| BackgroundAccording 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.ObjectiveIn 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.MethodsThirty-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 Results1.Expression of GRIM-19, Stat3 and it's downstream target gene Bcl-2,Cyclin B1,Cyclin D1 in lung adenocarcinoma and lung tissuesSince 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-A1For 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 .ConclusionIn 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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