Curcumin Inhibits Retinoblastoma Cell Proliferation by miR-26a Targeting the Tumor Suppressor Gene Rb1 in Y79 Cells

2020 ◽  
Vol 9 ◽  
pp. 63-71
Author(s):  
Jing Bai ◽  
Author(s):  
Liqin SHAO ◽  
Zhangxing SHENG ◽  
Yuefeng ZHU ◽  
Jianchao LI ◽  
Rufa MENG

Background: Effect of miR-215 on the expression of tumor suppressor gene retinoblastoma (Rb)1 in Rb cell lines was investigated. Methods: A total of 128 patients were selected. The expression of miR‑215 in cancer and adjacent healthy tissues of the 128 patients was detected by reverse transcription-quantitative PCR (RT-qPCR). HXO‑Rb44 and Y79 cell lines were transfected with miR‑215 analogs or miR‑215 inhibitors, and the expression of Rb1 protein in the cell lines was detected by western blotting. Results: The expression of miR-215 in the adjacent healthy tissues of patients was significantly lower than that in cancer tissues (P<0.001). The expression of miR-215 in Y79 and HXO-Rb44 cells was significantly higher than that in APRE-19 cells (P<0.001). The expression of miR-215 in HXO-Rb44 cells was significantly higher than that in Y79 cells (P<0.001). The expression of miR-215 was statistically different from the degree of differentiation and nerve infiltration (P<0.05). The expression of Rb1 in cancer tissues was significantly lower than that in adjacent tissues (P<0.001), the expression of APRE-19 was significantly higher than that in Y79 and HXO-Rb44 cells (P<0.001), and the expression of Rb1 in HXO-Rb44 cells was significantly higher than that in Y79 cells (P<0.05). There was a negative correlation between miR-215 and Rb1 in the tissues of patients, and Rb1 expression decreased with the increase of miR-215 (r=-0.576, P<0.001). Conclusion: miR‑215 is highly expressed in Rb cell lines, and is related to the clinicopathological features of this disease.


2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Yeqing Dong ◽  
Yongsheng Huang ◽  
Chengyan Fan ◽  
Liang Wang ◽  
Ran Zhang ◽  
...  

AbstractPheochromocytoma/paraganglioma (PPGL) is an endocrine tumor of the chromaffin cells in the adrenal medulla or the paraganglia. Currently, about 70% of PPGLs can be explained by germline or somatic mutations in several broadly expressed susceptibility genes including RET, VHL, and SDHB, while for the remaining, mainly sporadic cases, the pathogenesis is still unclear. Even for known susceptible genes, how mutations in these mostly ubiquitous genes result in tissue-specific pathogenesis remains unanswered, and why RET-mutated tumors almost always occur in the adrenal while SDHB-mutated tumors mostly occur extra-adrenal remains a mystery. By analyzing 22 sporadic PPGLs using SNP 6.0 genotyping arrays combined with expression profiling of 4 normal and 4 tumor tissues, we identified GIPC2, a gene located at 1p31.1 with preferential expression in adrenal and inducible by adrenal glucocorticoid, as a novel putative tumor suppressor gene for PPGLs. Copy number deletion and GIPC2 promoter hypermethylation but not GIPC2 mutation, accompanied with reduced GIPC2 expression, were observed in 39 of 55 PPGLs in our cohort. Examination of a published expression database consisting of 188 PPGLs found little GIPC2 expression in Cluster 1A (SDHx-associated) and Cluster 2A (NF1/RET-associated) tumors, but less pronounced reduction of GIPC2 expression in Cluster 1B (VHL-associated) and Cluster 2B/2C tumors. GIPC2 induced p27, suppressed MAPK/ERK and HIF-1ɑ pathways as well as cancer cell proliferation. Overexpressing GIPC2 in PC12 cells inhibited tumor growth in nude mice. We found GIPC2 interacted with the nucleoprotein NONO and both proteins regulated p27 transcription through the same GGCC box on p27 promoter. Significantly, low expression of both GIPC2 and p27 was associated with shorter disease-free survival time of PPGLs patients in the TCGA database. We found that PPGL-causing mutations in RET and in SDHB could lead to primary rat adrenal chromaffin cell proliferation, ERK activation, and p27 downregulation, all requiring downregulating GIPC2. Notably, the RET-mutant effect required the presence of dexamethasone while the SDHB-mutant effect required its absence, providing a plausible explanation for the tumor location preference. In contrast, the PPGL-predisposing VHL mutations had no effect on proliferation and GIPC2 expression but caused p53 downregulation and reduced apoptosis in chromaffin cells compared with wild-type VHL. Thus, our study raises the importance of cortical hormone in PPGL development, and GIPC2 as a novel tumor suppressor provides a unified molecular mechanism for the tumorigenesis of both sporadic and hereditary tumors of Clusters 1A and 2A concerning SDHB and RET, but not tumors of Cluster 1B concerning VHL and other clusters.


Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1279-1279
Author(s):  
Sanghoon Lee ◽  
Changhong Yin ◽  
Timmy O'Connell ◽  
Janet Ayello ◽  
Carmella van de Ven ◽  
...  

Abstract Background Pediatric Burkitt Lymphoma (BL) is the most common histological subtype of Non Hodgkin Lymphoma (NHL) in children and adolescents (Cairo et al, Blood, 2007; Miles/Cairo, BJHaem, 2012). We previously identified, in a subset analysis, that children with BL and a 13q deletion, particularly 13q14.3, had significantly poorer outcome and inferior overall survival (OS) despite aggressive short, intensive multi-agent chemotherapy (Poirel/Cairo et al, Leukemia, 2009; Nelson/Cairo/Sanger et al, BJHaem, 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is a BL classifier gene on 13q14.3 region (Dave et al, NEJM, 2006). DLEU1 has been implicated in regulating programmed cell death in patients by inducing TUBB2C and UBR1 expression. Sequence-specific Transcription Activator-Like Effector Nucleases (TALENs) technologies have been developed for precision targeted genome editing in in vitro and in vivo experiments with high efficiency as a new experimental and therapeutic tool (Sander et al, Nat Bio, 2011). We have previously reported TALENs mediated transient downregulation of DLEU1 in Raji cells (Figure 1), and demonstrated a decrease in the spontaneous apoptotic rate and an increase in cell proliferation in transiently DLEU1 down-regulated Raji cells (Lee/Cairo et al, ASH, 2012). Objectives We hypothesize that DLEU1 may act as a tumor suppressor gene and therefore investigated whether the silencing of DLEU1 results in changes in proliferation, programmed cell death and associated signaling pathways induced by Rituximab and Cyclophosphamide alone and in combination in TALEN-mediated DLEU1 knockout Raji BL cells. Methods A stable, single DLEU1 knockout clone (DLEU1 KO) was isolated from Raji cells transfected with DLEU1 TALENs (Lee/Cairo et al, ASH, 2012). Genomic DNA and total RNA were extracted using a DNA extraction kit (Promega) and Trizol (Invitrogen) for verification of endogenous DLEU1 gene disruption by TALENs. qRT-PCR was performed by CFX96 (Bio-rad) and the protein level was measured by western blotting. DLEU1 KO cells were plated (1x104 cells) into 48 or 96 well plates and then treated with Rituximab (0, 1, 10 and 100ug/ml) and/or Cyclophosphamide (0, 1, 5 and 10mM) and cell proliferation and apoptosis were measured by MTS (Promega) and Caspase 3/7 (Promega) assay. Statistical significance was determined by a one tailed paired Student t-test. Results DLEU1 KO (mono-allelic deletion) in Raji (BL) exhibited significantly reduced DLEU1 mRNA expression compared to mock control cells (83% reduction, p<0.006). There was significantly reduced expression of c-myc, TUBB2C and UBR1 mRNA (85%, p<0.001; 60%, p<0.04; 25%, p<0.006), respectively in DLEU1 KO compared to mock control cells. DLEU1-KO showed significant reduction of Caspase 3/7 activities (25% at 24hrs, p<0.007; 33% at 48hrs, p<0.03) and significant increase in cell proliferation (20% at 24hrs, p<0.04; 25% at 48hrs, p<0.03). DLEU1-KO showed a significant increase in expression of anti-apoptotic genes, Bcl-2, Bcl-xL and Mcl-1 mRNA (14%, p<0.03; 30%, p<0.04; 17%, p<0.04), respectively and a significantly a decrease in mRNA expression of pro-apoptotic genes, Bax and Bad (18%, p<0.04; 14%, p<0.04), respectively. There was a significant increase in phosphorylation of IkBa (2.6 fold, p<0.007) and Akt (1.5 fold, p<0.05) in DLEU1 KO. There was a significant reduction in Caspase-dependent apoptosis with RTX (15% reduction, p<0.05), CTX alone (24% reduction, p<0.03), and in combination (35% reduction, p<0.03) in DLEU1-KO compared to mock control. A significant increase in cell proliferation was observed with RTX (10% increase, p<0.04), CTX alone (9.4% increase, p<0.05), and combination (7.7% increase, p<0.02) in DLEU1-KO. RTX treated DLEU1-KO showed a significant increase in expression in Bcl-2 and Bcl-xL mRNA (1.3 fold, p<0.05 and 2.0 fold, p<0.02), respectively and a significant decrease in mRNA expression of Bax (1.2 fold, p<0.03). Conclusions We demonstrate that 1) TALENs mediated DLEU1 gene knockout resulted in downregulation of DLEU1 mRNA and consequently, inhibition of BL apoptosis and increase in cell proliferation, through several mechanism(s), and 2) the silencing of DLEU1 resulted in decrease in apoptosis in RTX, CTX alone and in combination treatment. We hypothesize that deletion of DLEU1 in childhood BL may result in chemotherapy resistance secondarily to a loss of a tumor suppressor gene. Disclosures: No relevant conflicts of interest to declare.


2006 ◽  
Vol 17 (8) ◽  
pp. 3534-3542 ◽  
Author(s):  
Minghua Wu ◽  
Chen Huang ◽  
Kai Gan ◽  
He Huang ◽  
Qiong Chen ◽  
...  

We have previously reported that the LRRC4 gene, which contains a conserved leucine-rich repeat (LRR) cassette and an immunoglobulin (Ig) IgC2 domain, is associated with glioma suppression both in vitro and in vivo. The present study provides evidence that the conspicuous absence of LRRC4 in high-grade gliomas directly contributes to the increasing tumor grade. The loss of LRRC4 in U251 cells is caused by the loss of homozygosity at chromosome 7q32-ter. It was also found that LRRC4 requires a functional LRR cassette domain to suppress U251 cell proliferation. In the LRR cassette domain, the third LRR motif of the core LRR is found to be indispensable for the function of LRRC4. The inhibitory effect of LRRC4 is accompanied by a decrease in the expression of pERK, pAkt, pNF-κBp65, signal transducer and activator of transcription protein-3 (STAT3), and mutant p53, and an increase in the expression of c-Jun NH2-terminal kinase (JNK)2 and p-c-Jun, suggesting that LRRC4 plays a major role in suppressing U251 cell proliferation by regulating the extracellular signal-regulated kinase (ERK)/Akt/NF-κBp65, STAT3, and JNK2/c-Jun pathways. In conclusion, LRRC4 may act as a novel candidate of tumor suppressor gene. Therefore, the loss of LRRC4 function may be an important event in the progression of gliomas.


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