Gene Associated With Retinoid-interferon-induced Mortality-19 Expression Research Articles

Interaction of M2 macrophages and endometrial cells induces downregulation of GRIM-19 in endometria of adenomyosis

Research questionDoes the aggregation of M2 macrophages affect the expression of gene associated with retinoid-interferon-induced mortality 19 (GRIM-19) in adenomyosis? DesignEndometrial tissues were collected from patients with (n = 15) and without (n = 15) adenomyosis. Tissues were analysed for GRIM-19 and toll-like receptor 4 (TLR4) expression by immunohistochemistry and western blotting. Apoptosis was analysed by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay. Human endometrial stromal cells (HESC) were transfected with GRIM-19 small interfering RNA (SiRNA) to knockdown GRIM-19 expression. The HESC were co-cultured with M2 macrophages to detect the influence of M2 macrophages in HESC cells. Analyses included GRIM-19, caspase-3 and TLR4 expression by western blotting, and GRIM-19 and TLR4 by quantitative real-time polymerase chain reaction. Apoptosis was measured by flow cytometry and TUNEL assay. Cell proliferation (Cell Counting Kit-8 assay) and migration assays were carried out. ResultsThe expression of GRIM-19 was significantly lower in adenomyosis lesions compared with controls (P < 0.001). Deficiency of GRIM-19 induced by siRNA decreased apoptosis and increased proliferation and migration in HESC. A significant decrease in GRIM-19 expression occurred in HESC after co-culture with M2 macrophages (P = 0.018). After co-culture with M2 macrophage, apoptosis decreased and proliferation and cell invasion in HESC increased. Protein (P = 0.006) and mRNA (P = 0.013) expression of TLR4 in HESC also reduced after this co-culture. Up-regulation of GRIM-19 occurred in HESC treated with the activator TLR4 (P = 0.016). Up-regulation of GRIM-19 was significantly reversed in cells treated with the TLR4 inhibitor (P = 0.011). ConclusionM2 macrophages may be involved in regulating the expression of GRIM-19 partly through the TLR4 signalling axis in adenomyosis.

GRIM-19 deficiency promotes clear cell renal cell carcinoma progression and is associated with high TNM stage and Fuhrman grade.

Clear cell renal cell carcinoma (ccRCC) exhibits the highest mortality among all urological malignancies. The investigation of the potential disease progression markers can improve ccRCC diagnosis and treatment. Gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) is involved in carcinogenesis and cancer progression in a variety of cancer types including RCC. While, its role in ccRCC remains unclear, this cancer type is considered the most aggressive RCC subtype. In the present study, RT-qPCR, western blotting and immunohistochemical (IHC) assays demonstrated that GRIM-19 protein and mRNA levels were downregulated in ccRCC tumor tissues compared with the corresponding levels noted in paracancerous non-tumor tissues. The deficiency of this protein contributed in relaxed and/or collapsed structures of the kidney tubules and collecting duct noted in tumor tissues. Moreover, the reduction in GRIM-19 expression was associated with high tumor, lymph nodes and metastasis (TNM) stage and Fuhrman grade of ccRCC tumors. The data suggested that GRIM-19 acted as a tumor suppressor and that its deficiency promoted ccRCC development and progression. GRIM-19 can be considered a potential tumor marker for ccRCC.

GRIM-19 Ameliorates Multiple Sclerosis in a Mouse Model of Experimental Autoimmune Encephalomyelitis with Reciprocal Regulation of IFNγ/Th1 and IL-17A/Th17 Cells.

The protein encoded by the Gene Associated with Retinoid-Interferon-Induced Mortality-19 (GRIM-19) is located in the mitochondrial inner membrane and is homologous to the NADH dehydrogenase 1-alpha subcomplex subunit 13 of the electron transport chain. Multiple sclerosis (MS) is a demyelinating disease that damages the brain and spinal cord. Although both the cause and mechanism of MS progression remain unclear, it is accepted that an immune disorder is involved. We explored whether GRIM-19 ameliorated MS by increasing the levels of inflammatory cytokines and immune cells; we used a mouse model of experimental autoimmune encephalomyelitis (EAE) to this end. Six-to-eight-week-old male C57BL/6, IFNγ-knockout (KO), and GRIM-19 transgenic mice were used; EAE was induced in all strains. A GRIM-19 overexpression vector (GRIM19 OVN) was electrophoretically injected intravenously. The levels of Th1 and Th17 cells were measured via flow cytometry, immunofluorescence, and immunohistochemical analysis. IL-17A and IFNγ expression levels were assessed via ELISA and quantitative PCR. IL-17A expression decreased and IFNγ expression increased in EAE mice that received injections of the GRIM19 OVN. GRIM-19 transgenic mice expressed more IFNγ than did wild-type mice; this inhibited EAE development. However, the effect of GRIM-19 overexpression on the EAE of IFNγ-KO mice did not differ from that of the empty vector. GRIM-19 expression was therapeutic for EAE mice, elevating the IFNγ level. GRIM-19 regulated the Th17/Treg cell balance.

Heterogeneity of GRIM-19 Expression in the Adult Mouse Brain.

Gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) is a subunit of the mitochondrial respiratory chain complex I that has a significant effect on ATP production. The brain is particularly susceptible to ATP deficiency due to its limited energy storage capability and its high rate of oxygen consumption. Thus, GRIM-19 might be involved in regulating ATP level in the brain or cell death caused by several neurological disorders. To understand the physiological and pathophysiological roles of GRIM-19 in the brain, a thorough investigation of the neuroanatomic distribution of GRIM-19 in the normal brain is necessary. Therefore, the present study examined the distribution patterns of GRIM-19 in the adult C57BL/6 mouse brain using immunohistochemistry and identified cell types expressing GRIM-19 using double immunofluorescence staining. We found that GRIM-19 was ubiquitously but not homogenously expressed throughout the brain. GRIM-19 immunoreactivity was predominantly observed in neurons, but not in astrocytes, microglia, or oligodendrocytes under normal physiological conditions. Following transient global cerebral ischemia, GRIM-19-positive immunoreactivity was, however, observed in neurons as well as glial cells including astrocytes in the hippocampus. Furthermore, GRIM-19 was weakly expressed in the hippocampal subgranular zone, in which neural stem and progenitor cells are abundant, but highly expressed in the immature and mature neuronal cells in the granular cell layer of the normal brain, suggesting an inverse correlation between expression of GRIM-19 and stemness activity. Collectively, our study demonstrating widespread and differential distribution of GRIM-19 in the adult mouse brain contributes to investigating the functional and pathophysiological roles of this protein.

OLFM4 Enhances STAT3 Activation and Promotes Tumor Progression by Inhibiting GRIM19 Expression in Human Hepatocellular Carcinoma.

Olfactomedin 4 (OLFM4) induces signal transducer and activator of transcription 3 (STAT3) activation by inhibiting gene associated with retinoid‐interferon‐induced mortality 19 (GRIM19), a strong STAT3 suppressor gene; however, the mechanisms of OLFM4 for regulating GRIM19‐STAT3 cascade in hepatocellular carcinoma (HCC) remain unclear. The functions and regulations of OLFM4, GRIM19, and STAT3 activation in HCC progression were evaluated using surgical specimens collected from 111 HCC patients or 2 HCC cell lines in vitro. Moreover, the cancer stem cell–like property of OLFM4 mediated by leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5), known as an intestinal stem cell marker, was investigated. OLFM4 was increased in HCC compared with adjacent liver tissue. The multivariate analysis revealed that high OLFM4 expression was an independent factor for poor prognosis. OLFM4 expression was negatively correlated with GRIM19 expression and positively correlated with STAT3 activation in HCC, thereby increasing cell cycle progression. OLFM4 knockdown in HCC cells increased GRIM19 expression and inhibited STAT3 activation; however, after double knockdown of GRIM19 and OLFM4, STAT3 activation decreased by OLFM4 knockdown was increased again. OLFM4 knockdown increased cell apoptosis, inhibited cell proliferation, and suppressed cancer stem cell–like property in HCC cells. The incidence of hematogenous recurrence was higher in HCC patients with high OLFM4 expression, suggesting that anoikis resistance of HCC was enhanced by OLFM4. In clinical cases, LGR5 expression and CD133 expression was correlated with OLFM4 expression in HCC, leading to poor patient prognosis. In vitro, LGR5 enhanced cancer stem cell–like property by up‐regulating OLFM4 through the Wnt signaling pathway. Conclusion: OLFM4 is induced by the LGR5‐Wnt signaling pathway and is strongly associated with aggressive tumor progression and poor prognosis in HCC by regulating STAT3‐induced tumor cell proliferation and cancer stem cell–like property. Therefore, OLFM4 is a novel prognostic predictor and a potential therapeutic target for patients with HCC.

GRIM-19 repressed hypoxia-induced invasion and EMT of colorectal cancer by repressing autophagy through inactivation of STAT3/HIF-1α signaling axis.

Hypoxia leads to cancer progression and promotes the metastatic potential of cancer cells. Thereby, the aim of the present study was to investigate the detailed effects of gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) in colorectal cancer (CRC) cell lines under hypoxia conditions and explore the potential molecular mechanisms. Here, we observed that GRIM-19 expression was downregulated in several CRC cell lines as well as in HCT116 and Caco-2 cells under a hypoxic microenvironment. Additionally, the introduction of GRIM-19 obviously suppressed cell invasive ability and epithelial-mesenchymal transition (EMT) through modulating EMT markers as reflected by the upregulation of E-cadherin along with the downregulation of vimentin and N-cadherin under hypoxic conditions. Moreover, the addition of GRIM-19 repressed hypoxia-induced autophagy through modulating autophagy associated proteins as reflected by the downregulation of LC3-II/LC3-I ratio and Beclin-1 expression, as well as the increased of p62 expression. Interestingly, overexpression of GRIM-19 markedly ameliorated the accumulation of HIF-1α triggered by hypoxia accompanied by an inhibition of vascular endothelial growth factor (VEGF) production and phospho-signal transducer and activator of transcription 3(p-STAT3) expression. Further data demonstrated that GRIM-19 have a negative feedback effect on the expression of HIF-1α. Mechanistically, re-expression of HIF-1α completely reversed the inhibitory effects of GRIM-19 on hypoxia-induced invasion and EMT. Taken all data together, our findings established that GRIM-19 suppresses hypoxia-triggered invasion and EMT by inhibiting hypoxia-induced autophagy through inactivation HIF-1α/STAT3 signaling pathway, indicating that GRIM-19 may serve as a potential predictive factor and therapeutic target for CRC treatment.

Repression of GRIM19 expression potentiates cisplatin chemoresistance in advanced bladder cancer cells via disrupting ubiquitination-mediated Bcl-xL degradation.

The mainstay of treatment for advanced bladder cancer (BC) is cisplatin (CDDP)-based systematic chemotherapy. However, acquired chemoresistance induced by as yet unidentified mechanisms is encountered frequently and often results in treatment failure and disease progression. The present study was designed to elucidate the expression and potential role of the gene associated with retinoid-interferon-induced mortality-19 (GRIM19) in the pathogenesis of CDDP resistance in BC. RT-qPCR and immunoblotting were employed to evaluate the expression profile of GRIM19 in clinical BC samples and in different BC cells. Using cell viability assay, apoptotic ELISA, xenografts mouse model, and Transwell assay, the effects of GRIM19 inhibition or GRIM19 overexpression on CDDP resistance were determined in different BC cells. Lastly, using co-immunoprecipitation, we provided the molecular evidence for the interaction between GRIM19 and Bcl-xL. Expression levels of GRIM19 were significantly down-regulated in recurrent BC specimens, and in experimentally induced CDDP-resistant BC cells. Functionally, overexpression of the exogenous GRIM19 potentiated CDDP sensitivity and suppressed the survival and invasion of BC cells in the presence of CDDP challenge. Mechanistically, the compromised CDDP chemosensitization induced by GRIM19 loss was at least partially attributed to the attenuation of Bcl-xL polyubiquitination and subsequent degradation, because (1) GRIM19 colocalized with Bcl-xL in the mitochondria of BC cells and (2) GRIM19 overexpression promoted the ubiquitination of Bcl-xL, and this event could be effectively reversed by pretreatment with inhibitors of p38-MAPK and JNK pathways, indicating that GRIM19 overexpression-induced Bcl-xL ubiquitination may achieve in a p38/JNK-dependent manner. Using the UMUC-3 cells stably depleted of endogenous GRIM19, we further show that inhibition of Bcl-xL rectified GRIM19 deficiency-caused CDDP resistance in BC cells. In addition, BCL2L1 mRNA levels were negatively correlated with GRIM19 mRNA levels in CDDP-associated clinical BC tissues. Disruption of GRIM19/Bcl-xL is a key mechanism of CDDP resistance in advanced BC. Therapeutically, enhancement of GRIM19 expression or employment of p38/JNK inhibitors may serve as resensitizing therapies for subgroups of CDDP-resistant or refractory BC patients.

The effect of a gene associated with retinoid-interferon-induced mortality 19 (GRIM-19) on STAT3-induced gene expression in renal carcinoma.

This study aimed to investigate the exact regulatory mechanisms of retinoid-interferon-induced mortality 19 (GRIM-19) in renal carcinoma. Tumour tissue samples from patients with renal carcinoma (n = 30, there were seven cases of Stage I, eight cases of Stage II, eight cases of Stage III, seven cases of Stage IV) and control subjects were selected from adjacent normal tissue (n = 10). Real-time quantitative PCR and western blotting were used to assess the level of GRIM-19, signal transducer and activator of transcription-3 (STAT3) and its downstream molecules. CD31 was detected by immunohistochemistry. The MTT assay was used to measure cell proliferation. The amount of apoptosis cells was analysed by Flow cytometry. The results showed that expression of GRIM-19 was decreased in renal carcinoma. However, in tumour tissue, STAT3 and its downstream signalling molecules showed the higher expression compared with control. Overexpression of GRIM-19, inhibited tumour growth apoptosis by mediating activators of STAT3 signal. In addition, interferon-β and all-trans-retinoic acid inhibited the renal carcinoma cell growth and induced apoptosis, and effect of drug combinations was particularly evident. In conclusion, GRIM-19 expression is associated with hyperactivation of STAT3-induced gene expression in renal carcinoma.

MiR-6743-5p, as a direct upstream regulator of GRIM-19, enhances proliferation and suppresses apoptosis in glioma cells.

Gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) has been recognized as a tumor suppressor protein, which regulates cell growth, apoptosis, and migration by signal transducer and activator of transcription 3 (STAT3) signaling pathway and non-STAT3 pathway in glioma cells. Here, we investigated the molecular mechanisms that regulated GRIM-19 expression in glioma cells. By the TargetScan algorithm, four miRNAs, hsa-miR-17-3p, hsa-miR-423-5p, hsa-miR-3184-5p, and hsa-miR-6743-5p, were identified with the potential to bind with 3′-UTR of GRIM-19. Further miRNA inhibitor transfection and luciferase assays revealed that miR-6743-5p was able to directly target the 3′-UTR of GRIM-19. Additionally, miR-6743-5p expression was inversely related with GRIM-19 expression in glioma specimens and cell lines. Moreover, the inhibition of miR-6743-5p caused a significant inhibition of cell proliferation and a marked promotion of cell apoptosis in glioma cells, and this phenotype was rescued by GRIM-19 knockdown. Finally, the inhibition of miR-6743-5p expression suppressed the phosphorylation of STAT3, and the mRNA expression of CyclinD1 and Bcl-2, two target genes of STAT3, while miR-6743-5p mimic had the inversed effects. Treatment with STAT3 inhibitor AG490 partially rescued the proliferation-promoting and anti-apoptosis effects of miR-6743-5p overexpression or GRIM-19 knockdown. Collectively, miR-6743-5p may act as an oncomiRNA in glioma by targetting GRIM-19 and STAT3.

Mitochondrial GRIM-19 as a potential therapeutic target for STAT3-dependent carcinogenesis of gastric cancer.

Aberrant STAT3 activation occurs in most human gastric cancers (GCs) and contributes to the malignant progression of GC, but mechanism(s) underlying aberrant STAT3 remain largely unknown. Here we demonstrated that the gene associated with retinoid interferon-induced mortality 19 (GRIM-19) was severely depressed or lost in GC and chronic atrophic gastritis (CAG) tissues and its loss contributed to GC tumorigenesis partly by activating STAT3 signaling. In primary human GC tissues, GRIM-19 was frequently depressed or lost and this loss correlated with advanced clinical stage, lymph node metastasis, H. pylori infection and poor overall survival of GC patients. In CAG tissues, GRIM-19 was progressively decreased along with its malignant transformation. Functionally, we indentified an oncogenic role of GRIM-19 loss in promoting GC tumorigenesis. Ectopic GRIM-19 expression suppressed GC tumor formation in vitro and in vivo by inducing cell cycle arrest and apoptosis. Moreover, we revealed that GRIM-19 inhibited STAT3 transcriptional activation and its downstream targets by reducing STAT3 nuclear distribution. Conversely, knockdown of GRIM-19 induced aberrant STAT3 activation and accelerated GC cell growth in vitro and in vivo, and this could be partly attenuated by the blockage of STAT3 activation. In addition, we observed subcellular redistributions of GRIM-19 characterized by peri-nuclear aggregates, non-mitochondria cytoplasmic distribution and nuclear invasion, which should be responsible for reduced STAT3 nuclear distribution. Our studies suggest that mitochondrial GRIM-19 could not only serve as an valuable prognostic biomarker for GC development, but also as a potential therapeutic target for STAT3-dependent carcinogenesis of GC.

Decreased expression of GRIM-19 by DNA hypermethylation promotes aerobic glycolysis and cell proliferation in head and neck squamous cell carcinoma.

To identify novel tumor suppressor genes that are down-regulated by promoter hypermethylation in head and neck squamous cell carcinoma (HNSCC), genome-wide methylation profiling was performed using a methylated DNA immunoprecipitation (MeDIP) array in HNSCC and normal mucosa tissue samples. Promoter hypermethylation of the candidate gene, gene associated with retinoid-interferon induced mortality-19 (GRIM-19), was confirmed in HNSCC cell lines. Multivariate regression analysis determined that GRIM-19 hypermethylation was an independent significant factor for HNSCC diagnosis (OR:125.562; P < 0.001). HNSCC patients with lower ratio of GRIM-19/ACTB hypermethylation had increased overall and disease free survival. Furthermore, the optimal cutoff provided 90% sensitivity and 77% specificity of GRIM-19 hypermethylation as a diagnostic marker for HNSCC. Ectopic expression of GRIM-19 in HNSCC cells led to increased oxygen consumption, reduced glycolysis and decreased cell proliferation. HNSCC cells ectopically expressing GRIM-19 displayed increased p53 activity as well as decreased Stat3 and HIF-1α activities. Moreover, GRIM-19 knockdown not only resulted in decreased oxygen consumption and increased aerobic glycolysis but also promoted cell proliferation and tumorigenic capacity in HNSCC cells. Our data indicate that decreased GRIM-19 expression due to promoter hypermethylation may be important in head and neck carcinogenesis by promoting cell proliferation and regulating metabolic activity.

Expression of gene associated with retinoid-interferon-induced mortality-19 in preimplantation embryo of mice.

To study the expression of gene associated with retinoid-interferon-induced mortality-19(GRIM-19) in preimplantation embryo of mice and explore its role in embryonic development. The protein and mRNA expressions of GRIM-19 in 2-cell, 4-cell, 8-cell, morula, and blastocyst phases of mice preimplantation embryo were detected by Western blot analysis and Real-time polymerase chain reaction (PCR). GRIM-19 was continuously expressed in every stage of preimplantation embryo of mice. Western blot analysis and Real-time PCR demonstrated a gradual increase of GRIM-19 expression from 2-cell, which reached a peak in 8-cell phase and then decreased progressively. The expression of GRIM-19 in mouse preimplantation embryos changes as at different developmental phases. GRIM-19 may play an important role during embryonic development.

Expression and clinical significance of GRIM-19 in lung cancer

We investigated the expression of gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) in lung cancer, a recently discovered cell death regulatory gene. Over-expression of GRIM-19 potentially suppresses proliferation and promotes tumor cell apoptosis. However, the expression of GRIM-19 in human lung cancer has not yet been thoroughly investigated. All of the specimens were obtained using CT-guided lung puncture or bronchial biopsy. The expression of GRIM-19 was investigated using immunohistochemistry. The expression level of GRIM-19 was significantly different between lung cancer and lung inflammation. A relatively lower GRIM-19 expression level was also found in small cell lung carcinomas compared to squamous cell carcinoma and adenocarcinoma. No significant difference between GRIM-19 expression in squamous cell carcinoma and adenocarcinoma was determined. Downregulation of GRIM-19 was found in non-small cell lung carcinomas stages III-IV compared to stages I-II, indicating a negative correlation between the expression level of GRIM-19 and the stage of the primary lesion (T). Furthermore, we found GRIM-19 to be primarily located in the cytoplasm in lung inflammation tissues, but located in the nucleus in lung cancer tissues. GRIM-19 expression occurs as an early phenomenon in the pathogenesis of lung cancer. Our study found that GRIM-19 expression in lung cancer is significantly lower compared to lung inflammation, exhibits a relationship with the histological type and clinical stage of lung cancer, and is a suitable target for the development of new lung cancer therapies.

Downregulation of GRIM-19 is associated with hyperactivation of p-STAT3 in hepatocellular carcinoma

Accumulating evidence has implicated that constitutive activation of signal transducer and activator of transcription protein 3 (STAT3) may be a major oncogenic factor involved in hepatocellular carcinoma (HCC) development. Gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) has been shown to be a tumor suppressor associated with growth control and suppression of STAT3 activity. The downregulation of GRIM-19 expression has been shown in a number of human tumor types, and it has been correlated with hyperactivation of STAT3. However, the role of GRIM-19 in the pathogenesis of HCC has not been evaluated. The aim of our study was to evaluate GRIM-19 expression levels and investigate their correlation with phosphorylated STAT3 (p-STAT3) levels in HCC. GRIM-19 and p-STAT3 expression levels were analyzed in HCC and adjacent nontumorous liver tissues (ANLT) by immunohistochemistry, western blot analysis, and RT-PCR. GRIM-19 protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only located in the cytoplasm in HCC tissues. HCC samples exhibited low levels of GRIM-19 and moderate to high levels of p-STAT3 expression. In contrast, ANLT was characterized by high levels of GRIM-19 and low levels of p-STAT3 expression. Downregulation of GRIM-19 was closely correlated with increased histological grade in HCC. GRIM-19 expression is closely correlated with histological grading and p-STAT3 in HCC. Thus, the potential role of GRIM-19 in HCC development may be through these correlations.

Down-Regulation of GRIM-19 Expression Is Associated With Hyperactivation of STAT3-Induced Gene Expression and Tumor Growth in Human Cervical Cancers

Cervical cancer is the most common malignant disease responsible for the deaths of a large number of women in the developing world. Although certain strains of human papillomavirus (HPV) have been identified as the cause of this disease, events that lead to formation of malignant tumors are not fully clear. STAT3 is a major oncogenic transcription factor involved in the development and progression of a number of human tumors. However, the mechanisms that result in loss of control over STAT3 activity are not understood. Gene associated with Retinoid-Interferon-induced Mortality-19 (GRIM-19) is a tumor-suppressive protein identified using a genetic technique in the interferon/retinoid-induced cell death pathway. Here, we show that reduction in GRIM-19 protein levels occur in a number of primary human cervical cancers. Consequently, these tumors tend to express a high basal level of STAT3 and its downstream target genes. More importantly, using a surrogate model, we show that restoration of GRIM-19 levels reestablishes the control over STAT3-dependent gene expression and tumor growth in vivo. GRIM-19 suppressed the expression of tumor invasion- and angiogenesis-associated factors to limit tumor growth. This study identifies another major novel molecular pathway inactivated during the development of human cervical cancer.

Point mutation in GRIM-19: a new genetic lesion in Hurthle cell thyroid carcinomas

Point mutation in GRIM-19: a new genetic lesion in Hurthle cell thyroid carcinomas