过氧化物酶体增殖物活化受体γ基因静默对肝癌生物学行为的影响
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摘要
第一部分PPARγ在肝细胞癌中的表达及其临床意义
目的探讨过氧化物酶体增殖物活化受体γ(peroxisome proliferators activated receptorγ, PPARγ)在肝癌中的表达及其临床意义。方法半定量RT-PCR及Western blot法、免疫组织化学法检测肝癌细胞株HepG2、HepG2.2.15、HCCLM3、肝细胞株L-02和34例手术切除的肝癌及相对应的癌旁组织PPARγmRNA、蛋白表达,应用CD34染色和CD34联合过碘酸雪夫氏反应(PAS)复合染色检测微血管密度(MVD)和血管生成拟态(VM),结合细胞株生物学特性及患者临床特征探讨PPARγ表达的临床意义。结果PPARγmRNA在细胞株L-02、HepG2、HepG2.2.15及HCCLM3中均有表达,与GAPDH的相对灰度值比分别为0.905±0.028,1.547±0.071,1.887±0.085和1.497±0.061, L-02与肝癌细胞株之间存在显著性差异(F=118.84,P<0.01);HepG2.2.15与HepG2、HCCLM3之间亦有差异(F=25.33,P<0.01);Western blot法检测结果与RT-PCR结果一致。79.4% (27/34)的肝癌组织PPARγmRNA表达高于癌旁组织; PPARγ蛋白在肝癌组织中均呈阳性表达,为胞浆和/或胞核染色,而癌旁组织为阴性或弱阳性表达,为胞浆浅染色,阳性表达分级之间存在显著差异(x2=39.62,P<0.05)。PPARγmRNA表达水平与蛋白表达分级与肝癌大小、包膜完整、高转移复发倾向和HBsAg感染及肝硬化背景密切相关,(P<0.05);但与患者年龄、性别、Okuda分期、pTNM分期、组织分化程度、AFP阳性和随访转移结果无关。PPARγmRNA及蛋白高表达与患者的总生存期负相关,Kaplan-Meier曲线分析的Log-rank检验x2=3.953和11.406,P=0.047和0.003。癌组织中MVD高于癌旁组织(t=18.925,P<0.05);高MVD与肿瘤>5cm(P=0.010)、有无包膜(P=0.001)、转移复发倾向(P=0.004)、随访有无转移(P=0.008)和分化程度(P=0.001)有关;7/34的癌中发现VM存在,VM阳性组MVD高(t=16.373,P<0.05);VM阳性与临床分期(P=0.001)、复发倾向(P=0.029)和分化程度(P=0.004)相关。PPARγ阳性表达与高MVD成正相关(r=0.519);PPARγ阳性,高MVD和VM阳性可判断患者生存率,Log-rank检验P分别为0.013,0. 036和0. 022。COX风险模型分析PPARγ阳性和高MVD均是独立的预测HCC预后的因子。结论过氧化物酶体增殖物活化受体γ在肝癌中过表达,与肝癌的发生发展有关,可能是判断肝癌预后的预测指标。
第二部分PPARγ短发夹状RNA的构建、鉴定及稳定克隆的筛选
目的构建靶向过氧化物酶体增殖物活化受体γ(PPARγ)基因的shRNA-DNA表达载体,观察体外转染肝癌细胞株HCCLM3细胞后,其对PPARγ活性的抑制效率。方法以人PPARγ1为靶基因设计并合成两条DNA序列,克隆到载体pBAsi-hU6-DNA中构建重组体pshPPARγ,无内毒素抽提,并测序鉴定。DOTAP脂质体法转染重组体至HCCLM3细胞,优化G418浓度、筛选,以裸质粒转染为阳性对照,正常培养细胞为阴性对照,半定量RT-PCR及Western blot观察转染40h、筛选的阳性克隆细胞中PPARγ表达变化。结果pshPPARγ经测序鉴定与设计目的序列完全一致。pshPPARγ转染HCCLM3细胞后40h、应用600μg/ml的G418筛选的阳性克隆细胞中PPARγmRNA表达降低,与对照组比较存在显著性差异(F=29.47和75.22,P<0.01),对照组之间无差异(t=0.526和0.703,P>0.05),蛋白水平的静默效率与mRNA水平一致,抑制效率均在80%以上。结论成功构建无内毒素的靶向PPARγ基因的shRNA重组质粒表达载体,能稳定抑制HCCLM3细胞中PPARγ活性,为利用RNAi技术在细胞和在体水平研究PPARγ在肝癌中的作用机制、探索HCC的基因治疗打下了基础。
第三部分PPARγ基因静默对肝细胞癌增殖与凋亡的影响
目的体外和活体内观察pshPPARγ抑制PPARγ活性对HCCLM3细胞增殖和凋亡的影响,并探讨可能的作用机制。方法应用pshPPARγ瞬时转染HCCLM3细胞,MTT法检测细胞的生长曲线,TUNEL法观察凋亡细胞形态,流式细胞仪分析凋亡率;应用稳定表达pshPPARγ的HCCLM3细胞建立裸鼠动物模型,观察肿瘤生长状况,应用TUNEL法观察移植瘤肿瘤细胞凋亡形态;应用免疫组织化学法检测HCCLM3细胞和移植瘤PCNA、wt-P53和整合素β1变化,RT-PCR检测MMP2、TIMP2和整合素β1改变。结果pshPPARγ转染HCCLM3后40h,MTT检测显示细胞增殖抑制率达71.5%。流式细胞仪检测示pshPPARγ组凋亡率为23.2±4.2%,裸质粒组与对照组细胞凋亡率分别为3.3±0.9%和3.1±0.7%,pshPPARγ组凋亡明显增多,存在显著差异(F=48.32,P<0.01)。TUNEL法原位凋亡指数结果与流式细胞仪结果类似。pshPPARγ组移植瘤内PPARγ蛋白和mRNA均下调,成瘤潜伏期延长,移植瘤生长减慢,瘤体积为1.86±0.65 cm3,而对照组为4.86±1.15 cm3,成瘤潜伏期、生长曲线及瘤体积与对照组比较,P均<0.05;pshPPARγ组原位凋亡明显增多。在细胞和动物模型中,pshPPARγ组PCNA表达减少,而野生型P53增多,与对照组比较,P<0.05;RT-PCR显示pshPPARγ组MMP2、整合素β1表达下调,TIMP2表达上调。结论PPARγ基因静默能抑制HCCLM3细胞增殖,促进凋亡;此作用可能经由整合素信号通路,调节MMP2/TIMP2平衡,降低PCNA表达和促进wt-P53起作用。
第四部分PPARγ基因静默对肝癌转移与移植瘤瘤内微循环的影响目的观察pshPPARγ抑制PPARγ活性对HCCLM3细胞转移和移植瘤瘤内微循环的影响,并探讨可能作用机制。方法应用Transwell小室建立肿瘤体外侵袭模型,观察pshPPARγ瞬时转染对HCCLM3细胞黏附、迁移、侵袭能力的影响,应用过河实验观察细胞移动能力的改变,应用HCCLM3细胞与ECV304细胞共培养,观察pshPPARγ对其形成管腔能力的影响。应用裸鼠肝癌皮下移植瘤模型的肺组织连续切片,HE染色和hepatocyte免疫组织化学鉴定肺转移灶并计数。应用免疫组织化学检测移植瘤VEGF、TSP1和肺转移灶中整合素β1蛋白表达,RT-PCR检测移植瘤VEGF、TSP1以及肺转移灶整合素β1 mRNA表达。结果pshPPARγ组跨过半透膜的细胞数为68±7,明显少于裸质粒组和对照组的280±10和302±14 (F=548.72,P<0.01);透过ECM的细胞数量为17±3,亦明显少于对照组(F=258.19,P<0.01);细胞越过划痕的时间为6.40±1.14d,与对照组存在显著性差异(F=17.58,P<0.01);细胞黏附能力亦显著下降,2h后pshPPARγ组粘附的细胞数为49±9,而对照组为83±13和88±11(F=17.39,P<0.01)。pshPPARγ明显降低ECV304细胞体外形成管腔样结构的能力。pshPPARγ组裸鼠肺转移灶小,整肺总转移灶数量显著少于对照组(t=18.69,P<0.05)。pshPPARγ组移植瘤的微血管(MV)少且多呈点状,而对照组密集且出现环形,两组MVD计数有显著性差异,P<0.05;移植瘤块中存在血管生成拟态现象(VM),pshPPARγ组稀疏分布,细小;VEGF在pshPPARγ组表达下调,而TSP1上调。整合素β1表达在肺转移灶无变化。结论PPARγ基因静默能抑制HCCLM3细胞侵袭及远处转移,减少肝癌血管生成;此作用亦可能经由整合素信号通路,调节MMP2/TIMP2平衡、调节VEGF/TSP分泌而起作用。
The First Section: The expression and significance of PPARγin hepatocellular carcinoma Objective To study the expression of peroxisome proliferators-activated receptor gamma (PPARγ) mRNA and protein in hepatocellular carcinoma (HCC) and its association with clinicopathologic features, angiogenesis, and prognosis of HCC. Methods PPARγmRNA and protein were detected with half quantitative reverse transcription PCR (RT-PCR) and Western blot in hepatocyte line L02 and hepatoma carcinoma cell lines HepG2, HepG2.2.15 and HCCLM3. Furthermore, PPARγmRNA and protein in 34 cases of HCC, including HCC tissues and corresponding peri-tumor tissues, were detected with RT-PCR and immunohistochemical method, anti-CD34 and multiplicity staining of anti-CD34 combined with periodic acid-schiff (PAS) were utilized to stain these specimens, and the significance of PPARγin HCC were analyzed. Results PPARγmRNA express in all cell lines, its relative gray scale score is 0.905±0.028, 1.547±0.071, 1.887±0.085 and 1.497±0.061 in L-02, HepG2, HepG2.2.15 and HCCLM3, respectively. It is statistically significant difference between L02 and hepatoma carcinoma cell lines (F=118.84, P<0.05), the expression of PPARγin HepG2.2.15 is highest, comparisons between the other HCC lines, it is statistically significant (F=25.33, P<0.05). PPARγprotein levels are coincidence with mRNA. 27 cases, PPARγmRNA in HCC tissues is higher than their paired non-tumor tissues; all of the HCC tumor cells were positive stained for PPARγprotein, expressed in cytoplasm and/or nucleus of hepatocytes. Hepatocytes of peri-tumor stained negative or weakly positive in cytoplasm, grade of positive staining in two groups is significant (x2=39.62, P<0.05). The clinical characteristic of HCC, such as tumor size, tumor capsule, tendency to recurrence, the background of HBV infection and cirrhosis, is correlated with PPARγmRNA levels and the grade of positive staining; but the patients’ages, gender, Okuda and pTNM stages, serum AFP levels, degrees of histological differentiation and metabasis by follow ups, is not associated with the expression of PPARγ. There is negative correlation between the expression of PPARγand the total life span in HCC patients, it is significant, P value tested by Log rank of Kaplan-Meier curve analysis in various mRNA levels and protein grading is 0.047 and 0.003, respectively. MVD (Microvessel density) in tumor tissues are higher than non-tumor tissues, it is statistically significance (t=18.925, P<0.05). High MVD is correlated with tumor size (P=0.010), tumor capsule breakdown (P=0.001), tendency to recurrence (P=0.004), metastases during follow up (P=0.008) and low differentiation type (P=0.001). The expression of VM (Vascularization mimicry) exists in 7/34 specimens of HCC, MVD in positive specimens of VM are higher (t=16.373, P<0.05). The expression of VM is correlated with clinical TNM stage (P=0.001), tendency to recurrence (P=0.029), and low differentiation type (P=0.001). There was a significant positive correlation between PPARγand high MVD expression (r=0.519, P<0.05), but there was no correlation between PPARγand MV expression (r=0.169, P<0.05). Kaplan-Meier analysis indicated that PPARγoverexpression, high MVD and VM expression were associated with patient’s worse disease-free survival; P value tested by Log-rank is 0.013, 0.036 and 0.022, respectively. According to Cox multivariate survival analysis, PPARγand MVD were the independent prognostic factors of HCC (both Pmuti<0.05). Conclusions PPARγover-expressed in HCC, it may be associated with the carcinogenesis and development of HCC; it may be an index to judge prognosis of HCC. [Key Words] PPARγ; Hepatocellular carcinoma; Survival analysis; Biological characteristic; Angiogenesis; Vascularization mimicry The Second Section: Construction, identification and screening of shRNA targeted on PPARγgene Objective To construct shRNA targeted on PPARγgene with pBAsi-hU6-DNA plasmids, and to observe the inhibition efficacy of PPARγactivation after HCCLM3 cells were transfected by shRNA. Methods Duplicate bands DNA sequence were synthesized in accordance with human PPARγ1 gene cDNA sequence, to construct recombinant of PPARγ(pshPPARγ) with pBAsi-hU6-DNA plasmids, then free endotoxin extracted, and assessed by sequencing. pshPPARγrecombinants were transfected into HCCLM3 cells by DOTAP hangosome methods, RT-PCR and Western blot was used to detect the inhibition efficacy of PPARγactivation in 40h and 4w post-transfection. Results pshPPARγis coincidence with targeted sequence, after 40h pshPPARγwere transfected into HCCLM3 cells, the expression of PPARγmRNA and protein were inhibited comparing with normal cells and cells transfected by neo-DNA plasmids (F=29.47, P<0.01). In positive clone cells selcted with 600μg/ml G418 (in 4w), PPARγmRNA and protein also were inhibited significantly by pshPPARγ(F=75.22, P<0.01), their inhibition rate in RT-PCR test is 80.5% and 82.3%, respectively. Conclusions pshPPARγcan lastingly inhibit PPARγactivation in HCCLM3 cells; it may be a tool to study the mechanisms of PPARγof hepatocellular carcinoma in vitro or in vivo. [Key Words] RNA interference; short hairpin RNA; transfection; expression; recombinant The Third Section: Gene silencing targeted on PPARγaffect proliferation and apoptosis of hepatocellular carcinoma Objective To observe the effect on proliferation and apoptosis of HCCLM3 cells with short hair-pin RNA targeted on PPARγgene in vitro and in vivo, and to explore the potential mechanisms. Methods After HCCLM3 cells were transient transfected with pshPPARγplasmid, growth curves were detected by MTT methods, apoptosis rate were analyzed by flow cytometry, and morphocytology of apoptotic HCCLM3 cells were observed by TUNEL methods. Then, animal models were constructed with HCCLM3 cells stable transfected with pshPPARγ, PPARγmRNA and protein in tumor tissues were detected with RT-PCR and Western blot or immuocytochemistry (ICC) methods, growth curves and volumes of tumor were observed, and morphocytology of apoptotic cells were detected by TUNEL method. Moreover, the expression of PCNA and wt-P53 were stained by ICC methods, and MMP2, TIMP2 andβ1 integrin were detected by RT-PCR in vitro and in vivo. Results Cell growth was inhibited in pshPPARγgroup by 71.5%, apoptosis also increased significantly, FCM show the apoptosis ratio is 23.2±4.2%, it is statistically significance between pshPPARγgroup and control group (3.3±0.9% vs 3.1±0.7 %) (F=48.32, P<0.01), morphocytology test is the same as FCM. PPARγmRNA and protein decreased significantly in tumor tissues of pshPPARγgroup, inhibition rate reach to 82%. Stage of latency lengthen, and growth were inhibited, volumes of tumors in pshPPARγgroups are 1.86±0.65 cm3, but 4.86±1.15 cm3 in control groups, apoptosis in situ of tumors increased, it is statistically significance, P<0.05. In vitro and in vivo, PCNA protein decreased, and wt-P53 protein increased. In HCCLM3 cells of pshPPARγgroup, MMP2 and integrinβ1 mRNA decreased significantly, but TIMP2 increased. Conclusions Inhibition of PPARγactivation can decrease the proliferation in HCC. It maybe regulate the balance of MMP2 and TIMP2 via integrin signal way, then decrease the expression of PCNA, and promote wild-type P53 to induce the apoptosis of HCCLM3 cells. Key Words Proliferation; Apoptosis; Proliferating cell nuclear antigen; Wild-type P53; Matrix metalloproteinase; Tissue inhibitor of metalloproteinase; Integrin The Fourth Section: Gene silencing targeted on PPARγinfluence metastasis and angiogenesis of hepatocellular carcinoma Objective To observe the effect on invasion, metastasis, and angiogenesis in HCC when PPARγactivation was inhibited by shRNA targeted at PPARγgene of HCCLM3 cells in vitro and in vivo, and to explore the potential mechanisms. Methods Boyden chambers were utilized to observe the effect on cell adhesion, migration, invasion and locomotion of HCCLM3 cells, meanwhile, the effect on lumens formed by ECV 304 cells was observed in models which HCCLM3 cells co-cultivated with ECV 304 cells post-transient transfected in vitro. Animal models of cancerometastasis were established by HCCLM3 cells stable expressed pshPPARγor not with nude mouse, pulmonary metastasis (PM) was assessed by HE staining and immuocytochemistry (ICC) staining with hepatocyte, graded and counted in serial sections of lung tissues. MVD and VM in transplantation tumor were stained by immuocytochemistry staining of CD34 and multiplicity staining with CD34 combined with periodic acid-schiff (PAS). Integrinβ1 expression in lung tissues, VEGF and TSP1 expression in tumors was detected by ICC staining and RT-PCR methods. Results Cells crawled over semipermeable membrane of boyden chambers were 68±7 in pshPPARγgroups, it is less than control groups (281±10 and 302±14), it is statistical significance (F=548.72, P<0.01). Cells permeated through ECM were 17±3; it is also less than control groups (F=258.19, P<0.01). Time when cells healed the wound were 6.40±1.14d, it is longer than control groups (F=17.58, P<0.01). Ability of cell adhesion decreased significantly, after 2h, cell counts are 49±9, 83±13 and 88±11 in pshPPARγgroup and two control groups, respectively, it is statistical significance (F=17.39, P<0.01). Lumens formed by ECV 304 cells were weakened in pshPPARγgroup. PM in pshPPARγgroup were smaller than in control group, and total amounts in lung tissues were also less (t=18.69, P<0.05). MVD in transplantation tumors are scarce and punctiform, while in control group, MVD are intensive and annuliform, it is statistically significance between two groups, P<0.05. VM exist in transplantation tumor, sparse and tiny in pshPPARγgroup, meanwhile, VEGF mRNA and protein decreased significantly, TSP1 increased. Integrinβ1 expression in lung tissues was not difference between two groups. Conclusions PPARγinhibition can decrease the ability of invasion, metastasis and angiogenesis in HCC; it may be via integrin signal way, to regulate the balance of MMP2 and TIMP2, and affect the secretion of VEGF/TSP1.
目的探讨过氧化物酶体增殖物活化受体γ(peroxisome proliferators activated receptorγ, PPARγ)在肝癌中的表达及其临床意义。方法半定量RT-PCR及Western blot法、免疫组织化学法检测肝癌细胞株HepG2、HepG2.2.15、HCCLM3、肝细胞株L-02和34例手术切除的肝癌及相对应的癌旁组织PPARγmRNA、蛋白表达,应用CD34染色和CD34联合过碘酸雪夫氏反应(PAS)复合染色检测微血管密度(MVD)和血管生成拟态(VM),结合细胞株生物学特性及患者临床特征探讨PPARγ表达的临床意义。结果PPARγmRNA在细胞株L-02、HepG2、HepG2.2.15及HCCLM3中均有表达,与GAPDH的相对灰度值比分别为0.905±0.028,1.547±0.071,1.887±0.085和1.497±0.061, L-02与肝癌细胞株之间存在显著性差异(F=118.84,P<0.01);HepG2.2.15与HepG2、HCCLM3之间亦有差异(F=25.33,P<0.01);Western blot法检测结果与RT-PCR结果一致。79.4% (27/34)的肝癌组织PPARγmRNA表达高于癌旁组织; PPARγ蛋白在肝癌组织中均呈阳性表达,为胞浆和/或胞核染色,而癌旁组织为阴性或弱阳性表达,为胞浆浅染色,阳性表达分级之间存在显著差异(x2=39.62,P<0.05)。PPARγmRNA表达水平与蛋白表达分级与肝癌大小、包膜完整、高转移复发倾向和HBsAg感染及肝硬化背景密切相关,(P<0.05);但与患者年龄、性别、Okuda分期、pTNM分期、组织分化程度、AFP阳性和随访转移结果无关。PPARγmRNA及蛋白高表达与患者的总生存期负相关,Kaplan-Meier曲线分析的Log-rank检验x2=3.953和11.406,P=0.047和0.003。癌组织中MVD高于癌旁组织(t=18.925,P<0.05);高MVD与肿瘤>5cm(P=0.010)、有无包膜(P=0.001)、转移复发倾向(P=0.004)、随访有无转移(P=0.008)和分化程度(P=0.001)有关;7/34的癌中发现VM存在,VM阳性组MVD高(t=16.373,P<0.05);VM阳性与临床分期(P=0.001)、复发倾向(P=0.029)和分化程度(P=0.004)相关。PPARγ阳性表达与高MVD成正相关(r=0.519);PPARγ阳性,高MVD和VM阳性可判断患者生存率,Log-rank检验P分别为0.013,0. 036和0. 022。COX风险模型分析PPARγ阳性和高MVD均是独立的预测HCC预后的因子。结论过氧化物酶体增殖物活化受体γ在肝癌中过表达,与肝癌的发生发展有关,可能是判断肝癌预后的预测指标。
第二部分PPARγ短发夹状RNA的构建、鉴定及稳定克隆的筛选
目的构建靶向过氧化物酶体增殖物活化受体γ(PPARγ)基因的shRNA-DNA表达载体,观察体外转染肝癌细胞株HCCLM3细胞后,其对PPARγ活性的抑制效率。方法以人PPARγ1为靶基因设计并合成两条DNA序列,克隆到载体pBAsi-hU6-DNA中构建重组体pshPPARγ,无内毒素抽提,并测序鉴定。DOTAP脂质体法转染重组体至HCCLM3细胞,优化G418浓度、筛选,以裸质粒转染为阳性对照,正常培养细胞为阴性对照,半定量RT-PCR及Western blot观察转染40h、筛选的阳性克隆细胞中PPARγ表达变化。结果pshPPARγ经测序鉴定与设计目的序列完全一致。pshPPARγ转染HCCLM3细胞后40h、应用600μg/ml的G418筛选的阳性克隆细胞中PPARγmRNA表达降低,与对照组比较存在显著性差异(F=29.47和75.22,P<0.01),对照组之间无差异(t=0.526和0.703,P>0.05),蛋白水平的静默效率与mRNA水平一致,抑制效率均在80%以上。结论成功构建无内毒素的靶向PPARγ基因的shRNA重组质粒表达载体,能稳定抑制HCCLM3细胞中PPARγ活性,为利用RNAi技术在细胞和在体水平研究PPARγ在肝癌中的作用机制、探索HCC的基因治疗打下了基础。
第三部分PPARγ基因静默对肝细胞癌增殖与凋亡的影响
目的体外和活体内观察pshPPARγ抑制PPARγ活性对HCCLM3细胞增殖和凋亡的影响,并探讨可能的作用机制。方法应用pshPPARγ瞬时转染HCCLM3细胞,MTT法检测细胞的生长曲线,TUNEL法观察凋亡细胞形态,流式细胞仪分析凋亡率;应用稳定表达pshPPARγ的HCCLM3细胞建立裸鼠动物模型,观察肿瘤生长状况,应用TUNEL法观察移植瘤肿瘤细胞凋亡形态;应用免疫组织化学法检测HCCLM3细胞和移植瘤PCNA、wt-P53和整合素β1变化,RT-PCR检测MMP2、TIMP2和整合素β1改变。结果pshPPARγ转染HCCLM3后40h,MTT检测显示细胞增殖抑制率达71.5%。流式细胞仪检测示pshPPARγ组凋亡率为23.2±4.2%,裸质粒组与对照组细胞凋亡率分别为3.3±0.9%和3.1±0.7%,pshPPARγ组凋亡明显增多,存在显著差异(F=48.32,P<0.01)。TUNEL法原位凋亡指数结果与流式细胞仪结果类似。pshPPARγ组移植瘤内PPARγ蛋白和mRNA均下调,成瘤潜伏期延长,移植瘤生长减慢,瘤体积为1.86±0.65 cm3,而对照组为4.86±1.15 cm3,成瘤潜伏期、生长曲线及瘤体积与对照组比较,P均<0.05;pshPPARγ组原位凋亡明显增多。在细胞和动物模型中,pshPPARγ组PCNA表达减少,而野生型P53增多,与对照组比较,P<0.05;RT-PCR显示pshPPARγ组MMP2、整合素β1表达下调,TIMP2表达上调。结论PPARγ基因静默能抑制HCCLM3细胞增殖,促进凋亡;此作用可能经由整合素信号通路,调节MMP2/TIMP2平衡,降低PCNA表达和促进wt-P53起作用。
第四部分PPARγ基因静默对肝癌转移与移植瘤瘤内微循环的影响目的观察pshPPARγ抑制PPARγ活性对HCCLM3细胞转移和移植瘤瘤内微循环的影响,并探讨可能作用机制。方法应用Transwell小室建立肿瘤体外侵袭模型,观察pshPPARγ瞬时转染对HCCLM3细胞黏附、迁移、侵袭能力的影响,应用过河实验观察细胞移动能力的改变,应用HCCLM3细胞与ECV304细胞共培养,观察pshPPARγ对其形成管腔能力的影响。应用裸鼠肝癌皮下移植瘤模型的肺组织连续切片,HE染色和hepatocyte免疫组织化学鉴定肺转移灶并计数。应用免疫组织化学检测移植瘤VEGF、TSP1和肺转移灶中整合素β1蛋白表达,RT-PCR检测移植瘤VEGF、TSP1以及肺转移灶整合素β1 mRNA表达。结果pshPPARγ组跨过半透膜的细胞数为68±7,明显少于裸质粒组和对照组的280±10和302±14 (F=548.72,P<0.01);透过ECM的细胞数量为17±3,亦明显少于对照组(F=258.19,P<0.01);细胞越过划痕的时间为6.40±1.14d,与对照组存在显著性差异(F=17.58,P<0.01);细胞黏附能力亦显著下降,2h后pshPPARγ组粘附的细胞数为49±9,而对照组为83±13和88±11(F=17.39,P<0.01)。pshPPARγ明显降低ECV304细胞体外形成管腔样结构的能力。pshPPARγ组裸鼠肺转移灶小,整肺总转移灶数量显著少于对照组(t=18.69,P<0.05)。pshPPARγ组移植瘤的微血管(MV)少且多呈点状,而对照组密集且出现环形,两组MVD计数有显著性差异,P<0.05;移植瘤块中存在血管生成拟态现象(VM),pshPPARγ组稀疏分布,细小;VEGF在pshPPARγ组表达下调,而TSP1上调。整合素β1表达在肺转移灶无变化。结论PPARγ基因静默能抑制HCCLM3细胞侵袭及远处转移,减少肝癌血管生成;此作用亦可能经由整合素信号通路,调节MMP2/TIMP2平衡、调节VEGF/TSP分泌而起作用。
The First Section: The expression and significance of PPARγin hepatocellular carcinoma Objective To study the expression of peroxisome proliferators-activated receptor gamma (PPARγ) mRNA and protein in hepatocellular carcinoma (HCC) and its association with clinicopathologic features, angiogenesis, and prognosis of HCC. Methods PPARγmRNA and protein were detected with half quantitative reverse transcription PCR (RT-PCR) and Western blot in hepatocyte line L02 and hepatoma carcinoma cell lines HepG2, HepG2.2.15 and HCCLM3. Furthermore, PPARγmRNA and protein in 34 cases of HCC, including HCC tissues and corresponding peri-tumor tissues, were detected with RT-PCR and immunohistochemical method, anti-CD34 and multiplicity staining of anti-CD34 combined with periodic acid-schiff (PAS) were utilized to stain these specimens, and the significance of PPARγin HCC were analyzed. Results PPARγmRNA express in all cell lines, its relative gray scale score is 0.905±0.028, 1.547±0.071, 1.887±0.085 and 1.497±0.061 in L-02, HepG2, HepG2.2.15 and HCCLM3, respectively. It is statistically significant difference between L02 and hepatoma carcinoma cell lines (F=118.84, P<0.05), the expression of PPARγin HepG2.2.15 is highest, comparisons between the other HCC lines, it is statistically significant (F=25.33, P<0.05). PPARγprotein levels are coincidence with mRNA. 27 cases, PPARγmRNA in HCC tissues is higher than their paired non-tumor tissues; all of the HCC tumor cells were positive stained for PPARγprotein, expressed in cytoplasm and/or nucleus of hepatocytes. Hepatocytes of peri-tumor stained negative or weakly positive in cytoplasm, grade of positive staining in two groups is significant (x2=39.62, P<0.05). The clinical characteristic of HCC, such as tumor size, tumor capsule, tendency to recurrence, the background of HBV infection and cirrhosis, is correlated with PPARγmRNA levels and the grade of positive staining; but the patients’ages, gender, Okuda and pTNM stages, serum AFP levels, degrees of histological differentiation and metabasis by follow ups, is not associated with the expression of PPARγ. There is negative correlation between the expression of PPARγand the total life span in HCC patients, it is significant, P value tested by Log rank of Kaplan-Meier curve analysis in various mRNA levels and protein grading is 0.047 and 0.003, respectively. MVD (Microvessel density) in tumor tissues are higher than non-tumor tissues, it is statistically significance (t=18.925, P<0.05). High MVD is correlated with tumor size (P=0.010), tumor capsule breakdown (P=0.001), tendency to recurrence (P=0.004), metastases during follow up (P=0.008) and low differentiation type (P=0.001). The expression of VM (Vascularization mimicry) exists in 7/34 specimens of HCC, MVD in positive specimens of VM are higher (t=16.373, P<0.05). The expression of VM is correlated with clinical TNM stage (P=0.001), tendency to recurrence (P=0.029), and low differentiation type (P=0.001). There was a significant positive correlation between PPARγand high MVD expression (r=0.519, P<0.05), but there was no correlation between PPARγand MV expression (r=0.169, P<0.05). Kaplan-Meier analysis indicated that PPARγoverexpression, high MVD and VM expression were associated with patient’s worse disease-free survival; P value tested by Log-rank is 0.013, 0.036 and 0.022, respectively. According to Cox multivariate survival analysis, PPARγand MVD were the independent prognostic factors of HCC (both Pmuti<0.05). Conclusions PPARγover-expressed in HCC, it may be associated with the carcinogenesis and development of HCC; it may be an index to judge prognosis of HCC. [Key Words] PPARγ; Hepatocellular carcinoma; Survival analysis; Biological characteristic; Angiogenesis; Vascularization mimicry The Second Section: Construction, identification and screening of shRNA targeted on PPARγgene Objective To construct shRNA targeted on PPARγgene with pBAsi-hU6-DNA plasmids, and to observe the inhibition efficacy of PPARγactivation after HCCLM3 cells were transfected by shRNA. Methods Duplicate bands DNA sequence were synthesized in accordance with human PPARγ1 gene cDNA sequence, to construct recombinant of PPARγ(pshPPARγ) with pBAsi-hU6-DNA plasmids, then free endotoxin extracted, and assessed by sequencing. pshPPARγrecombinants were transfected into HCCLM3 cells by DOTAP hangosome methods, RT-PCR and Western blot was used to detect the inhibition efficacy of PPARγactivation in 40h and 4w post-transfection. Results pshPPARγis coincidence with targeted sequence, after 40h pshPPARγwere transfected into HCCLM3 cells, the expression of PPARγmRNA and protein were inhibited comparing with normal cells and cells transfected by neo-DNA plasmids (F=29.47, P<0.01). In positive clone cells selcted with 600μg/ml G418 (in 4w), PPARγmRNA and protein also were inhibited significantly by pshPPARγ(F=75.22, P<0.01), their inhibition rate in RT-PCR test is 80.5% and 82.3%, respectively. Conclusions pshPPARγcan lastingly inhibit PPARγactivation in HCCLM3 cells; it may be a tool to study the mechanisms of PPARγof hepatocellular carcinoma in vitro or in vivo. [Key Words] RNA interference; short hairpin RNA; transfection; expression; recombinant The Third Section: Gene silencing targeted on PPARγaffect proliferation and apoptosis of hepatocellular carcinoma Objective To observe the effect on proliferation and apoptosis of HCCLM3 cells with short hair-pin RNA targeted on PPARγgene in vitro and in vivo, and to explore the potential mechanisms. Methods After HCCLM3 cells were transient transfected with pshPPARγplasmid, growth curves were detected by MTT methods, apoptosis rate were analyzed by flow cytometry, and morphocytology of apoptotic HCCLM3 cells were observed by TUNEL methods. Then, animal models were constructed with HCCLM3 cells stable transfected with pshPPARγ, PPARγmRNA and protein in tumor tissues were detected with RT-PCR and Western blot or immuocytochemistry (ICC) methods, growth curves and volumes of tumor were observed, and morphocytology of apoptotic cells were detected by TUNEL method. Moreover, the expression of PCNA and wt-P53 were stained by ICC methods, and MMP2, TIMP2 andβ1 integrin were detected by RT-PCR in vitro and in vivo. Results Cell growth was inhibited in pshPPARγgroup by 71.5%, apoptosis also increased significantly, FCM show the apoptosis ratio is 23.2±4.2%, it is statistically significance between pshPPARγgroup and control group (3.3±0.9% vs 3.1±0.7 %) (F=48.32, P<0.01), morphocytology test is the same as FCM. PPARγmRNA and protein decreased significantly in tumor tissues of pshPPARγgroup, inhibition rate reach to 82%. Stage of latency lengthen, and growth were inhibited, volumes of tumors in pshPPARγgroups are 1.86±0.65 cm3, but 4.86±1.15 cm3 in control groups, apoptosis in situ of tumors increased, it is statistically significance, P<0.05. In vitro and in vivo, PCNA protein decreased, and wt-P53 protein increased. In HCCLM3 cells of pshPPARγgroup, MMP2 and integrinβ1 mRNA decreased significantly, but TIMP2 increased. Conclusions Inhibition of PPARγactivation can decrease the proliferation in HCC. It maybe regulate the balance of MMP2 and TIMP2 via integrin signal way, then decrease the expression of PCNA, and promote wild-type P53 to induce the apoptosis of HCCLM3 cells. Key Words Proliferation; Apoptosis; Proliferating cell nuclear antigen; Wild-type P53; Matrix metalloproteinase; Tissue inhibitor of metalloproteinase; Integrin The Fourth Section: Gene silencing targeted on PPARγinfluence metastasis and angiogenesis of hepatocellular carcinoma Objective To observe the effect on invasion, metastasis, and angiogenesis in HCC when PPARγactivation was inhibited by shRNA targeted at PPARγgene of HCCLM3 cells in vitro and in vivo, and to explore the potential mechanisms. Methods Boyden chambers were utilized to observe the effect on cell adhesion, migration, invasion and locomotion of HCCLM3 cells, meanwhile, the effect on lumens formed by ECV 304 cells was observed in models which HCCLM3 cells co-cultivated with ECV 304 cells post-transient transfected in vitro. Animal models of cancerometastasis were established by HCCLM3 cells stable expressed pshPPARγor not with nude mouse, pulmonary metastasis (PM) was assessed by HE staining and immuocytochemistry (ICC) staining with hepatocyte, graded and counted in serial sections of lung tissues. MVD and VM in transplantation tumor were stained by immuocytochemistry staining of CD34 and multiplicity staining with CD34 combined with periodic acid-schiff (PAS). Integrinβ1 expression in lung tissues, VEGF and TSP1 expression in tumors was detected by ICC staining and RT-PCR methods. Results Cells crawled over semipermeable membrane of boyden chambers were 68±7 in pshPPARγgroups, it is less than control groups (281±10 and 302±14), it is statistical significance (F=548.72, P<0.01). Cells permeated through ECM were 17±3; it is also less than control groups (F=258.19, P<0.01). Time when cells healed the wound were 6.40±1.14d, it is longer than control groups (F=17.58, P<0.01). Ability of cell adhesion decreased significantly, after 2h, cell counts are 49±9, 83±13 and 88±11 in pshPPARγgroup and two control groups, respectively, it is statistical significance (F=17.39, P<0.01). Lumens formed by ECV 304 cells were weakened in pshPPARγgroup. PM in pshPPARγgroup were smaller than in control group, and total amounts in lung tissues were also less (t=18.69, P<0.05). MVD in transplantation tumors are scarce and punctiform, while in control group, MVD are intensive and annuliform, it is statistically significance between two groups, P<0.05. VM exist in transplantation tumor, sparse and tiny in pshPPARγgroup, meanwhile, VEGF mRNA and protein decreased significantly, TSP1 increased. Integrinβ1 expression in lung tissues was not difference between two groups. Conclusions PPARγinhibition can decrease the ability of invasion, metastasis and angiogenesis in HCC; it may be via integrin signal way, to regulate the balance of MMP2 and TIMP2, and affect the secretion of VEGF/TSP1.
引文
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