GRIM-19抑制肺腺癌细胞株SPC-A1生长及作用机制体内外实验研究
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摘要
背景:
肺癌是当今世界各国常见的恶性肿瘤,已成为绝大多数国家癌症死亡的主要原因,目前被认为是对人类健康和生命威胁最大的恶性肿瘤。按组织学分类,分为非小细胞肺癌和小细胞肺癌,其中非小细胞肺癌分为腺癌、鳞癌、大细胞肺癌、鳞腺癌。其中腺癌目前占第一位。干扰素/维甲酸诱导凋亡相关基因-19(gene associated with retinoid-IFN-induced mortality-19,GRIM-19),是2000年由Angell等应用反义基因敲除的方法分离发现的一种细胞凋亡调节因子。在一些原发性肾细胞肿瘤和泌尿生殖系肿瘤中GRIM-19表达缺失或被严重抑制,用肾脏恶性肿瘤细胞系的研究证实:GRIM-19的下调通过增强依赖Stat3(signal transducers and activators of transcription 3)基因的表达而促进肿瘤生长,并且首次表明GRIM-19具有肿瘤抑制因子作用。对于GRIM-19在肺腺癌的表达情况、是否抑制其生长及抑制机制目前还没有见到相关报道。我们先期已经对GRIM-19在肺癌组织中的表达一般情况与正常肺组织表达情况做了一个对比研究,但肺腺癌中表达具体情况没有做进一步研究分析。本研究对GRIM-19和Stat3基因在肺腺癌组织与肺组织中表达作对比,采用体内外实验研究,验证GRIM-19抑制肺腺癌生长和初步探讨其作用机制。
目的:
本研究以人肺癌组织及对应的肺组织为材料,检测GRIM-19、Stat3及其下游靶基因Bcl-2、Cyclin B1、Cyclin D1在肺腺癌及正常肺组织中的表达情况;对pIRES-Puro2-GRIM-19-Myc质粒进行测序,分别将pIRES-Puro2-GRIM-19-Myc和pIRES-Puro2-GRIM-19-Myc真核表达质粒转染SPC-A1细胞中,建立稳转细胞株SPC-A1/GRIM-19和SPC-A1/CON,将上述两种细胞分别种植于不同组的BALB/C裸鼠皮下,28天杀死裸鼠,取出皮下肿瘤组织,分别检测GRIM-19、Stat3及其下游靶基因Bcl-2、Cyclin B1、Cyclin D1等的表达。在体内外验证GRIM-19抑制肺腺癌生长及其作用机制。
方法:
收集肺腺癌和对应的肺组织标本,用免疫组化方法比较肺腺癌组织和对应肺组织中GRIM-19蛋白表达。再用Western blot法比较肺腺癌和肺组织GRIM-19、STAT3蛋白的表达。分别抽取37例患者肺腺癌组织及肺组织中的总RNA,然后采用RT-PCR法比较肺腺癌组织和正常肺组织中GRIM-19、Stat3及其下游靶基因Bcl-2、Cyclin B1、Cyclin D1的mRNA表达。
中国科技大学生命科学学院肖卫华教授馈赠PIRES-Puro2-GRIM-19-Myc和PIRES-Puro2- Myc真核表达质粒,上海生工对此进行测序,判断是否有基因突变。Lipofectamine将真核表达质粒pIRES-Puro2-GRIM-19-Myc和pIRES-Puro2-Myc转染肺腺癌细胞株SPC-A1中,建立稳转细胞株。用MTT方法检测GRIM-19是否对肺腺癌细胞株SPC-A1的生长影响。用Western blot法比较稳转细胞株SPC-A1/GRIM-19和SPC-A1/CON的GRIM-19和STAT3蛋白表达。然后采用RT-PCR法分别比较稳转细胞株SPC-A1/GRIM-19和SPC-A1/CON的GRIM-19、Stat3及其下游靶基因Bcl-2、Cyclin B1、Cyclin D1、VEGF的mRNA表达。通过上述方法,在体外实验判断GRIM-19是否抑制肺腺癌细胞株的生长及其机制。
分别取SPC-A1/GRIM-19, SPC-A1/CON (2×106)种植于5周的BALB/C裸鼠(雌性),观察4周,每隔7天检测一次其体积变化,用体内实验判断GRIM-19是否对肺腺癌细胞株SPC-A1的生长影响。第28天分别取不同组BALB/C裸鼠的皮下肿瘤组织,用Western blot法比较其GRIM-19、STAT3和MMP2蛋白表达。分别提取不同组BALB/C裸鼠的皮下肿瘤组织的总RNA,然后采用RT-PCR法比较其GRIM-19、Stat3及其下游靶基因Bcl-2、Cyclin B1、Cyclin D1、VEGF的mRNA表达情况。通过上述方法,在体内实验判断GRIM-19是否抑制肺腺癌细胞株的生长及其机制。
结果:
一、GRIM-19、STAT3及其下游靶基因在肺腺癌组织和肺组织中的表达用免疫组化检测结果显示:GRIM-19蛋白在肺腺癌组织表达明显低于在肺组织表达。Western Blot结果表明:肺腺癌组与肺组织组相比,GRIM-19蛋白表达水平在肺腺癌中明显低于肺组织,差异有统计学意义(p<0.01)。STAT3蛋白表达与之相反。RT-PCR结果表明:肺腺癌组与肺组织相比,GRIM-19基因表达水平明显减低,差异有统计学意义(p<0.01);与之相反,RT-PCR结果发现Stat3及其下游靶基因Bcl-2、Cyclin B1、Cyclin D1的表达在肺腺癌组织中高于其肺组织中。
二、GRIM-19抑制肺腺癌细胞株SPC-A1生长体外实验结果
上海生工对对PIRES-Puro2-GRIM-19-Myc真核表达质粒测序无基因突变。用Lipofectamine将真核表达质粒pIRES-Puro2-GRIM-19-Myc和pIRES-Puro2-Myc转染肺腺癌细胞株SPC-A1中, G418(浓度0.60μg/mL)持续筛选6周建立稳转细胞株: SPC-A1/GRIM-19和SPC-A1/CON。Western Blot结果表明:SPC-A1/GRIM-19细胞株与SPC-A1/CON细胞株相比, SPC-A1/CON细胞株中的GRIM-19蛋白水平表达明显低于SPC-A1/GRIM-19细胞中的表达,差异有统计学意义(p<0.01)。STAT3蛋白表达与之相反。RT-PCR结果表明:SPC-A1/GRIM-19细胞株与SPC-A1/CON细胞株相比, SPC-A1/CON细胞株中的GRIM-19表达水平明显低于SPC-A1/GRIM-19细胞株中的表达水平,差异有统计学意义(p<0.01),RT-PCR结果发现Stat3及其下游靶基因Bcl-2、CyclinB1、CyclinD1,VEGF的mRNA表达与之相反。
三、GRIM-19抑制肺腺癌细胞株SPC-A1生长体内实验结果
把SPC-A1/GRIM-19和SPC-A1/CON细胞分别种植于BALB/C裸鼠皮下,每周检测其肿瘤大小,构建其生长曲线,结果显示:种植SPC-A1/GRIM-19细胞的BALB/C裸鼠的皮下肿瘤的体积明显减小(p<0.01)。4周后分别取不同组BALB/C裸鼠的皮下肿瘤组织用Western blot法检测其GRIM-19、STAT3和MMP2蛋白,结果表明:种植SPC-A1/GRIM-19细胞的BALB/C裸鼠的皮下肿瘤组织与种植SPC-A1/CON细胞的BALB/C裸鼠的皮下肿瘤组织相比: GRIM-19的蛋白水平在SPC-A1/CON细胞的瘤组织中的表达明显低于SPC-A1/GRIM-19细胞的瘤组织的表达,差异有统计学意义(p<0.01)。STAT3、MMP2的蛋白表达与之相反。RT-PCR结果表明:种植SPC-A1/GRIM-19细胞的BALB/C裸鼠的皮下肿瘤组织与种植SPC-A1/CON细胞的BALB/C裸鼠的皮下肿瘤组织相比:GRIM-19的mRNA表达水平明显升高,差异有统计学意义(p<0.01); Stat3及其下游靶基因Bcl-2、Cyclin B1、Cyclin D1,VEGF的mRNA表达水平与之相反。
结论
1.肺腺癌组GRIM-19的mRNA水平及蛋白水平明显低于其在相对应的正常肺组织的表达,并且基因表达水平的降低伴随有蛋白表达的相应下降;肺腺癌组Stat3的mRNA水平及蛋白及其下游靶基因Bcl-2、Cyclin B1、CyclinD1、VEGF基因表达水平均高于其在肺组织中的表达。
2.通过建立肺腺癌稳转细胞株SPC-A1/GRIM-19和SPC-A1/C0N,在体外验证GRIM-19抑制肺腺癌生长。
3.把SPC-A1/GRIM-19和SPC-A1/CON稳转细胞株分别种植不同组BALB/C裸鼠的皮下,在体内验证GRIM19抑制肺腺癌生长。
Background
According to the World Health Organization,lung cancer is currently the leading cause of cancer-related death in the world. Recently,a number of studies have shown that the family of proteins known as gene associated with retinoid- IFN- induced mortality (GRIMs) may represent novel types of tumor suppressors.Gene associated with retinoid interferon-induced mortality-19(GRIM-19), was originally identified as a cell death regulatory gene using the antisense technical knockout approach by Angell et al. In a number of primary RCC and in some urinogenital tumors.the expression of GRIM-19 is lost or severely depressed .By using an RCC cell line, researchers show that down regulation of GRIM-19 promotes tumor growth via an augmentation of Stat3-dependent gene expression. These studies for the first time show a tumor suppressor like activity of GRIM-19. Maximo et al confirmed that Hürthle cell thyroid carcinomas exist mutation of GRIM-19. In this study, we revealed that GRIM-19 were significantly reduced at mRNA and protein levels in lung adenocarcinoma tissue . STAT3 and its downstream target genes were tended to express a higher basal level in lung adenocarcinoma tissues. Overexpression of GRIM-19 was also found to suppress lung adenocancinoma tumor growth both in vitro and in vivo.Taken together,these finding will likely contribute to the future development of GRIM-19-based gene therapy approaches to treat lung adenocancinoma.
Objective
In the present study, we used lung adenocarcinoma tissues and lung tissues to investigate the expression of GRIM-19, Stat3 and it’s downstream target gene Bcl-2, Cyclin B1,Cyclin D1,Analyzing the relationship between GRIM-19 and Stat3, and to further explore the effects of GRIM-19 on SPC-A1 tumor cell growth in vitro and vivo.
Methods
Thirty-seven lung adenocarcinoma tumor samples and thirty-seven lung tissues were collected for expression of GRIM-19 in immunohistochemistry ; the expressions of GRIM-19 and STAT3 were measured by Western Blot. Total RNA were extracted from 37 cases lung adenocarcinoma tumor samples and lung tissues, respectively, and the expression of GRIM-19, Stat3 and its downstream target genes Bcl-2, Cyclin B1, Cyclin D1 was measured by RT-PCR.
SPC-A1 cells were transfected with either pIRES-Puro2-Myc or pIRES-Puro2-GRIM-19-Myc into cells using Lipofectamine (Invitrogen, Carlsbad,CA). For stable expression of GRIM-19-Myc, transfectants were selected in growth medium containing 0.60μg/mL puromycin. The established stable lines were designated as SPC-A1 /Control and SPC-A1 /GRIM-19, respectively. SPC-A1 /Control and SPC-A1 /GRIM-19 cells were harvested,the expressions of GRIM-19 and STAT3 were measured by Western Blot. Total RNA were extracted from SPC-A1 /Control and SPC-A1 /GRIM-19, respectively. and the expression of GRIM-19, Stat3 and its downstream target genes Bcl-2, Cyclin B1, Cyclin D1 ,VEGF was measured by RT-PCR.
Female BALB/c nude mice (Shanghai Institute of Experimental Animals) were divided into 2 groups. Each group contained 4 mouses. All mouses were housed in a pathogen-free environment.SPC-A1/GRIM-19, SPC-A1/Control (2×106) cells in 0.1 mL saline containing 50% Matrigel were implanted subcutaneously on the dorsal flank of 5-week-old female athymic nude mice. Mice were sacrificed on day 28, and tumor sizes were determined. Various tissues (lung, liver,spleen, kidney, heart, and tumor) were harvested from these mice for monitoring metastases. Tumors were excised. Tissues were processed for Western blot(GRIM-19,STAT3,MMP2), RT-PCR(Bcl-2, Cyclin B1, Cyclin D1 ,VEGF) .Tumor growth was monitored by measuring tumor dimensions with a caliper and the volume was calculated using the formula V= (4/3)×πa2b, where 2a = minor axis, 2b = major axis of prolate spheroid
Results
1.Expression of GRIM-19, Stat3 and it’s downstream target gene Bcl-2、Cyclin B1、Cyclin D1 in lung adenocarcinoma and lung tissues
Since GRIM-19 was identified as a potential tumor suppressor in recent studies, we checked the expression status of GRIM-19 in lung adenocarcinoma and normal lung tissues. GRIM-19 was highly expressed in normal lung tissues and decreased in lung adenocarcinoma tumor samples.
While normal lung tissues showed high levels of GRIM-19, they generally had a low expression levels of STAT3. In contrast, a significantly reduced expression of GRIM-19 and increased expression of STAT3 were observed in lung adenocarcinoma tissues . Interestingly, a negative correlation was found between the expression levels of GRIM-19 and STAT3 expression both in normal and lung adenocarcinoma tissues.After expressions of GRIM-19 and STAT3 were detected, we further examined mRNA expression of some STAT3-regulated genes such as Cyclin B1, CyclinD1, Bcl-2. Consistent with the increase of STAT3 and the reduction in GRIM-19 levels, the STAT3-regulated genes expressions at mRNA level were elevated in lung adenocarcinoma tissues compared to lung tissues.
2.GRIM-19 suppresses cell proliferation and reduces STAT3 and it’s downstream target gene expression in lung adenocarcinoma cell line SPC-A1
For testifing the effect of GRIM-19 on cell proliferation, we established a GRIM-19 stabilized expression lung adenocarcinoma cell line, SPC-A1/GRIM-19 and SPC-A1/Control cell line. SPC-A1 cells expressed a low basal level of GRIM-19. A significant increase in GRIM-19 expression and a down-regulation of STAT3 were observed after transfection with pIRES-Puro2-GRIM-19-Myc.The growth of SPC-A1 /GRIM-19 cells, as indicated by MTT assay, was markedly inhibited on day 3 and day 4compared to the SPC-A1 / CON cells (P < 0.05 respectively).
STAT3 is known to up-regulate the expression of genes associated with angiogenesis , antiapoptosis ,increasing tumor cell proliferation such as the vascular endothelial growth factor (VEGF) , Bcl-2 ,Cyclin B1 and CyclinD1. We examined if SPC-A1 /GRIM-19 cells had reduced expression of these genes compared to SPC-A1 /CON cells.The expression of GRIM-19 significantly suppressed the expression of VEGF (an angiogenic growth factor) , Bcl-2(antiapoptosis) and Cyclin B1 and CyclinD1 (increasing tumor cell proliferation).
3.GRIM-19 suppress human lung adenocarcinoma cell line SPC-A1 growth in vivo.
We next determined if the GRIM-19 could inhibit tumor growth using a xenograft tumor model. we transplanted SPC-A1/GRIM-19 and SPC-A1/Control cells into athymic nude mice. Cells(106)were transplanted subcutaneously into the right flank(n=4 per group) and tumor growth was monitored for 28 days. On day 28, animals were sacrificed and final tumor weights and volumes were determined.Tumor weights and volumes were significantly smaller in SPC-A1/GRIM-19 group than SPC-A1/Control group(P<0.01)). We next examined the expression of STAT3, and GRIM-19 in tumors derived from mice using Western blot analysis. As expected,GRIM-19 expression levels were higher in SPC-A1/GRIM-19 group than in SPC-A1/Control group(P<0.01).The levels of STAT3 were strongly reduced in SPC-A1/GRIM-19 group (P<0.01) . STAT3 is also known to up-regulate the expression of genes associated with tumor invasion such as matrix metalloproteases(MMPS),We,therefore, examined if SPC-A1/GRIM-19 group had reduced expression of these genes. MMP-2 was suppressed in the tumors treated with SPC-A1/GRIM-19 .We examined if SPC-A1/GRIM-19 group had reduced expression of STAT3-dependent growth–associated genes compared to SPC-A1 /CON group. The expression level of all these gene transcripts was decreased in SPC-A1/GRIM-19 cells compared to SPC-A1/Control. Tumor infiltration into muscle or lymph node metastasis was not observed in all 8 mice .
Conclusion
In summary, we have revealed that GRIM-19 levels were significantly reduced at the mRNA and protein levels in lung adenocarcinoma tissues; furthermore, STAT3 levels were increased and accompanied by changes in related downstream target genes. Overexpression of GRIM-19 was found to cause suppression of lung adenocarcinoma tumor growth, both in vitro and in vivo, which may contribute to the future development of gene therapy for lung adenocarcinoma using a GRIM-19-based approach.
肺癌是当今世界各国常见的恶性肿瘤,已成为绝大多数国家癌症死亡的主要原因,目前被认为是对人类健康和生命威胁最大的恶性肿瘤。按组织学分类,分为非小细胞肺癌和小细胞肺癌,其中非小细胞肺癌分为腺癌、鳞癌、大细胞肺癌、鳞腺癌。其中腺癌目前占第一位。干扰素/维甲酸诱导凋亡相关基因-19(gene associated with retinoid-IFN-induced mortality-19,GRIM-19),是2000年由Angell等应用反义基因敲除的方法分离发现的一种细胞凋亡调节因子。在一些原发性肾细胞肿瘤和泌尿生殖系肿瘤中GRIM-19表达缺失或被严重抑制,用肾脏恶性肿瘤细胞系的研究证实:GRIM-19的下调通过增强依赖Stat3(signal transducers and activators of transcription 3)基因的表达而促进肿瘤生长,并且首次表明GRIM-19具有肿瘤抑制因子作用。对于GRIM-19在肺腺癌的表达情况、是否抑制其生长及抑制机制目前还没有见到相关报道。我们先期已经对GRIM-19在肺癌组织中的表达一般情况与正常肺组织表达情况做了一个对比研究,但肺腺癌中表达具体情况没有做进一步研究分析。本研究对GRIM-19和Stat3基因在肺腺癌组织与肺组织中表达作对比,采用体内外实验研究,验证GRIM-19抑制肺腺癌生长和初步探讨其作用机制。
目的:
本研究以人肺癌组织及对应的肺组织为材料,检测GRIM-19、Stat3及其下游靶基因Bcl-2、Cyclin B1、Cyclin D1在肺腺癌及正常肺组织中的表达情况;对pIRES-Puro2-GRIM-19-Myc质粒进行测序,分别将pIRES-Puro2-GRIM-19-Myc和pIRES-Puro2-GRIM-19-Myc真核表达质粒转染SPC-A1细胞中,建立稳转细胞株SPC-A1/GRIM-19和SPC-A1/CON,将上述两种细胞分别种植于不同组的BALB/C裸鼠皮下,28天杀死裸鼠,取出皮下肿瘤组织,分别检测GRIM-19、Stat3及其下游靶基因Bcl-2、Cyclin B1、Cyclin D1等的表达。在体内外验证GRIM-19抑制肺腺癌生长及其作用机制。
方法:
收集肺腺癌和对应的肺组织标本,用免疫组化方法比较肺腺癌组织和对应肺组织中GRIM-19蛋白表达。再用Western blot法比较肺腺癌和肺组织GRIM-19、STAT3蛋白的表达。分别抽取37例患者肺腺癌组织及肺组织中的总RNA,然后采用RT-PCR法比较肺腺癌组织和正常肺组织中GRIM-19、Stat3及其下游靶基因Bcl-2、Cyclin B1、Cyclin D1的mRNA表达。
中国科技大学生命科学学院肖卫华教授馈赠PIRES-Puro2-GRIM-19-Myc和PIRES-Puro2- Myc真核表达质粒,上海生工对此进行测序,判断是否有基因突变。Lipofectamine将真核表达质粒pIRES-Puro2-GRIM-19-Myc和pIRES-Puro2-Myc转染肺腺癌细胞株SPC-A1中,建立稳转细胞株。用MTT方法检测GRIM-19是否对肺腺癌细胞株SPC-A1的生长影响。用Western blot法比较稳转细胞株SPC-A1/GRIM-19和SPC-A1/CON的GRIM-19和STAT3蛋白表达。然后采用RT-PCR法分别比较稳转细胞株SPC-A1/GRIM-19和SPC-A1/CON的GRIM-19、Stat3及其下游靶基因Bcl-2、Cyclin B1、Cyclin D1、VEGF的mRNA表达。通过上述方法,在体外实验判断GRIM-19是否抑制肺腺癌细胞株的生长及其机制。
分别取SPC-A1/GRIM-19, SPC-A1/CON (2×106)种植于5周的BALB/C裸鼠(雌性),观察4周,每隔7天检测一次其体积变化,用体内实验判断GRIM-19是否对肺腺癌细胞株SPC-A1的生长影响。第28天分别取不同组BALB/C裸鼠的皮下肿瘤组织,用Western blot法比较其GRIM-19、STAT3和MMP2蛋白表达。分别提取不同组BALB/C裸鼠的皮下肿瘤组织的总RNA,然后采用RT-PCR法比较其GRIM-19、Stat3及其下游靶基因Bcl-2、Cyclin B1、Cyclin D1、VEGF的mRNA表达情况。通过上述方法,在体内实验判断GRIM-19是否抑制肺腺癌细胞株的生长及其机制。
结果:
一、GRIM-19、STAT3及其下游靶基因在肺腺癌组织和肺组织中的表达用免疫组化检测结果显示:GRIM-19蛋白在肺腺癌组织表达明显低于在肺组织表达。Western Blot结果表明:肺腺癌组与肺组织组相比,GRIM-19蛋白表达水平在肺腺癌中明显低于肺组织,差异有统计学意义(p<0.01)。STAT3蛋白表达与之相反。RT-PCR结果表明:肺腺癌组与肺组织相比,GRIM-19基因表达水平明显减低,差异有统计学意义(p<0.01);与之相反,RT-PCR结果发现Stat3及其下游靶基因Bcl-2、Cyclin B1、Cyclin D1的表达在肺腺癌组织中高于其肺组织中。
二、GRIM-19抑制肺腺癌细胞株SPC-A1生长体外实验结果
上海生工对对PIRES-Puro2-GRIM-19-Myc真核表达质粒测序无基因突变。用Lipofectamine将真核表达质粒pIRES-Puro2-GRIM-19-Myc和pIRES-Puro2-Myc转染肺腺癌细胞株SPC-A1中, G418(浓度0.60μg/mL)持续筛选6周建立稳转细胞株: SPC-A1/GRIM-19和SPC-A1/CON。Western Blot结果表明:SPC-A1/GRIM-19细胞株与SPC-A1/CON细胞株相比, SPC-A1/CON细胞株中的GRIM-19蛋白水平表达明显低于SPC-A1/GRIM-19细胞中的表达,差异有统计学意义(p<0.01)。STAT3蛋白表达与之相反。RT-PCR结果表明:SPC-A1/GRIM-19细胞株与SPC-A1/CON细胞株相比, SPC-A1/CON细胞株中的GRIM-19表达水平明显低于SPC-A1/GRIM-19细胞株中的表达水平,差异有统计学意义(p<0.01),RT-PCR结果发现Stat3及其下游靶基因Bcl-2、CyclinB1、CyclinD1,VEGF的mRNA表达与之相反。
三、GRIM-19抑制肺腺癌细胞株SPC-A1生长体内实验结果
把SPC-A1/GRIM-19和SPC-A1/CON细胞分别种植于BALB/C裸鼠皮下,每周检测其肿瘤大小,构建其生长曲线,结果显示:种植SPC-A1/GRIM-19细胞的BALB/C裸鼠的皮下肿瘤的体积明显减小(p<0.01)。4周后分别取不同组BALB/C裸鼠的皮下肿瘤组织用Western blot法检测其GRIM-19、STAT3和MMP2蛋白,结果表明:种植SPC-A1/GRIM-19细胞的BALB/C裸鼠的皮下肿瘤组织与种植SPC-A1/CON细胞的BALB/C裸鼠的皮下肿瘤组织相比: GRIM-19的蛋白水平在SPC-A1/CON细胞的瘤组织中的表达明显低于SPC-A1/GRIM-19细胞的瘤组织的表达,差异有统计学意义(p<0.01)。STAT3、MMP2的蛋白表达与之相反。RT-PCR结果表明:种植SPC-A1/GRIM-19细胞的BALB/C裸鼠的皮下肿瘤组织与种植SPC-A1/CON细胞的BALB/C裸鼠的皮下肿瘤组织相比:GRIM-19的mRNA表达水平明显升高,差异有统计学意义(p<0.01); Stat3及其下游靶基因Bcl-2、Cyclin B1、Cyclin D1,VEGF的mRNA表达水平与之相反。
结论
1.肺腺癌组GRIM-19的mRNA水平及蛋白水平明显低于其在相对应的正常肺组织的表达,并且基因表达水平的降低伴随有蛋白表达的相应下降;肺腺癌组Stat3的mRNA水平及蛋白及其下游靶基因Bcl-2、Cyclin B1、CyclinD1、VEGF基因表达水平均高于其在肺组织中的表达。
2.通过建立肺腺癌稳转细胞株SPC-A1/GRIM-19和SPC-A1/C0N,在体外验证GRIM-19抑制肺腺癌生长。
3.把SPC-A1/GRIM-19和SPC-A1/CON稳转细胞株分别种植不同组BALB/C裸鼠的皮下,在体内验证GRIM19抑制肺腺癌生长。
Background
According to the World Health Organization,lung cancer is currently the leading cause of cancer-related death in the world. Recently,a number of studies have shown that the family of proteins known as gene associated with retinoid- IFN- induced mortality (GRIMs) may represent novel types of tumor suppressors.Gene associated with retinoid interferon-induced mortality-19(GRIM-19), was originally identified as a cell death regulatory gene using the antisense technical knockout approach by Angell et al. In a number of primary RCC and in some urinogenital tumors.the expression of GRIM-19 is lost or severely depressed .By using an RCC cell line, researchers show that down regulation of GRIM-19 promotes tumor growth via an augmentation of Stat3-dependent gene expression. These studies for the first time show a tumor suppressor like activity of GRIM-19. Maximo et al confirmed that Hürthle cell thyroid carcinomas exist mutation of GRIM-19. In this study, we revealed that GRIM-19 were significantly reduced at mRNA and protein levels in lung adenocarcinoma tissue . STAT3 and its downstream target genes were tended to express a higher basal level in lung adenocarcinoma tissues. Overexpression of GRIM-19 was also found to suppress lung adenocancinoma tumor growth both in vitro and in vivo.Taken together,these finding will likely contribute to the future development of GRIM-19-based gene therapy approaches to treat lung adenocancinoma.
Objective
In the present study, we used lung adenocarcinoma tissues and lung tissues to investigate the expression of GRIM-19, Stat3 and it’s downstream target gene Bcl-2, Cyclin B1,Cyclin D1,Analyzing the relationship between GRIM-19 and Stat3, and to further explore the effects of GRIM-19 on SPC-A1 tumor cell growth in vitro and vivo.
Methods
Thirty-seven lung adenocarcinoma tumor samples and thirty-seven lung tissues were collected for expression of GRIM-19 in immunohistochemistry ; the expressions of GRIM-19 and STAT3 were measured by Western Blot. Total RNA were extracted from 37 cases lung adenocarcinoma tumor samples and lung tissues, respectively, and the expression of GRIM-19, Stat3 and its downstream target genes Bcl-2, Cyclin B1, Cyclin D1 was measured by RT-PCR.
SPC-A1 cells were transfected with either pIRES-Puro2-Myc or pIRES-Puro2-GRIM-19-Myc into cells using Lipofectamine (Invitrogen, Carlsbad,CA). For stable expression of GRIM-19-Myc, transfectants were selected in growth medium containing 0.60μg/mL puromycin. The established stable lines were designated as SPC-A1 /Control and SPC-A1 /GRIM-19, respectively. SPC-A1 /Control and SPC-A1 /GRIM-19 cells were harvested,the expressions of GRIM-19 and STAT3 were measured by Western Blot. Total RNA were extracted from SPC-A1 /Control and SPC-A1 /GRIM-19, respectively. and the expression of GRIM-19, Stat3 and its downstream target genes Bcl-2, Cyclin B1, Cyclin D1 ,VEGF was measured by RT-PCR.
Female BALB/c nude mice (Shanghai Institute of Experimental Animals) were divided into 2 groups. Each group contained 4 mouses. All mouses were housed in a pathogen-free environment.SPC-A1/GRIM-19, SPC-A1/Control (2×106) cells in 0.1 mL saline containing 50% Matrigel were implanted subcutaneously on the dorsal flank of 5-week-old female athymic nude mice. Mice were sacrificed on day 28, and tumor sizes were determined. Various tissues (lung, liver,spleen, kidney, heart, and tumor) were harvested from these mice for monitoring metastases. Tumors were excised. Tissues were processed for Western blot(GRIM-19,STAT3,MMP2), RT-PCR(Bcl-2, Cyclin B1, Cyclin D1 ,VEGF) .Tumor growth was monitored by measuring tumor dimensions with a caliper and the volume was calculated using the formula V= (4/3)×πa2b, where 2a = minor axis, 2b = major axis of prolate spheroid
Results
1.Expression of GRIM-19, Stat3 and it’s downstream target gene Bcl-2、Cyclin B1、Cyclin D1 in lung adenocarcinoma and lung tissues
Since GRIM-19 was identified as a potential tumor suppressor in recent studies, we checked the expression status of GRIM-19 in lung adenocarcinoma and normal lung tissues. GRIM-19 was highly expressed in normal lung tissues and decreased in lung adenocarcinoma tumor samples.
While normal lung tissues showed high levels of GRIM-19, they generally had a low expression levels of STAT3. In contrast, a significantly reduced expression of GRIM-19 and increased expression of STAT3 were observed in lung adenocarcinoma tissues . Interestingly, a negative correlation was found between the expression levels of GRIM-19 and STAT3 expression both in normal and lung adenocarcinoma tissues.After expressions of GRIM-19 and STAT3 were detected, we further examined mRNA expression of some STAT3-regulated genes such as Cyclin B1, CyclinD1, Bcl-2. Consistent with the increase of STAT3 and the reduction in GRIM-19 levels, the STAT3-regulated genes expressions at mRNA level were elevated in lung adenocarcinoma tissues compared to lung tissues.
2.GRIM-19 suppresses cell proliferation and reduces STAT3 and it’s downstream target gene expression in lung adenocarcinoma cell line SPC-A1
For testifing the effect of GRIM-19 on cell proliferation, we established a GRIM-19 stabilized expression lung adenocarcinoma cell line, SPC-A1/GRIM-19 and SPC-A1/Control cell line. SPC-A1 cells expressed a low basal level of GRIM-19. A significant increase in GRIM-19 expression and a down-regulation of STAT3 were observed after transfection with pIRES-Puro2-GRIM-19-Myc.The growth of SPC-A1 /GRIM-19 cells, as indicated by MTT assay, was markedly inhibited on day 3 and day 4compared to the SPC-A1 / CON cells (P < 0.05 respectively).
STAT3 is known to up-regulate the expression of genes associated with angiogenesis , antiapoptosis ,increasing tumor cell proliferation such as the vascular endothelial growth factor (VEGF) , Bcl-2 ,Cyclin B1 and CyclinD1. We examined if SPC-A1 /GRIM-19 cells had reduced expression of these genes compared to SPC-A1 /CON cells.The expression of GRIM-19 significantly suppressed the expression of VEGF (an angiogenic growth factor) , Bcl-2(antiapoptosis) and Cyclin B1 and CyclinD1 (increasing tumor cell proliferation).
3.GRIM-19 suppress human lung adenocarcinoma cell line SPC-A1 growth in vivo.
We next determined if the GRIM-19 could inhibit tumor growth using a xenograft tumor model. we transplanted SPC-A1/GRIM-19 and SPC-A1/Control cells into athymic nude mice. Cells(106)were transplanted subcutaneously into the right flank(n=4 per group) and tumor growth was monitored for 28 days. On day 28, animals were sacrificed and final tumor weights and volumes were determined.Tumor weights and volumes were significantly smaller in SPC-A1/GRIM-19 group than SPC-A1/Control group(P<0.01)). We next examined the expression of STAT3, and GRIM-19 in tumors derived from mice using Western blot analysis. As expected,GRIM-19 expression levels were higher in SPC-A1/GRIM-19 group than in SPC-A1/Control group(P<0.01).The levels of STAT3 were strongly reduced in SPC-A1/GRIM-19 group (P<0.01) . STAT3 is also known to up-regulate the expression of genes associated with tumor invasion such as matrix metalloproteases(MMPS),We,therefore, examined if SPC-A1/GRIM-19 group had reduced expression of these genes. MMP-2 was suppressed in the tumors treated with SPC-A1/GRIM-19 .We examined if SPC-A1/GRIM-19 group had reduced expression of STAT3-dependent growth–associated genes compared to SPC-A1 /CON group. The expression level of all these gene transcripts was decreased in SPC-A1/GRIM-19 cells compared to SPC-A1/Control. Tumor infiltration into muscle or lymph node metastasis was not observed in all 8 mice .
Conclusion
In summary, we have revealed that GRIM-19 levels were significantly reduced at the mRNA and protein levels in lung adenocarcinoma tissues; furthermore, STAT3 levels were increased and accompanied by changes in related downstream target genes. Overexpression of GRIM-19 was found to cause suppression of lung adenocarcinoma tumor growth, both in vitro and in vivo, which may contribute to the future development of gene therapy for lung adenocarcinoma using a GRIM-19-based approach.
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