ABSTRACT
Long noncoding RNAs (lncRNAs) play a critical role in the regulation of atherosclerosis. Here, we investigated the role of the lncRNA growth arrest-specific 5 (lncR-GAS5) in atherogenesis. We found that the enforced expression of lncR-GAS5 contributed to the development of atherosclerosis, which presented as increased plaque size and reduced collagen content. Moreover, impaired autophagy was observed, as shown by a decreased LC3II/LC3I protein ratio and an elevated P62 level in lncR-GAS5-overexpressing human aortic endothelial cells. By contrast, lncR-GAS5 knockdown promoted autophagy. Moreover, serine/arginine-rich splicing factor 10 (SRSF10) knockdown increased the LC3II/LC3I ratio and decreased the P62 level, thus enhancing the formation of autophagic vacuoles, autolysosomes, and autophagosomes. Mechanistically, lncR-GAS5 regulated the downstream splicing factor SRSF10 to impair autophagy in the endothelium, which was reversed by the knockdown of SRSF10. Further results revealed that overexpression of the lncR-GAS5-targeted gene miR-193-5p promoted autophagy and autophagic vacuole accumulation by repressing its direct target gene, SRSF10. Notably, miR-193-5p overexpression decreased plaque size and increased collagen content. Altogether, these findings demonstrate that lncR-GAS5 partially contributes to atherogenesis and plaque instability by impairing endothelial autophagy. In conclusion, lncR-GAS5 overexpression arrested endothelial autophagy through the miR-193-5p/SRSF10 signaling pathway. Thus, miR-193-5p/SRSF10 may serve as a novel treatment target for atherosclerosis.
Subject(s)
Humans , Atherosclerosis/genetics , Autophagy/genetics , Cell Cycle Proteins/metabolism , Endothelial Cells/metabolism , Endothelium/metabolism , MicroRNAs/metabolism , Repressor Proteins/metabolism , RNA Splicing Factors , Serine-Arginine Splicing Factors/genetics , RNA, Long Noncoding/metabolismABSTRACT
@#Objective To investigate the effects of long non-coding RNA(LncRNA) growth arrest specific transcript 5(GAS5) negatively regulating nucleophosmin 1(NPM1) on cisplatin(DDP) resistance of gastric cancer cells.Methods The normal human gastric mucosa cell line GES-1 and human gastric cancer cell lines BG3-823,MGC-803 and AGS were selected as the research objects,of which the level of LncRNA GAS5 in each cell was measured by qRT-PCR.The drug resistance of AGS cells to DDP(AGS/DDP) was induced,and the experiment was divided into control group,empty plasmid group(BC group),GAS5 nonsense interference group(pLJM-GAS5 NC group) and GAS5 overexpression group(pLJM-GAS5 group).MTT method was used to determine the effect of DDP on the proliferation of AGS and AGS/DDP cells;and the levels of NPM1,multidrug resistance 1(MDR1),excision repair cross complementation group 1(ERCC1),multidrug resistance-associated protein 1(MRP1) and N-cadherin in AGS and AGS/DDP cells were measured by Western blot.Results Compared with the normal gastric mucosa GES-1 cells,the level of LncRNA GAS5 in BG3-823 and AGS cells decreased significantly,and among them,the level of LncRNA GAS5 in AGS cells was the lowest,so AGS cells were used for the follow-up experiments.Compared with the control group,the level of LncRNA GAS5 in AGS cells of BC group and pLJM-GAS5 NC group decreased significantly,while the levels of NPM1,MDRl,ERCC1,MRP1 and N-cadherin increased significantly;compared with BC group and pLJM-GAS5 NC group,the level of LncRNA GAS5 in AGS/DDP cells of pLJM-GAS5 group increased significantly,while the levels of NPM1,MDR1,ERCC1,MRP1 and N-cadherin decreased significantly;after treatment with DDP of the same concentration(except 0 μmol/L),compared with the control group,the inhibition rate of AGS/DDP cell proliferation in BC group and pLJM-GAS5 NC group decreased significantly;compared with BC group and pLJM-GAS5 NC group,the inhibition rate of AGS/DDP cell proliferation in pLJM-GAS5group was significantly higher.The semi inhibitory concentration(IC_(50)) of DDP on AGS/DDP cells in pLJM-GAS5 group for 48 h was(65.38±5.04) μmol/L,which was significantly lower than(120.74±4.17) μmol/L and(120.24±4.29) μmol/L in BC group and pLJM-GAS5 NC group.Conclusion Up-regulating the level of LncRNA GAS5 in AGS/DDP cells can reverse the drug resistance of AGS/DDP cells,which may be related to the down-regulation of NPM1expression
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Objective:To investigate the role and mechanism of long non-coding RNA GAS5 in the targeted regulation of miR-29, miR-96, and miR-208 in promoting insulin secretion of pancreatic β-cells.Methods:Q-PCR was used to detect the expression of miR-29, miR-96, and miR-208 in sera of 122 healthy subjects and 88 type 2 diabetic patients; and so of long non-coding RNA GAS5 and miR-208 in the rat islet cell tumor strain ins-1832/13. Effects of silencing and overexpressing GAS5 on insulin secretion of islet β-cells by lentiviral vector construction were observed. Bioinformatics was used to predict that GAS5 had complementary binding sites with miR-29, miR-96, and miR-208, which was further verified by luciferase reporting system. GAS5 siRNA was co-transfected with miR-29, miR-96, and miR-208 inhibitors, and the effect of GAS5 on insulin receptor (INSR), insulin receptor substrate (irs-1) and PI3K levels was detected by the above method, so as to reveal the effect of GAS5 on insulin secretion in islet cells.Results:The expression of GAS5 in serum of T2DM patients was lower than that of healthy control group ( t=4.632, P<0.01), and expression of miR-29, miR-96, and miR-208 were higher than those of healthy control group ( t were 7.832, 9.164, and 12.359, all P<0.01). GAS5 level was negatively correlated with miR-29, miR-96, and miR-208 ( r were -0.50, -0.47, and -0.70, respectively). GAS5 expression was significantly decreased in serum of type 2 diabetic patients compared with that of in healthy subjects. Overexpression of GAS5 by lentivirus resulted in increased glucose-stimulated insulin secretion and increased insulin concentration compared to negative control. In contrast, knockdown of GAS5 led to significant reduction of glucose-stimulated insulin secretion and insulin concentration. GAS5 levels were negatively correlated with miR-29, miR-96, and miR-208 in serum samples of type-2 diabetes patients. GAS5 can negatively regulate the expression of miR-96, miR-29, and miR-208. By bioinformatics tools, we screened miR-29, miR-96 and miR-208 as targets of GAS5, and their interaction was validated with dual luciferase reporter gene assay. shGAS5 significantly decreased the expressions of INSR, IRS-1 and PI3K( P were 0.022, 0.038, and 0.009), while overexpressed GAS5 significantly upregulated the expressions of INSR, IRS-1 and PI3K at both mRNA and protein levels( P were 0.024, 0.045, and 0.016). Conclusion:GAS5 could stimulate insulin secretion of islet cell through its inhibitry regulationor of expressions of miR-29, miR-96, and miR-208, therely up-regulating INSR, IRS-1, and PI3K that may be the potential targets of these miRNAs, and stimulate insulin secretion of islet cells.
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Objective@#To investigate the characteristics of polymorphism distribution at the functional insertion/deletion locus rs145204276I/D in the promoter region of LncRNA GAS5 (growth-arrest specific transcript 5) gene in population of Guangxi district, and analyze the differences of polymorphism distribution of rs145204276I/D in the populations between Guangxi and other regions. @*Methods@#SNPscan high-throughput sequencing technique was used to detect rs145204276I/D locus in genotype of GAS5 gene of 289 subjects from Guangxi district, and the distribution frequencies of genotypes and alleles between different genders were analyzed. The differences of polymorphism distribution were compared with those in the database from the population of European (EUR), Japanese in Tokyo (JPT), South Asian (SAS), Admixed American (AMR), African (AFR), Chinese Han in Beijing (CHB), Nanjing, Jilin, Chongqing and Kunming which were published by 1000 genome project or reported in literatures. @*Results@#The frequencies of I/I, I/D and D/D genotypes of rs145204276I/D in GAS5 were 48.4%, 43.6% and 8.0%, respectively. The frequencies of I and D alleles were 70.2% and 29.8%, respectively. No significant difference of genotype and allele frequencies of rs145204276I/D was observed between different genders in Guangxi population ( P ＞0.05). The genotype and allele frequencies of rs145204276I/D in Guangxi population were significantly different from JPT, EUR, AFR, SAS and AMR populations ( P ＜0.05), but were not significantly different from those of Chinese Han population in Beijing, Nanjing, Jilin, Chongqing and Kunming ( P ＞0.05). @*Conclusion@#The distribution of LncRNA GAS5 gene rs145204276I/D polymorphism in Guangxi population was not different between men and women, and the polymorphism of LncRNA GAS5 gene was different from those of other regions in the world.
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Objective To explore the effect of lncRNA of growth arrest-specific 5 (lncRNA GAS5) on the radiosensitivity of colon cancer cells by targeting miR-223.Methods The expressions of lncRNA GAS5 in a few of colon cancer cell lines were detected by real-time quantitative PCR (qPCR).The cell lines with low expression level of lncRNA GAS5 were selected for subsequent study.The effect of overexpression of lncRNA GAS5 on the radiosensitivity of colon cancer SW480 cells was detected by cell cloning experiments.The target gene miR-223 of lncRNA GAS5 was predicted and validated by the bioinformatics database starBase and dual luciferase reporter assays.qPCR was used to detect the expression of miR-223 in various colon cancer cell lines and the influence of lncRNA GAS5 overexpression on the expression of miR-223 in SW480 cells.Results Compared with normal human colonic epithelial cells (NCM460),the expressions of lncRNA GAS5 in the colon cancer SW480,LOVO,HT-29 and SW620 cell lines were significantly lower(t =15.25,8.69,14.42,11.62,P ＜ 0.05),with the lowest level in SW480 cells.Both overexpression of lncRNA GAS5 and down-regulation of miR-223 significantly increased the radiosensitivity of colon cancer cells by decreasing cell survival fraction (at 8 Gy,lncRNA GAS5,t =13.51,P ＜ 0.05;anti-miR-223,t =14.93,P ＜ 0.05)and promoting apoptosis (lncRNA GAS5,t =8.30,P ＜ 0.05;anti-miR-223,t =7.32,P ＜ 0.05).Bioinformatics analysis showed that the 3'sequence of lncRNA GAS5 contained the binding sites with miR-223.After overexpression or downregulation of lncRNA GAS5,the expression of miR-223 was enhanced or reduced.Conclusions The lncRNA GAS5 promotes the apoptosis of colon cancer cells and inhibits its survival by targeting miR-223 expression,thereby increases the radiosensitivity of colon cancer cells.
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Objective To study the expression and diagnostic values of long non-coding RNA(IncRNA)metastasis-associated lung adenocarcinoma transcript 1 (Malat-1),p21 and growth arrest-specific 5 (GAS5)in colorectal cancer tissue.Methods Coloreetal cancer of early stage and advanced stage(including paracareinoma tissue),precancerous lesion,benign tumor and noncancer lesion groups were collected,33 cases in each group.The levels of Malat-1,p21 and GAS5 in colorectal tissue of the 6 groups were detected by reverse transcription-polymerase chain reaction(RT-PCR).Serum carcinoembryonic antigen(CEA)and carbohydrate antigen19-9(CA19-9)were detected by ELISA.Results RT-PCR showed the levels of Malat-1,p21 and GAS5 were significantly higher in colorectal cancer of early and advanced stage and precancerous lesion than in the paracarcinoma lesion,benign tumor and non-cancer lesion(P＜0.05).Malat-1,p21 and GAS5 were the highest in the advanced stage group(P＜0.05).The levels of CEA and CA19-9 in non-cancer lesion were the least(P＜0.05).The AUC of ROC analysis operator characteristic curve(ROC)of Malat-1,p21 and GAS5 was comparable(P＞0.05).Pathological changes showed the pathological changes in colorectal cancer of early stage,advanced stage and precancerous lesion were more significant than those in the paracarcinoma lesion,benign tumor and non-cancer lesion (P＜ 0.05),which were the most significant in the advanced stage group.Conclusion The levels of Malat-1,p21 and GAS5.in colorectal cancer tissues could be good indicators for colorectal cancer diagnosis.
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Objective:To detect the expression of long non-coding RNA (Long non-coding RNA,IncRNA) maternally expressed gene 3 (maternally expressed gene 3,MEG3),specific growth inhibitor 5 (Growth arrest-specific 5,GAS5) and to analyze the correlation between GAS5 and cyclin P21,E2F1,so as to investigate the mechanism of GAS5.Methods:From December 2014 to December 2015,63 consecutive Patients with colorectal cancer admitted to Qingdao Municipal Hospital for surgical treatment were included into our study.We collect colorectal cancer tissues and paired normal tissues.We tested the relative expression of MEG3,GAS5 genes by real-time quantitative PCR (RT-PCR),and Western blot was used to detect the expression of P21 and E2F1 in colorectal cancer and to evaluate their correlations with GAS5.Results:The expression of MEG3 in cancer (7.76 ± 1.38) was lower than that in normal tissues(9.52 ± 1.31)P＜0.052.The expression of GAS5 was decreased in cancer tissues (3.98 ± 1.15) relative to the normal (6.21 ± 1.33)P＜0.05.3.It showed that there were positive relationships between P21 and GAS5(r=0.410,P=0.001),and there were negative relationships between E2F1 and GAS5(r=-0.366,P=0.003).Conclusions:MEG3 and GAS5 were decreased in colorectal cancer,suggesting that they play inhibiting effects on the cancer.P21 and E2F1 are important target spots that GAS5 give play to the role of tumor suppressor.
ABSTRACT
[Objective]Dysregulated long noncoding RNAs(lncRNAs)have been found involved in human diseases,including cancers. Long non-coding RNA growth arrest-specific 5(GAS5)was reported to be dysregulated in different types of cancers. Howev-er,the role of GAS5 in ovarian cancer remains elusive.[Methods]In the present study,the expression of GAS5 was detected in 108 ovarian cancer tissues and compared adjacent normal tissues by quantitative real-time PCR(qRT-PCR).[Results]The results showed that the expression levels of lncRNA GAS5 were significantly decreased in cancer tissues(P=0.0004),and it was negatively correlated with tumor size(5 cm,P<0.0001),invasion depth(T1-T2 vs. T3-T4,P=0.0021),and tumor grade(Ⅰ~Ⅱgrades vsⅢ~Ⅳgrades,P=0.0086)in ovarian cancer patients. Kaplan-Meier analysis demonstrated that decreased lncRNA GAS5 expression contributed to poor disease-free survival and overall survival.[Conclusion]In conclusion ,our study suggested that decreased lncRNA GAS5 expression may beidentified as a potential poor prognostic biomarker in ovarian cancer.