肝(癌)组织中表达有差异,其可能参与肝细胞肝癌的发生和侵袭转移。
第二部分转录因子KLF8在肝癌组织中的表达与肝癌临床病理特征及预后的关系
目的:研究转录因子KLF8在肝细胞肝癌组织中的表达,探讨KLF8与肝细胞肝癌临床病理特征和预后的关系。方法:应用Real-time PCR检测50例肝细胞肝癌组织中KLF8 mRNA的表达;Western Blot检测10例肝细胞肝癌组织中KLF8蛋白的表达;检测由314例肝细胞肝癌组织构建的组织芯片中KLF8蛋白的表达,SSPS11.5统计分析其与肝细胞肝癌临床病理特征及预后的关系。结果:在肝癌组织样本中,相对于无复发的肝细胞肝癌病人标本,有复发的病人标本具有较高的KLF8 mRNA表达水平(p=0.001);Western blot分析显示KLF8蛋白在5例有复发的病人标本中高表达,而在5例无复发的病人标本中仅1例高表达,其他的表达很弱或无表达。KLF8~-组的5-年总生存率OS和肿瘤复发率是60.3%和41.9%,而KLF8~+组是48.7%和59.8%,p=0.038和p=0.005。分层分析显示:在TNM stageⅠ的病人中KLF8~+病人的5-年肿瘤复发率相对于KLF8~-病人来说是显著增加的(54.6%vs.35.3%,p=0.008);同样的结果存在于BCLC 0+A期的病人中(57.0%vs.37.9%,p=0.009);在AFP≤20 ng/ml组中,KLF8~-和KLF8~+病人的5-年肿瘤复发率分别是41.5%和60.3%(p=0.027);而在Edmondson stageⅠ-Ⅱ组中,KLF8~-和KLF8~+病人的5-年肿瘤复发率分别是36.5%和54.3%(p=0.005)。KLF8过表达在病人早期复发中具有预测作用(p=0.001),而对于病人晚期复发无预测作用(p=0.623)。KLF8~+与肝癌的早期复发相关(p=0.008);单变量分析示KLF8对总生存率OS(p=0.040)和无复发生存时间TTR(p=0.006)有预后作用。多变量分析显示KLF8是OS(p=0.037)和TTR(p=0.018)的一个独立预后因素。结论:KLF8过表达与肝癌的早期复发相关,对总生存率OS和无复发生存时间TTR的不良预后有预测作用,是肝细胞肝癌病人的OS和TTR的独立预后因素。在TNM stage I、BCLC 0+A期、AFP≤20 ng/ml、Edmondson stageⅠ-Ⅱ病人中KLF8过表达提示高复发概率,KLF8过表达病人早期复发的概率高。
第三部分下调KLF8表达抑制HCCLM3增殖和侵袭能力的体外实验及其可能的机理研究
目的:考察KLF8表达的下调对HCCLM3细胞增殖和侵袭能力的影响并探讨其可能的机理。方法:siRNA下调KLF8后,MTT实验观察HCCLM3的增殖能力的改变;Transwell侵袭实验观察其侵袭能力的改变;基因芯片技术考察下调KLF8及未下调KLF8的HCCLM3之间的差异表达基因,荧光定量PCR验证部分差异表达的基因;RT-PCR、Western blot、免疫组织化学考察部分差异表达基因。结果:siRNA下调KLF8后HCCLM3的增殖(p＜0.0001)和侵袭能力(p＜0.0001)显著降低;基因芯片结果提示许多参与肿瘤发生发展的基因表达发生改变,KLF8参与多条肿瘤信号通路。结论:KLF8通过参与多条信号通路来调控HCCLM3的增殖及侵袭能力;其在肝细胞肝癌的发生发展中具有重要作用。
第四部分下调KLF8的HCCLM3在裸鼠体内的成瘤及转移能力研究
目的:研究KLF8的下调对HCCLM3在裸鼠体内的成瘤及转移能力的影响。方法:构建KLF8shRNA慢病毒载体,包装成慢病毒颗粒后感染HCCLM3细胞,接种雄性裸鼠,观察其对HCCLM3在裸鼠体内的成瘤与肺转移能力的作用。结果:对照组和实验组均可在裸鼠皮下成瘤;对照组老鼠的肿瘤重量显著大于KLF8下调组老鼠的肿瘤重量(3.67±0.75g vs.1.02±0.78g,p＜0.001);KLF8下调组的老鼠相对于对照组老鼠其肿瘤组织中具有更多的坏死灶,对照组老鼠和KLF8下调组老鼠间肿瘤肺转移的发生率分别是100%(6/6)和16.7%(1/6)(p=0.015)。KLF8下调组的肿瘤转移灶大小局限于I级而大多数的对照组里的转移灶是Ⅲ-Ⅳ级。结论:KLF8的下调抑制HCCLM3在裸鼠体内的成瘤及肺转移能力,KLF8可以作为HCC的一个治疗靶点。
Hepatocellular carcinoma(HCC) is the third most common cause of cancer-related death.Although survival of patients with HCC has been improved by advances in surgical techniques and perioperative management, long-term survival following surgical resection remains unsatisfactory due to the high rate of recurrence and metastasis.Advances in treatment of this disease are likely to stem from a better understanding of its biology and behavior.As biological and clinical behaviors of cancer are affected by multiple molecular pathways that are under the control of transcription factors,improved understanding of how these transcription factors affect cancer biology may lead to an improved ability to predict clinical outcomes and the discovery of novel therapeutic strategies. Kr(u|¨)ppel-like factors(KLFs) constitute a family of nuclear proteins that modulate gene transcription.Emerging evidence suggests that KLFs may be critical factors in tumor development,growth,and metastasis.One of these family members,KLF8,is a CACCC-box binding protein that associates with C-terminal binding protein(CtBP) to repress transcription.Recent studies in breast cancer indicated that KLF8 participates in oncogenic transformation and induces the epithelial-to-mesenchymal transition(EMT),which is an important mechanism during tumor invasion transformation.We found that KLF8 is highly expressed in HCC tissues compared with peritumoral tissues based on transcript profiles,and this result suggested that KLF8 may play an important role in hepatocarcinogenesis.Until now,no studies have reported a role of KLF8 in HCC or the clinicopathological significance of this factor.Here,we examined KLF8 expression in stepwise metastatic potential human HCC cell lines and tumor tissues by quantitative real-time polymerase chain reaction(qRT-PCR),western blot,and immunochemistry analyses.To investigate the possible mechanism,KLF8 expression was knocked down in a HCC cell line with high metastatic potential(HCCLM3 cells) with small-interfering RNA(siRNA),and then the differential gene profiles were investigated by cDNA microarray analysis.The effects of KLF8 depletion were observed in vitro and in vivo.To explore the clinical effects of this factor,KLF8 expression was detected in 314 HCC patients using tissue microarrays(TMAs).Together,our data indicates that KLF8 promotes HCC cell proliferation,invasion,and migration.High expression of KLF8 is strongly associated with early recurrence and poor prognosis in HCC patients after curative resection.
Part one Expression of KLF8 in different liver cell lines and in HCC tissues
Objective:This study investigated expression of KLF8 in differenr liver cell lines and in HCC tissues to evaluate the role of KLF8 in HCC development and progression.Methods:The KLF8 level in different liver cell lines was respectively determined by quantitative real-time PCR, Immunocytochemistry and Western blot.The KLF8 mRNA level in different HCC tissues was determined by RT-PCR.Using immunohistochemistry,we investigated KLF8 expression patterns in Hepatocellular carcinoma(HCC) tissue specimens,metastases,normal liver tissue specimens,and liver cirrhosis tissue specimens.Results:We found that KLF8 protein expressed in the nuclei of cancer cells,strong KLF8 expression was detected in tumor cells,whereas no or very weak KLF8 expression was detected in normal cells surrounding or within the tumors.Metastatic potential HCC cell lines have higher KLF8 mRNA and protein level than primary HCC cell lines.The same results appeared in KLF8 mRNA level of different HCC tissues.Metastasis specimens exhibited the highest level of KLF8 expression(83.3%strong positive;P＜0.001),and primary tumor tissue specimens had higher level of KLF8 expression than normal and liver cirrhosis tissue specimens(P＜0.001).Conclusion:The overexpression of KLF8 may be related to the carcinogenesis and development of HCC.
Part two Clinical significance of overexpression of KLF8 in hepatocellular carcinoma
Objective:To investigate the clinical significance of overexpression of KLF8 in hepatocellular carcinoma.Methods:The qRT-PCR and westernblot were used to detect the expression of ELF8 in HCC tissues,the TMA was used to detect the expression of KLF8 in 314 HCC patients,then the clinical significance of overexpression of KLF8 was analyzed by SPSS11.5.
Results:In tissue samples,patients suffering HCC recurrence exhibited higher KLF8 mRNA expression levels than those without recurrence (p=0.001).According to western blot analysis,KLF8 was highly expressed in five patients with recurrence,KLF8 is also present in 3 of 5 without recurrence,but only one of these the level of expression is high.The expression of KLF8 was classified as strong positive in 162 cases(51.6%, 162/314) and weak positive or not stained in 152 cases(48.4%, 152/314).KLF8 was found to be prognostic for OS(p=0.040) and TTR (p=0.006).The 5-year OS rate and tumor recurrence probabily in the KLF8-negative group were 60.3%and 41.9%,compared with 48.7%and 59.8% for the KLF8-positive group(p=0.038 and p=0.005).Multivariate analysis indicated that KLF8 was an independent prognosticator for OS (p=0.037) and TTR(p=0.018).We further investigated the predictive value of KLF8 within clinical subgroups(AFP≤20 ng/ml vs.＞20 ng/ml,TNM stageⅠvs.Ⅱ-Ⅲ,BCLC stage O+A vs.B+C,EdmondsonⅠ-Ⅱvs.Ⅲ-Ⅳ,and early recurrence vs.later recurrence).The prognostic significance of KLF8 occurred in HCC patients with TNM stageⅠ,BCLC stage O+A,AFP≤20ng/ml and Edmondson stageⅠ-Ⅱ.The 5-year tumor recurrence probabily of KLF8-positive patients was significantly increased compared with KLF8-negative patients in TNM stageⅠ(54.6%vs.35.3%,p=0.008).The 5-year tumor recurrence probabily of KLF8-positive patients was significantly increased compared with KLF8-negative patients in BCLC stage O+A(57.0%vs.37.9%,p=0.009).In the AFP≤20 ng/ml group,the 5-year tumor recurrence probabily were 41.5%and 60.3%in the KLF8-neagtive and KLF8-positve samples,respectively(p=0.027).In the Edmondson stageⅠ-Ⅱgroup,the 5-year tumor recurrence probabily were 36.5%and 54.3%for the KLF8-neagtive and KLF8-positve samples, respectively(p=0.005).According to the recurrence time,the prognostic significance of KLF8 was useful in the early recurrence group (p=0.001) but not in the later recurrence group(p=0.623).KLF8 positve status was positively correlated with early recurrence(p=0.008).
Conclusion:KLF8 may be a new target for prognostic prediction and adjuvant treatment of HCC patients HCC after operation.
Part three Role of KLF8 in HCCLM3 cells proliferation and invasion in vitro,and its possible mechanism.
Objective:To investigate the function of KLF8 in HCC development and progression and its potential mechanism.Methods:KLF8 level in HCCLM3 was down regulated by small interference RNA,then proliferation and invasiveness of HCCLM3 was measured by MTT reduction assay and Matrigel invasion assay;we performed a microarray assay to compare the differential gene expression profiles between KLF8 siRNA-treated and scrambled siRNA-treated HCCLM3 cells.To verify the microarray findings, several differentially expressed genes were further analyzed by qRT-PCR. Other related genes were observed by RT-PCR and Western blot.Results: In western blots,the expression of endogenous KLF8 was knocked down by 90%after KLF8 siRNA treatment for 72 h.Based on trans-well matrigel invasion assays,the number of invasive cells in the KLF8 siRNA-treated group decreased significantly compared with those in the control group (p＜0.001).The MTT assay revealed that cell proliferation was significantly suppressed from 3 day post-transfection (p＜0.001).According to microarray analysis,453 genes were differentially expressed between the KLF8 siRNA-treated HCCLM3 cells and the scrambled siRNA-treated HCCLM3 cells.Of these,229 genes were upregulated(ratio＞2.0),and 224 genes were downregulated(ratio＜0.5). Many of these genes were related to tumor metastasis and regulation of cell migration.The RT-PCR results correspond well to the microarray data. Conclusion:The KLF8 may promote proliferation and invasion of HCCLM3, and involved in many pathways which are important in tumor development and progression.
Part four Effects of KLF8 depletion in vivo
Objective:To investigate the effects of KLF8 depletion on HCCLM3 carciogenesis and lung metastasis in nude mice.Methods:pGCSIL-KLF8 small hairpin RNA(shRNA),a KLFS-RNAi lentiviral vector was constructed, GFP-lentiviral vector(pGCSIL-GFP) was used as a negative control.The lentiviral vectors and pHelper were co-transfected into 293T cells.The culture supernatants were collected,concentrated,and used as a virus stock.The lentivirus was transfected into the HCCLM3 cells.HCCLM3 cells (5×10~6) that had been transfected with KLF8-RNAi lentivirus were implanted subcutaneously into the flanks of nude mice.HCCLM3 cells transfected with GFP-lentivirus were used as a negative control.All the mice were sacrificed 6 weeks later,and the weight of tumors was calculated. Lungs were removed and embedded in paraffin.The paraffin blocks were consecutively sectioned and stained with haematoxylin and eosin.Based on the cell number in the maximal section of the metastatic lesion,the lung metastases were classified into four grades:GradeⅠ,HCCLM3 cells≤20;GradeⅡ,20-50;GradeⅢ,50-100;and GradeⅣ,＞100.Results: The tumors in the KLF8 siRNA-treated group had more necrotic foci than those in the control group.The tumor weights in the control group were significantly greater than that in the KLF8 siRNA group(3.67±0.75g vs.1.02±0.78g,p＜0.001).The incidences of lung metastasis in the KLF8 siRNA group and the control group were 16.7%and 100%,respectively (p=0.015).The metastatic foci of the KLF8 siRNA group were GradeⅠ,while most of metastatic loci in the controls were GradeⅢ-Ⅳ.Conclusion:KLF8 depletion could inhibit HCCLM3 carcinogenesis and lung metastasis in nude mice in vivo.
Kr(u|¨)ppel-like factor 8(KLF8)最初是作为Kr(u|¨)ppel样Cys2/His2锌指转录因子蛋白质家族的一个转录抑制蛋白而被识别的,通过与辅抑制子碳端结合蛋白(CtBP)相联系抑制基因表达。有研究表明KLF8的表达在卵巢癌、肾癌以及乳腺癌的细胞系以及卵巢癌的原发肿瘤组织中是升高的,KLF8是v-Src诱导的致癌转换所必需的,在人类肿瘤的进展中发挥作用。最近研究显示KLF8是一个很强的上皮间质转换(EMT)和侵袭的诱导蛋白。而其在肝(癌)细胞中的表达及其与HCC发生、侵袭转移及临床预后等的关系尚无相关研究。
本课题旨在通过对比和分析不同转移潜能人HCC细胞系的KLF8表达及由314例有完整的临床和随访资料的HCC组织构建的组织芯片、有和无转移HCC组织标本中KLF8的表达,研究KLF8在HCC中的表达情况及其过表达与HCC临床预后的关系;通过靶向KLF8的小干扰RNA和构建表达shRNA-KLF8的慢病毒载体pGCSIL-KLF8并将其包装成慢病毒颗粒来(转染)感染HCCLM3细胞,在体外和体内探索下调KLF8基因表达对HCC生长及侵袭转移的作用,并通过比较下调和未下调KLF8的HCCLM3之间基因转录谱的差异初步探讨KLF8促进HCC生长与侵袭转移的作用机制,为HCC转移的基因治疗提供新的靶点。
第一部分转录因子KLF8在不同肝癌细胞系及组织中的表达
目的:观察正常肝细胞系L-02、不同转移潜能的肝癌细胞系及不同肝(癌)组织中KLF8的表达,初步探讨KLF8表达在肝细胞肝癌中的意义。方法:Real-timePCR、western blot、免疫细胞化学检测不同肝(癌)细胞系中KLF8的mRNA和蛋白质水平;RT-PCR检测不同肝(癌)组织中的KLF8 mRNA水平;免疫组织化学检测不同肝(癌)组织中的KLF8蛋白表达。结果:在不同的肝癌细胞系中KLF8的mRNA及蛋白质水平随着肝癌细胞系侵袭转移潜能的增强而显著升高(P＜0.05),KLF8主要表达于癌细胞的核内;KLF8的mRNA水平在正常肝组织中低,而在肝细胞肝癌组织中较高,在有明确转移的肝细胞肝癌组织中更高;KLF8蛋白表达的阳性率在正常肝组织和肝硬化组织中低而在肝细胞肝癌组织中很高,在肝癌转移灶中最高(强阳性率达83.3%,p＜0.001)。结论:KLF8在不同转移潜能的肝癌细胞系及不同肝(癌)组织中表达有差异,其可能参与肝细胞肝癌的发生和侵袭转移。
第二部分转录因子KLF8在肝癌组织中的表达与肝癌临床病理特征及预后的关系
目的:研究转录因子KLF8在肝细胞肝癌组织中的表达,探讨KLF8与肝细胞肝癌临床病理特征和预后的关系。方法:应用Real-time PCR检测50例肝细胞肝癌组织中KLF8 mRNA的表达;Western Blot检测10例肝细胞肝癌组织中KLF8蛋白的表达;检测由314例肝细胞肝癌组织构建的组织芯片中KLF8蛋白的表达,SSPS11.5统计分析其与肝细胞肝癌临床病理特征及预后的关系。结果:在肝癌组织样本中,相对于无复发的肝细胞肝癌病人标本,有复发的病人标本具有较高的KLF8 mRNA表达水平(p=0.001);Western blot分析显示KLF8蛋白在5例有复发的病人标本中高表达,而在5例无复发的病人标本中仅1例高表达,其他的表达很弱或无表达。KLF8~-组的5-年总生存率OS和肿瘤复发率是60.3%和41.9%,而KLF8~+组是48.7%和59.8%,p=0.038和p=0.005。分层分析显示:在TNM stageⅠ的病人中KLF8~+病人的5-年肿瘤复发率相对于KLF8~-病人来说是显著增加的(54.6%vs.35.3%,p=0.008);同样的结果存在于BCLC 0+A期的病人中(57.0%vs.37.9%,p=0.009);在AFP≤20 ng/ml组中,KLF8~-和KLF8~+病人的5-年肿瘤复发率分别是41.5%和60.3%(p=0.027);而在Edmondson stageⅠ-Ⅱ组中,KLF8~-和KLF8~+病人的5-年肿瘤复发率分别是36.5%和54.3%(p=0.005)。KLF8过表达在病人早期复发中具有预测作用(p=0.001),而对于病人晚期复发无预测作用(p=0.623)。KLF8~+与肝癌的早期复发相关(p=0.008);单变量分析示KLF8对总生存率OS(p=0.040)和无复发生存时间TTR(p=0.006)有预后作用。多变量分析显示KLF8是OS(p=0.037)和TTR(p=0.018)的一个独立预后因素。结论:KLF8过表达与肝癌的早期复发相关,对总生存率OS和无复发生存时间TTR的不良预后有预测作用,是肝细胞肝癌病人的OS和TTR的独立预后因素。在TNM stage I、BCLC 0+A期、AFP≤20 ng/ml、Edmondson stageⅠ-Ⅱ病人中KLF8过表达提示高复发概率,KLF8过表达病人早期复发的概率高。
第三部分下调KLF8表达抑制HCCLM3增殖和侵袭能力的体外实验及其可能的机理研究
目的:考察KLF8表达的下调对HCCLM3细胞增殖和侵袭能力的影响并探讨其可能的机理。方法:siRNA下调KLF8后,MTT实验观察HCCLM3的增殖能力的改变;Transwell侵袭实验观察其侵袭能力的改变;基因芯片技术考察下调KLF8及未下调KLF8的HCCLM3之间的差异表达基因,荧光定量PCR验证部分差异表达的基因;RT-PCR、Western blot、免疫组织化学考察部分差异表达基因。结果:siRNA下调KLF8后HCCLM3的增殖(p＜0.0001)和侵袭能力(p＜0.0001)显著降低;基因芯片结果提示许多参与肿瘤发生发展的基因表达发生改变,KLF8参与多条肿瘤信号通路。结论:KLF8通过参与多条信号通路来调控HCCLM3的增殖及侵袭能力;其在肝细胞肝癌的发生发展中具有重要作用。
第四部分下调KLF8的HCCLM3在裸鼠体内的成瘤及转移能力研究
目的:研究KLF8的下调对HCCLM3在裸鼠体内的成瘤及转移能力的影响。方法:构建KLF8shRNA慢病毒载体,包装成慢病毒颗粒后感染HCCLM3细胞,接种雄性裸鼠,观察其对HCCLM3在裸鼠体内的成瘤与肺转移能力的作用。结果:对照组和实验组均可在裸鼠皮下成瘤;对照组老鼠的肿瘤重量显著大于KLF8下调组老鼠的肿瘤重量(3.67±0.75g vs.1.02±0.78g,p＜0.001);KLF8下调组的老鼠相对于对照组老鼠其肿瘤组织中具有更多的坏死灶,对照组老鼠和KLF8下调组老鼠间肿瘤肺转移的发生率分别是100%(6/6)和16.7%(1/6)(p=0.015)。KLF8下调组的肿瘤转移灶大小局限于I级而大多数的对照组里的转移灶是Ⅲ-Ⅳ级。结论:KLF8的下调抑制HCCLM3在裸鼠体内的成瘤及肺转移能力,KLF8可以作为HCC的一个治疗靶点。
Hepatocellular carcinoma(HCC) is the third most common cause of cancer-related death.Although survival of patients with HCC has been improved by advances in surgical techniques and perioperative management, long-term survival following surgical resection remains unsatisfactory due to the high rate of recurrence and metastasis.Advances in treatment of this disease are likely to stem from a better understanding of its biology and behavior.As biological and clinical behaviors of cancer are affected by multiple molecular pathways that are under the control of transcription factors,improved understanding of how these transcription factors affect cancer biology may lead to an improved ability to predict clinical outcomes and the discovery of novel therapeutic strategies. Kr(u|¨)ppel-like factors(KLFs) constitute a family of nuclear proteins that modulate gene transcription.Emerging evidence suggests that KLFs may be critical factors in tumor development,growth,and metastasis.One of these family members,KLF8,is a CACCC-box binding protein that associates with C-terminal binding protein(CtBP) to repress transcription.Recent studies in breast cancer indicated that KLF8 participates in oncogenic transformation and induces the epithelial-to-mesenchymal transition(EMT),which is an important mechanism during tumor invasion transformation.We found that KLF8 is highly expressed in HCC tissues compared with peritumoral tissues based on transcript profiles,and this result suggested that KLF8 may play an important role in hepatocarcinogenesis.Until now,no studies have reported a role of KLF8 in HCC or the clinicopathological significance of this factor.Here,we examined KLF8 expression in stepwise metastatic potential human HCC cell lines and tumor tissues by quantitative real-time polymerase chain reaction(qRT-PCR),western blot,and immunochemistry analyses.To investigate the possible mechanism,KLF8 expression was knocked down in a HCC cell line with high metastatic potential(HCCLM3 cells) with small-interfering RNA(siRNA),and then the differential gene profiles were investigated by cDNA microarray analysis.The effects of KLF8 depletion were observed in vitro and in vivo.To explore the clinical effects of this factor,KLF8 expression was detected in 314 HCC patients using tissue microarrays(TMAs).Together,our data indicates that KLF8 promotes HCC cell proliferation,invasion,and migration.High expression of KLF8 is strongly associated with early recurrence and poor prognosis in HCC patients after curative resection.
Part one Expression of KLF8 in different liver cell lines and in HCC tissues
Objective:This study investigated expression of KLF8 in differenr liver cell lines and in HCC tissues to evaluate the role of KLF8 in HCC development and progression.Methods:The KLF8 level in different liver cell lines was respectively determined by quantitative real-time PCR, Immunocytochemistry and Western blot.The KLF8 mRNA level in different HCC tissues was determined by RT-PCR.Using immunohistochemistry,we investigated KLF8 expression patterns in Hepatocellular carcinoma(HCC) tissue specimens,metastases,normal liver tissue specimens,and liver cirrhosis tissue specimens.Results:We found that KLF8 protein expressed in the nuclei of cancer cells,strong KLF8 expression was detected in tumor cells,whereas no or very weak KLF8 expression was detected in normal cells surrounding or within the tumors.Metastatic potential HCC cell lines have higher KLF8 mRNA and protein level than primary HCC cell lines.The same results appeared in KLF8 mRNA level of different HCC tissues.Metastasis specimens exhibited the highest level of KLF8 expression(83.3%strong positive;P＜0.001),and primary tumor tissue specimens had higher level of KLF8 expression than normal and liver cirrhosis tissue specimens(P＜0.001).Conclusion:The overexpression of KLF8 may be related to the carcinogenesis and development of HCC.
Part two Clinical significance of overexpression of KLF8 in hepatocellular carcinoma
Objective:To investigate the clinical significance of overexpression of KLF8 in hepatocellular carcinoma.Methods:The qRT-PCR and westernblot were used to detect the expression of ELF8 in HCC tissues,the TMA was used to detect the expression of KLF8 in 314 HCC patients,then the clinical significance of overexpression of KLF8 was analyzed by SPSS11.5.
Results:In tissue samples,patients suffering HCC recurrence exhibited higher KLF8 mRNA expression levels than those without recurrence (p=0.001).According to western blot analysis,KLF8 was highly expressed in five patients with recurrence,KLF8 is also present in 3 of 5 without recurrence,but only one of these the level of expression is high.The expression of KLF8 was classified as strong positive in 162 cases(51.6%, 162/314) and weak positive or not stained in 152 cases(48.4%, 152/314).KLF8 was found to be prognostic for OS(p=0.040) and TTR (p=0.006).The 5-year OS rate and tumor recurrence probabily in the KLF8-negative group were 60.3%and 41.9%,compared with 48.7%and 59.8% for the KLF8-positive group(p=0.038 and p=0.005).Multivariate analysis indicated that KLF8 was an independent prognosticator for OS (p=0.037) and TTR(p=0.018).We further investigated the predictive value of KLF8 within clinical subgroups(AFP≤20 ng/ml vs.＞20 ng/ml,TNM stageⅠvs.Ⅱ-Ⅲ,BCLC stage O+A vs.B+C,EdmondsonⅠ-Ⅱvs.Ⅲ-Ⅳ,and early recurrence vs.later recurrence).The prognostic significance of KLF8 occurred in HCC patients with TNM stageⅠ,BCLC stage O+A,AFP≤20ng/ml and Edmondson stageⅠ-Ⅱ.The 5-year tumor recurrence probabily of KLF8-positive patients was significantly increased compared with KLF8-negative patients in TNM stageⅠ(54.6%vs.35.3%,p=0.008).The 5-year tumor recurrence probabily of KLF8-positive patients was significantly increased compared with KLF8-negative patients in BCLC stage O+A(57.0%vs.37.9%,p=0.009).In the AFP≤20 ng/ml group,the 5-year tumor recurrence probabily were 41.5%and 60.3%in the KLF8-neagtive and KLF8-positve samples,respectively(p=0.027).In the Edmondson stageⅠ-Ⅱgroup,the 5-year tumor recurrence probabily were 36.5%and 54.3%for the KLF8-neagtive and KLF8-positve samples, respectively(p=0.005).According to the recurrence time,the prognostic significance of KLF8 was useful in the early recurrence group (p=0.001) but not in the later recurrence group(p=0.623).KLF8 positve status was positively correlated with early recurrence(p=0.008).
Conclusion:KLF8 may be a new target for prognostic prediction and adjuvant treatment of HCC patients HCC after operation.
Part three Role of KLF8 in HCCLM3 cells proliferation and invasion in vitro,and its possible mechanism.
Objective:To investigate the function of KLF8 in HCC development and progression and its potential mechanism.Methods:KLF8 level in HCCLM3 was down regulated by small interference RNA,then proliferation and invasiveness of HCCLM3 was measured by MTT reduction assay and Matrigel invasion assay;we performed a microarray assay to compare the differential gene expression profiles between KLF8 siRNA-treated and scrambled siRNA-treated HCCLM3 cells.To verify the microarray findings, several differentially expressed genes were further analyzed by qRT-PCR. Other related genes were observed by RT-PCR and Western blot.Results: In western blots,the expression of endogenous KLF8 was knocked down by 90%after KLF8 siRNA treatment for 72 h.Based on trans-well matrigel invasion assays,the number of invasive cells in the KLF8 siRNA-treated group decreased significantly compared with those in the control group (p＜0.001).The MTT assay revealed that cell proliferation was significantly suppressed from 3 day post-transfection (p＜0.001).According to microarray analysis,453 genes were differentially expressed between the KLF8 siRNA-treated HCCLM3 cells and the scrambled siRNA-treated HCCLM3 cells.Of these,229 genes were upregulated(ratio＞2.0),and 224 genes were downregulated(ratio＜0.5). Many of these genes were related to tumor metastasis and regulation of cell migration.The RT-PCR results correspond well to the microarray data. Conclusion:The KLF8 may promote proliferation and invasion of HCCLM3, and involved in many pathways which are important in tumor development and progression.
Part four Effects of KLF8 depletion in vivo
Objective:To investigate the effects of KLF8 depletion on HCCLM3 carciogenesis and lung metastasis in nude mice.Methods:pGCSIL-KLF8 small hairpin RNA(shRNA),a KLFS-RNAi lentiviral vector was constructed, GFP-lentiviral vector(pGCSIL-GFP) was used as a negative control.The lentiviral vectors and pHelper were co-transfected into 293T cells.The culture supernatants were collected,concentrated,and used as a virus stock.The lentivirus was transfected into the HCCLM3 cells.HCCLM3 cells (5×10~6) that had been transfected with KLF8-RNAi lentivirus were implanted subcutaneously into the flanks of nude mice.HCCLM3 cells transfected with GFP-lentivirus were used as a negative control.All the mice were sacrificed 6 weeks later,and the weight of tumors was calculated. Lungs were removed and embedded in paraffin.The paraffin blocks were consecutively sectioned and stained with haematoxylin and eosin.Based on the cell number in the maximal section of the metastatic lesion,the lung metastases were classified into four grades:GradeⅠ,HCCLM3 cells≤20;GradeⅡ,20-50;GradeⅢ,50-100;and GradeⅣ,＞100.Results: The tumors in the KLF8 siRNA-treated group had more necrotic foci than those in the control group.The tumor weights in the control group were significantly greater than that in the KLF8 siRNA group(3.67±0.75g vs.1.02±0.78g,p＜0.001).The incidences of lung metastasis in the KLF8 siRNA group and the control group were 16.7%and 100%,respectively (p=0.015).The metastatic foci of the KLF8 siRNA group were GradeⅠ,while most of metastatic loci in the controls were GradeⅢ-Ⅳ.Conclusion:KLF8 depletion could inhibit HCCLM3 carcinogenesis and lung metastasis in nude mice in vivo.
引文
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