沉默信息调节因子SIRT1对胰腺癌生物学行为的影响及机制研究
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摘要
研究目的：
探讨SIRT1在胰腺癌组织中的表达及其与胰腺癌临床病例学特征之间的关系；以胰腺癌细胞系PANC-1为研究对象,构建针对人SIRT1基因的shRNA真核表达质粒,应用短发夹RNA (shRNA)技术抑制SIRT1基因表达,通过稳定转染获得靶向SIRT1基因沉默的胰腺癌细胞,研究SIRT1基因表达水平对人胰腺癌PANC-1细胞增殖能力,非锚定依赖性,成瘤性,化疗敏感性和侵袭能力等生物学行为特性的影响,并初步探讨机制。
研究方法：
用免疫组织化学染色法和实时荧光定量聚合酶链式反应(PCR)法检测49例胰腺癌组织和相应癌旁正常胰腺组织中SIRT1的表达,比较SIRT1表达与胰腺癌临床病理学特征之间的相关性；用实时荧光定量PCR和免疫印迹(western blot)法检测比较胰腺癌细胞系PANC-1, AsPC-1, BxPC-3中SIRT1的表达水平,选择SIRT1表达水平最高的细胞系作为RNA干扰研究对象。针对SIRT1基因的mRNA序列设计,分别构建3个shRNA真核表达质粒和阴性对照、阳性对照真核表达质粒,经大肠杆菌扩增,酶切、PCR、测序鉴定,转染胰腺癌PANC-1细胞,荧光显微镜观察转染效果,实时荧光定量PCR和western blot检测SIRT1 mRNA和蛋白的被抑制情况,通过优化转染条件实现最佳干扰效果,并选取干扰效果最佳的1个shRNA真核表达质粒和阴性对照质粒转染PANC-1细胞,并通过G418筛选稳定转染SIRT1-shRNA的细胞株,流式细胞术检测稳转细胞绿色荧光蛋白纯度。在建立稳定转染细胞PANC-1-Negative细胞和PANC-1-SIRT1-RNAi细胞基础上,MTT法绘制细胞生长曲线,流式细胞术检测细胞周期变化,软琼脂克隆形成实验检测细胞非锚定依赖性生长能力,TUNEL法检测细胞凋亡比例,实时荧光定量PCR和western blot检测凋亡相关基因p53、FOXO3a、Bax/Bcl-2的表达水平,BALB/c裸鼠成瘤实验鉴定各细胞的成瘤能力,活体荧光成像及移植瘤内SIRT1免疫组织化学染色检测SIRT1在体内移植水平,TUNEL法检测移植瘤内细胞凋亡比例,此外,运用MTT法检测PANC-1细胞,PANC-1-Negative细胞,PANC-1-SIRT1-RNAi细胞对化疗药物5-FU和吉西他滨敏感性的变化,并计算各组细胞的IC50；Transwell小室法检测各组细胞侵袭能力的改变,并通过实时荧光定量PCR和western blot检测各组细胞E-cadherin,MMP-2和MMP-9的表达变化。
实验结果：
SIRT1蛋白在胰腺癌组织和癌旁正常胰腺组织中的阳性表达率分别为75.51%和22.45%, SIRT1蛋白和mRNA表达水平基本一致,在胰腺癌组织中表达水平明显高于癌旁正常胰腺组织(P<0.01)。SIRT1表达水平与胰腺癌患者年龄、肿瘤大小、TNM分期、淋巴转移、肝转移相关(P<0.01),与患者性别、肿瘤组织分化程度、肿瘤部位、血管浸润、神经侵犯无关(P>0.05)。胰腺癌细胞系PANC-1的SIRT1表达水平在三株细胞系中最高,可作为RNA干扰试验对象。经测序证实,成功构建SIRT1-shRNA真核表达质粒,插入的DNA片段的序列与设计序列完全一致。重组质粒转染PANC-1细胞后,荧光显微镜下可观察到绿色荧光蛋白表达,实时荧光定量PCR和western blot检测显示SIRT1 mRNA和蛋白水平明显下调；其中以1号重组质粒干扰效应最强。经1号重组质粒和阴性对照质粒转染,G418筛选稳定的细胞株分别命名为PANC-1-SIRT1-RNAi细胞和PANC-1-Negative细胞。PANC-1-SIRT1-RNAi细胞中SIRT1 mRNA和蛋白水平的抑制率分别为95.8%和86.0%。PANC-1细胞、PANC-1-Negative细胞、PANC-1-SIRT1-RNAi细胞的倍增时间分别为(42.76±1.28)h,(44.21±1.95)h,(80.32±6.44)h；与PANC-1细胞和PANC-1-Negative细胞相比,PANC-1-SIRT1-RNAi细胞周期出现显著G0／G1期阻滞,细胞生长受到抑制,非锚定依赖性生长能力亦明显减弱,细胞凋亡比例增高,凋亡基因FOXO3a, Bax的表达明显升高,而p53的表达则无明显变化,抗凋亡基因Bcl-2的表达明显下调,各基因mRNA和蛋白水平表达基本一致,细胞成瘤能力显著下降,且移植瘤中SIRT1的表达持续抑制,细胞凋亡比例显著增高,差异具有统计学意义(P<0.01), PANC-1细胞、PANC-1-Negative细胞、PANC-1-SIRT1-RNAi细胞对5-FU的IC50分别为117.42±31.19μg/mL,104.54±29.22μg/mL和51.37±18.94μg/mL,对吉西他滨的IC50分别为78.32±15.49μg/mL,72.68±20.11μg/mL和21.47±7.53μg/mL, PANC-1-SIRT1-RNAi细胞的化疗敏感性显著提高(P<0.01)。Transwell小室法检测各组细胞侵袭能力发现,PANC-1细胞、PANC-1-Negative细胞、PANC-1-SIRT1-RNAi细胞的穿膜细胞数分别为(59±13)个,(61±10)个和(22±6)个,PANC-1-SIRT1-RNAi细胞侵袭能力显著下降(P<0.01),与PANC-1细胞和PANC-1-Negative细胞比较,PANC-1-SIRT1-RNAi细胞E-cadherin表达水平升高,MMP-2和MMP-9的表达水平则明显抑制(P<0.01),而PANC-1细胞和PANC-1-Negative细胞之间,各项生物学特性无显著性差异(P>0.05)。
实验结论：
1. SIRT1在胰腺癌组织中的表达水平显著升高,与患者年龄,肿瘤增殖水平密切相关,并参与胰腺癌的侵袭转移,检测SIRT1的表达有助于判断胰腺癌恶性程度及预后。
2.成功构建携带以SIRT1为靶向的shRNA重组质粒；以SIRT1表达水平最高的胰腺癌PANC-1细胞作为RNA干扰研究对象,通过稳定转染SIRT1靶向RNA干扰的SIRT1-shRNA质粒和阴性对照control-shRNA质粒,获得PANC-1-SIRT1-RNAi细胞和PANC-1-Negative细胞；为进一步研究SIRT1的功能和肿瘤的基因治疗奠定了基础。
3.通过SIRT1-shRNA稳定抑制SIRT1在胰腺癌PANC-1细胞中的表达,可显著抑制细胞增殖,减弱非锚定依赖性生长能力,增加细胞凋亡比例,降低细胞成瘤能力,其可能通过凋亡相关基因FOXO3a, Bcl-2/Bax的调节发挥作用。
4. SIRT1表达水平的降低,使PANC-1-SIRT1-RNAi细胞对5-FU和吉西他滨的化疗敏感性显著增加,并可能通过下调MMP-2和MMP-9的表达水平及上调E-cadherin的表达水平,抑制胰腺癌细胞的体外侵袭能力。
Objective:To investigate the expression and significance of SIRT1 in human pancreatic carcinoma and their association with the clinical pathologic characters. To detect the expression of SIRT1 in three human pancreatic carcinoma cell lines for experimental foundation of SIRT1 RNA interference. Taking human pancreatic carcinoma PANC-1 cells as investigative object, to construct three short hairpin RNA (shRNA) interference expression plasmid vectors of human SIRT1 gene, to assay the expression of SIRT1 in PANC-1 cells after transfecting with recombinant plasmids, and to detect the RNAi effect of shRNA. To investigate the effects and mechanism of SIRT1 gene expression inhibited by shRNA in proliferation, anchorage-independence, tumorigenic ability, chemosensitivity and invasion of PANC-1 cells.
Methods:Immunohistochemistry staining and real-time quantitative polymerase chain reaction (PCR) were used to detect the expression of SIRT1 in 49 pancreatic carcinoma tissues and homologous nontumorous normal pancreatic tissues. The correlations among the variety of expression of SIRT1 and pancreatic carcinoma clinical pathologic characters were statistically analyze. Real-time quantitative PCR and western blot were were used to compare the expression levels of human pancreatic carcinoma cell lines PANC-1, AsPC-1 and BxPC-3. The cell line with the highest SIRT1 expression was chosed for RNA interference. Three plasmid expression vectors coding for shRNA targeting SIRT1 gene sequence, a positive control vector and a negative control vector were constructed. The recombinant plasmids were amplified in Ecoli. DH5 and identified by restriction digestion, PCR and sequencing. The vectors were transfected into PANC-1 cells. The green fluorescent protein (GFP) was observed by fluorescence microscope. SIRT1 expression was assayed with real-time quantitative PCR and western blot. The vector with the best interference effect and the negative control-shRNA vector were stable transfected into PANC-1 cells by G418 screening. The stable transfection cells of PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells had been obtained. The growth curves of PANC-1 cells, PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells were drawed by MTT method. The cell cycles were detected by flow cytometry. Colony forming efficiency in soft agar was used for anchorage-independent growth. The ratios of cell apoptosis were detected by TUNEL (TdT-mediated dUTP nick end labeling) test. The expressions of p53, FOXO3a, Bax and Bcl-2 were assayed by real-time quantitative PCR and western blot. Xenografts in BALB/c nude mice were used for evaluating the tumorigenic ability. The expression of SIRT1 in xenografts was detected by flourescence image formation in vivo and immunohistochemistry staining and the ratios of cell apoptosis in xenografts were detected by TUNEL test. The chemosensitivities of PANC-1 cells, PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells to 5-FU and gemcitabine hydrochloride were detected by MTT, and each ICsowas calculated. Invasive abilities of PANC-1 cells, PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells were observed with Transwell cell culture chambers, and the expressions of E-cadherin, MMP-2 and MMP-9 were assayed by real-time quantitative PCR and western blot.
Results:The SIRT1 protein expression rate of the tumor tissues was 75.51%, significantly higher than that of homologous nontumorous normal pancreatic tissues (22.45%), which was coincident with the SIRT1 mRNA expression. The SIRT1 expression was significantly associated with patients'age, tumor size, TNM stage, nodes invasion and hepatic metastasis (P<0.01), and there was no rela tionship between SIRT1 expression and patients'sex, histologic grading, location of the tumor and blood vessel or nerves invasion (P>0.05). The SIRT1 expression of cell line PANC-1 was the highest in three human pancreatic carcinoma cell lines. The successful construction of recombinant plasmids was confirmed by DNA sequencing of the inserted segments. Transfection of shRNA plasmids significantly down-regulated SIRT1 expression in PANC-1cells. Recombinant plasmid 1 had the strongest effect. The stable transfections of recombinant plasmid of No.l and recombinant plasmid of negative control-shRNA were named PANC-1-SIRT1-RNAi cells and PANC-1-Negative cells, respectively. PANC-1-SIRT1-RNAi cells had SIRT1 inhibition ratio of 95.8% at the mRNA level and 86.0% at the protein level. The doubling generation time of PANC-1, PANC-1-Negative and PANC-1-SIRT1-RNAi cells was (42.76±1.28) h, (44.21±1.95) h and (80.32±6.44) h, respectively. Compared with PANC-1 and PANC-1-Negative cells, the proliferation of PANC-1-SIRTl-RNAi cells was inhibited with G0/G1 stage blocking, the anchorage-independence was weaken obviously, the ratio of cell apoptosis were raised up. Proapoptotic factors such as FOXO3a and Bax were up-regulated in PANC-1-SIRT1-RNAi cells, but p53 was not affectted. In the other hand, antiapoptotic Bcl-2 was down-regulated reciprocally. The expression of these factors was coincident in mRNA and protein levels. The tumorigenic ability of PANC-1-SIRTl-RNAi cells was decreased significantly. The apoptosis cell ratios of PANC-1, PANC-1-Negative and PANC-1-SIRT1-RNAi xenografts were (4.16±2.44)%, (5.23±1.41)% and (58.84±10.86)%, respectively (P<0.01). The 5-FU IC50s of PANC-1, PANC-1-Negative and PANC-1-SIRT1-RNAi cells were 117.42±31.19μg/mL,104.54±29.22μg/mL and 51.37±18.94μg/mL, respectively, and the gemcitabine IC50S were 78.32±15.49μg/mL, 72.68±20.11μg/mL and 21.47±7.53μg/mL, respectively. PANC-1-SIRT1-RNAi cells were much more chemosensitive to 5-FU and gemcitabine hydrochloride than PANC-1 cells and PANC-1-Negative cells (P<0.01). The Transwell invasive chambers found that the numbers of PANC-1 cells, PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells were (59±13), (61±10) and (22±6), respectively. The invasive ability of PANC-1-SIRTl-RNAi cells decreased dramatically (P<0.01) through promoting expression of E-cadherin and inhibiting expressions of MMP-2 and MMP-9 in mRNA and protein levels. There was no difference between PANC-1 cells and PANC-1-Negative cells in above-mentioned bionomics (P>0.05).
Conclusion:
1. The SIRT1 expression was raised up in pancreatic carcinoma tissues, significantly associated with the patients'age, tumor proliferation, and could participate in invasion and metastasis of pancreatic carcinoma. SIRT1 expression may be regarded as a parameter of determining the degree of malignancy and prognosis of pancreatic carcinoma.
2. The pancreatic carcinoma cell line PANC-1 was chosed for experiments of RNA interference. Plasmid vector expressing shRNA against SIRT1 has been successfully constructed and it can down-regulate SIRT1 expression after stable transfected into PANC-1 cells, which could facilitate further studies of SIRT1 functions and its application in tumour gene therapy.
3. It could inhibit proliferation, weaken anchorage-independence, induce apoptosis and depress tumorigenic ability in PANC-1 cells by inhibitting the expression of SIRT1 gene stably.
4. PANC-1-SIRT1-RNAi cells which the expression of SIRT1 gene were significantly inhibited were enhanced chemosensitivity to 5-FU and gemcitabine hydrochloride. Furthermore, the invasion of PANC-1-SIRT1-RNAi cells were inhibited obviously in vitro.
探讨SIRT1在胰腺癌组织中的表达及其与胰腺癌临床病例学特征之间的关系；以胰腺癌细胞系PANC-1为研究对象,构建针对人SIRT1基因的shRNA真核表达质粒,应用短发夹RNA (shRNA)技术抑制SIRT1基因表达,通过稳定转染获得靶向SIRT1基因沉默的胰腺癌细胞,研究SIRT1基因表达水平对人胰腺癌PANC-1细胞增殖能力,非锚定依赖性,成瘤性,化疗敏感性和侵袭能力等生物学行为特性的影响,并初步探讨机制。
研究方法：
用免疫组织化学染色法和实时荧光定量聚合酶链式反应(PCR)法检测49例胰腺癌组织和相应癌旁正常胰腺组织中SIRT1的表达,比较SIRT1表达与胰腺癌临床病理学特征之间的相关性；用实时荧光定量PCR和免疫印迹(western blot)法检测比较胰腺癌细胞系PANC-1, AsPC-1, BxPC-3中SIRT1的表达水平,选择SIRT1表达水平最高的细胞系作为RNA干扰研究对象。针对SIRT1基因的mRNA序列设计,分别构建3个shRNA真核表达质粒和阴性对照、阳性对照真核表达质粒,经大肠杆菌扩增,酶切、PCR、测序鉴定,转染胰腺癌PANC-1细胞,荧光显微镜观察转染效果,实时荧光定量PCR和western blot检测SIRT1 mRNA和蛋白的被抑制情况,通过优化转染条件实现最佳干扰效果,并选取干扰效果最佳的1个shRNA真核表达质粒和阴性对照质粒转染PANC-1细胞,并通过G418筛选稳定转染SIRT1-shRNA的细胞株,流式细胞术检测稳转细胞绿色荧光蛋白纯度。在建立稳定转染细胞PANC-1-Negative细胞和PANC-1-SIRT1-RNAi细胞基础上,MTT法绘制细胞生长曲线,流式细胞术检测细胞周期变化,软琼脂克隆形成实验检测细胞非锚定依赖性生长能力,TUNEL法检测细胞凋亡比例,实时荧光定量PCR和western blot检测凋亡相关基因p53、FOXO3a、Bax/Bcl-2的表达水平,BALB/c裸鼠成瘤实验鉴定各细胞的成瘤能力,活体荧光成像及移植瘤内SIRT1免疫组织化学染色检测SIRT1在体内移植水平,TUNEL法检测移植瘤内细胞凋亡比例,此外,运用MTT法检测PANC-1细胞,PANC-1-Negative细胞,PANC-1-SIRT1-RNAi细胞对化疗药物5-FU和吉西他滨敏感性的变化,并计算各组细胞的IC50；Transwell小室法检测各组细胞侵袭能力的改变,并通过实时荧光定量PCR和western blot检测各组细胞E-cadherin,MMP-2和MMP-9的表达变化。
实验结果：
SIRT1蛋白在胰腺癌组织和癌旁正常胰腺组织中的阳性表达率分别为75.51%和22.45%, SIRT1蛋白和mRNA表达水平基本一致,在胰腺癌组织中表达水平明显高于癌旁正常胰腺组织(P<0.01)。SIRT1表达水平与胰腺癌患者年龄、肿瘤大小、TNM分期、淋巴转移、肝转移相关(P<0.01),与患者性别、肿瘤组织分化程度、肿瘤部位、血管浸润、神经侵犯无关(P>0.05)。胰腺癌细胞系PANC-1的SIRT1表达水平在三株细胞系中最高,可作为RNA干扰试验对象。经测序证实,成功构建SIRT1-shRNA真核表达质粒,插入的DNA片段的序列与设计序列完全一致。重组质粒转染PANC-1细胞后,荧光显微镜下可观察到绿色荧光蛋白表达,实时荧光定量PCR和western blot检测显示SIRT1 mRNA和蛋白水平明显下调；其中以1号重组质粒干扰效应最强。经1号重组质粒和阴性对照质粒转染,G418筛选稳定的细胞株分别命名为PANC-1-SIRT1-RNAi细胞和PANC-1-Negative细胞。PANC-1-SIRT1-RNAi细胞中SIRT1 mRNA和蛋白水平的抑制率分别为95.8%和86.0%。PANC-1细胞、PANC-1-Negative细胞、PANC-1-SIRT1-RNAi细胞的倍增时间分别为(42.76±1.28)h,(44.21±1.95)h,(80.32±6.44)h；与PANC-1细胞和PANC-1-Negative细胞相比,PANC-1-SIRT1-RNAi细胞周期出现显著G0／G1期阻滞,细胞生长受到抑制,非锚定依赖性生长能力亦明显减弱,细胞凋亡比例增高,凋亡基因FOXO3a, Bax的表达明显升高,而p53的表达则无明显变化,抗凋亡基因Bcl-2的表达明显下调,各基因mRNA和蛋白水平表达基本一致,细胞成瘤能力显著下降,且移植瘤中SIRT1的表达持续抑制,细胞凋亡比例显著增高,差异具有统计学意义(P<0.01), PANC-1细胞、PANC-1-Negative细胞、PANC-1-SIRT1-RNAi细胞对5-FU的IC50分别为117.42±31.19μg/mL,104.54±29.22μg/mL和51.37±18.94μg/mL,对吉西他滨的IC50分别为78.32±15.49μg/mL,72.68±20.11μg/mL和21.47±7.53μg/mL, PANC-1-SIRT1-RNAi细胞的化疗敏感性显著提高(P<0.01)。Transwell小室法检测各组细胞侵袭能力发现,PANC-1细胞、PANC-1-Negative细胞、PANC-1-SIRT1-RNAi细胞的穿膜细胞数分别为(59±13)个,(61±10)个和(22±6)个,PANC-1-SIRT1-RNAi细胞侵袭能力显著下降(P<0.01),与PANC-1细胞和PANC-1-Negative细胞比较,PANC-1-SIRT1-RNAi细胞E-cadherin表达水平升高,MMP-2和MMP-9的表达水平则明显抑制(P<0.01),而PANC-1细胞和PANC-1-Negative细胞之间,各项生物学特性无显著性差异(P>0.05)。
实验结论：
1. SIRT1在胰腺癌组织中的表达水平显著升高,与患者年龄,肿瘤增殖水平密切相关,并参与胰腺癌的侵袭转移,检测SIRT1的表达有助于判断胰腺癌恶性程度及预后。
2.成功构建携带以SIRT1为靶向的shRNA重组质粒；以SIRT1表达水平最高的胰腺癌PANC-1细胞作为RNA干扰研究对象,通过稳定转染SIRT1靶向RNA干扰的SIRT1-shRNA质粒和阴性对照control-shRNA质粒,获得PANC-1-SIRT1-RNAi细胞和PANC-1-Negative细胞；为进一步研究SIRT1的功能和肿瘤的基因治疗奠定了基础。
3.通过SIRT1-shRNA稳定抑制SIRT1在胰腺癌PANC-1细胞中的表达,可显著抑制细胞增殖,减弱非锚定依赖性生长能力,增加细胞凋亡比例,降低细胞成瘤能力,其可能通过凋亡相关基因FOXO3a, Bcl-2/Bax的调节发挥作用。
4. SIRT1表达水平的降低,使PANC-1-SIRT1-RNAi细胞对5-FU和吉西他滨的化疗敏感性显著增加,并可能通过下调MMP-2和MMP-9的表达水平及上调E-cadherin的表达水平,抑制胰腺癌细胞的体外侵袭能力。
Objective:To investigate the expression and significance of SIRT1 in human pancreatic carcinoma and their association with the clinical pathologic characters. To detect the expression of SIRT1 in three human pancreatic carcinoma cell lines for experimental foundation of SIRT1 RNA interference. Taking human pancreatic carcinoma PANC-1 cells as investigative object, to construct three short hairpin RNA (shRNA) interference expression plasmid vectors of human SIRT1 gene, to assay the expression of SIRT1 in PANC-1 cells after transfecting with recombinant plasmids, and to detect the RNAi effect of shRNA. To investigate the effects and mechanism of SIRT1 gene expression inhibited by shRNA in proliferation, anchorage-independence, tumorigenic ability, chemosensitivity and invasion of PANC-1 cells.
Methods:Immunohistochemistry staining and real-time quantitative polymerase chain reaction (PCR) were used to detect the expression of SIRT1 in 49 pancreatic carcinoma tissues and homologous nontumorous normal pancreatic tissues. The correlations among the variety of expression of SIRT1 and pancreatic carcinoma clinical pathologic characters were statistically analyze. Real-time quantitative PCR and western blot were were used to compare the expression levels of human pancreatic carcinoma cell lines PANC-1, AsPC-1 and BxPC-3. The cell line with the highest SIRT1 expression was chosed for RNA interference. Three plasmid expression vectors coding for shRNA targeting SIRT1 gene sequence, a positive control vector and a negative control vector were constructed. The recombinant plasmids were amplified in Ecoli. DH5 and identified by restriction digestion, PCR and sequencing. The vectors were transfected into PANC-1 cells. The green fluorescent protein (GFP) was observed by fluorescence microscope. SIRT1 expression was assayed with real-time quantitative PCR and western blot. The vector with the best interference effect and the negative control-shRNA vector were stable transfected into PANC-1 cells by G418 screening. The stable transfection cells of PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells had been obtained. The growth curves of PANC-1 cells, PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells were drawed by MTT method. The cell cycles were detected by flow cytometry. Colony forming efficiency in soft agar was used for anchorage-independent growth. The ratios of cell apoptosis were detected by TUNEL (TdT-mediated dUTP nick end labeling) test. The expressions of p53, FOXO3a, Bax and Bcl-2 were assayed by real-time quantitative PCR and western blot. Xenografts in BALB/c nude mice were used for evaluating the tumorigenic ability. The expression of SIRT1 in xenografts was detected by flourescence image formation in vivo and immunohistochemistry staining and the ratios of cell apoptosis in xenografts were detected by TUNEL test. The chemosensitivities of PANC-1 cells, PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells to 5-FU and gemcitabine hydrochloride were detected by MTT, and each ICsowas calculated. Invasive abilities of PANC-1 cells, PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells were observed with Transwell cell culture chambers, and the expressions of E-cadherin, MMP-2 and MMP-9 were assayed by real-time quantitative PCR and western blot.
Results:The SIRT1 protein expression rate of the tumor tissues was 75.51%, significantly higher than that of homologous nontumorous normal pancreatic tissues (22.45%), which was coincident with the SIRT1 mRNA expression. The SIRT1 expression was significantly associated with patients'age, tumor size, TNM stage, nodes invasion and hepatic metastasis (P<0.01), and there was no rela tionship between SIRT1 expression and patients'sex, histologic grading, location of the tumor and blood vessel or nerves invasion (P>0.05). The SIRT1 expression of cell line PANC-1 was the highest in three human pancreatic carcinoma cell lines. The successful construction of recombinant plasmids was confirmed by DNA sequencing of the inserted segments. Transfection of shRNA plasmids significantly down-regulated SIRT1 expression in PANC-1cells. Recombinant plasmid 1 had the strongest effect. The stable transfections of recombinant plasmid of No.l and recombinant plasmid of negative control-shRNA were named PANC-1-SIRT1-RNAi cells and PANC-1-Negative cells, respectively. PANC-1-SIRT1-RNAi cells had SIRT1 inhibition ratio of 95.8% at the mRNA level and 86.0% at the protein level. The doubling generation time of PANC-1, PANC-1-Negative and PANC-1-SIRT1-RNAi cells was (42.76±1.28) h, (44.21±1.95) h and (80.32±6.44) h, respectively. Compared with PANC-1 and PANC-1-Negative cells, the proliferation of PANC-1-SIRTl-RNAi cells was inhibited with G0/G1 stage blocking, the anchorage-independence was weaken obviously, the ratio of cell apoptosis were raised up. Proapoptotic factors such as FOXO3a and Bax were up-regulated in PANC-1-SIRT1-RNAi cells, but p53 was not affectted. In the other hand, antiapoptotic Bcl-2 was down-regulated reciprocally. The expression of these factors was coincident in mRNA and protein levels. The tumorigenic ability of PANC-1-SIRTl-RNAi cells was decreased significantly. The apoptosis cell ratios of PANC-1, PANC-1-Negative and PANC-1-SIRT1-RNAi xenografts were (4.16±2.44)%, (5.23±1.41)% and (58.84±10.86)%, respectively (P<0.01). The 5-FU IC50s of PANC-1, PANC-1-Negative and PANC-1-SIRT1-RNAi cells were 117.42±31.19μg/mL,104.54±29.22μg/mL and 51.37±18.94μg/mL, respectively, and the gemcitabine IC50S were 78.32±15.49μg/mL, 72.68±20.11μg/mL and 21.47±7.53μg/mL, respectively. PANC-1-SIRT1-RNAi cells were much more chemosensitive to 5-FU and gemcitabine hydrochloride than PANC-1 cells and PANC-1-Negative cells (P<0.01). The Transwell invasive chambers found that the numbers of PANC-1 cells, PANC-1-Negative cells and PANC-1-SIRT1-RNAi cells were (59±13), (61±10) and (22±6), respectively. The invasive ability of PANC-1-SIRTl-RNAi cells decreased dramatically (P<0.01) through promoting expression of E-cadherin and inhibiting expressions of MMP-2 and MMP-9 in mRNA and protein levels. There was no difference between PANC-1 cells and PANC-1-Negative cells in above-mentioned bionomics (P>0.05).
Conclusion:
1. The SIRT1 expression was raised up in pancreatic carcinoma tissues, significantly associated with the patients'age, tumor proliferation, and could participate in invasion and metastasis of pancreatic carcinoma. SIRT1 expression may be regarded as a parameter of determining the degree of malignancy and prognosis of pancreatic carcinoma.
2. The pancreatic carcinoma cell line PANC-1 was chosed for experiments of RNA interference. Plasmid vector expressing shRNA against SIRT1 has been successfully constructed and it can down-regulate SIRT1 expression after stable transfected into PANC-1 cells, which could facilitate further studies of SIRT1 functions and its application in tumour gene therapy.
3. It could inhibit proliferation, weaken anchorage-independence, induce apoptosis and depress tumorigenic ability in PANC-1 cells by inhibitting the expression of SIRT1 gene stably.
4. PANC-1-SIRT1-RNAi cells which the expression of SIRT1 gene were significantly inhibited were enhanced chemosensitivity to 5-FU and gemcitabine hydrochloride. Furthermore, the invasion of PANC-1-SIRT1-RNAi cells were inhibited obviously in vitro.
引文
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