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Objective:To explore the role of N 6-methyladenosine (m6A) and its regulator METTL3 in the non-coding RNA of endometrial cancer.Methods:The expression levels of m6A and METTL3 were quantified in 20 paired carcinoma and adjacent clinical tissue samples from patients at from Jul. 2016 to Dec. 2020. HEC-1-A cell lines were constructed with METTL3 overexpression and knockdown. Western blot was used to detect the phosphorylation levels of key molecules in METTL3 and Akt/mTOR. The quantitative detection of mRNA levels were used qRT-PCR. The binding level of m6A to its receptor DGCR8 was determined by RNA immunoprecipitation.Results:The results of the m6A RNA methylation quantification kit showed that m6A (1.0±0.15) vs (1.7±0.34) ( P<0.01) and METTL3 levels were elevated in endometrial cancer cells, and METTL3 (1.0±0.13) vs (2.5±0.45) ( P<0.05) levels were elevated in endometrial cancer cells. Western blot and qRT-PCR detection of miR-17-92 cell clusters overexpressing METTL3, METTL3 overexpression significantly increased m6A modification on pri-miR-17-92 ( P<0.05) . Phosphorylation levels of AKT/mTOR pathway-related proteins were upregulated. In addition, RIP test results indicated that the binding of DGCR8 to pri-miR-17-92 was significantly facilitated. Conclusion:METTL3 modification of m6A facilitates the processing of pri-miR-1792 into the miR-17-92 clusters via m6A/DGCR8-dependent mechanism, which in turn activated the AKT/mTOR pathway.
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Objective To investigate the distribution of the polymorphisms rs1491033A/C and rs1813389A/G in the promoter region of the miR-17-92 gene cluster in a normal population of Guangxi and compare this distribution with that in different ethnic groups. Methods We detected the genotypes of rs1491033A/C and rs1813389A/G of 275 people from Guangxi using the SNaPshot technique and DNA sequencing. We then analyzed the genotype and allele frequency distribution differences among different genders and groups. Results Three genotypes,AA,AC,and CC,were found for rs1491033A/C with frequency distributions of 21. 1％,53. 1％,and 25. 8％, respectively. There were significant differences in genotype and allele frequencies of rs1491033A/C between the Guangxi population and those of Europe and Africa,as published in the International HapMap project (P < 0. 05). Three genotypes,AA,AG,and GG were observed for rs1813389A/G with frequency distributions of 40. 0％,50. 2％,and 9. 8％,respectively. There were significant differences in the genotype and allele frequencies of rs1813389A/G between the Guangxi population and those of Europe,Beijing,Japan,and Africa (P <0. 05). However,there were no significant differences in genotype or allele frequencies of the rs1491033A/C or rs1813389A/G polymorphisms between genders in the Guangxi population (P > 0. 05). Conclusion There are different distributions of the rs1491033A/C and rs1813389A/G polymorphisms in the miR-17-92 gene cluster in different races and regions.
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Objective:To investigate the distribution characteristics of polymorphisms of rs9515692C/T and rs1352743A/G in the promoter region of miR-17-92 gene cluster in Guangxi people and compare them with those of other ethnic groups and explore the association of its polymorphisms and lymphocytes.Methods:The rs9515692C/T and rs1352743A/G of miR-17-92 gene cluster were genotyped by using SNaPshot technique and DNA sequencing.Detection of the number of lymphocytes using flow cytometry.The differences of polymorphisms between groups were analyzed statistically.Results:No significant differences of genotype and allele frequency in the two SNPs was observed between different gender in the Guangxi population(P>0.05).However,there were significant differences in the distribution frequencies of genotype and allele of Europeans,Japanese and Africans in rs9515692C/T and rs1352743A/G (P<0.05).Conclusion:Polymorphisms of rs9515692C/T and rs1352743A/G are different in different people.In addition,rs9515692C/T polymorphism may be associated with the number of B cells.
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Background and purpose:miR-17-92 gene cluster overexpression has been observed in various cancers, such as lung cancer, liver cancer, gastric cancer and prostate cancer. In this study, we established the stable cell line overexpressingmiR-17-92 to explore the inlfuence ofmiR-17-92 on the migration, invasion abilities and cisplatin resistance of the prostate cancer DU145 cells.Methods:miR-17-92 overexpression vectors were constructed. DU145 cells were infected with the viral supernatants produced by Phoenix A packaging system. Real-time lfuorescent quanti-tative polymerase chain reaction (RTFQ-PCR) was conducted to detect the expression level of miR-17-92 in the cells. The migration and invasion abilities were measured by a real-time xCELLigence system. The scratch healing assay was carried out to investigate the migration abilities. The expression of integrin β1 was detected by Western blot, and the activities of matrix metalloprotein-2 (MMP-2) and matrix metalloprotein-9 (MMP-9) were measured by gelatin zymography experiment. The cell growth of the two cell lines after the treatment of cisplatin was detected by a real-time xCELLigence system. The mRNA expression ofERCC1 was measured by RTFQ-PCR. Western blot was conducted to investigate the protein expressions of ERCC1, ERK1/2 and pERK1/2.Results:DU145-miR-17-92 cells migrated faster than DU145-control cells during the 24 h continuous monitoring (P<0.01). The scratch healing assay indicated that DU145-miR-17-92 cells migrated from the edge towards the scratch center faster than DU145-control cells. DU145-miR-17-92 cells invaded through matrigel markedly faster than DU145-control cells (P<0.01). The protein expression level of integrin β1 and the MMP-9 activities in DU145-miR-17-92 cells were increased than those in DU145-control cells. After the treatment of cisplatin, DU145-miR-17-92 cells grew faster than DU145-control cells, presenting cisplatin resistance (P<0.01). The phosphorylation of ERK1/2 in DU145-miR-17-92 cells was constantly at a high level regard-less of the treatment of cisplatin. Compared with DU145-control cells, the expression of drug resistance-related gene ERCC1 was dramatically increased in DU145-miR-17-92 cells after the treatment of cisplatin.Conclusion:miR-17-92 overexpression increases the migration and invasion abilities of the prostate cancer DU145 cells, which is associated with the upregulated expression of integrin β1 and the increased activity of MMP-9. Besides,miR-17-92 overexpression enhances the cisplatin resistance of DU145, which is correlated with the increased phosphorylation level of ERK and the upregulated expression of ERCC1 at both the mRNA and protein levels.
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Objective@#To explore the effect and mechanism of over-expression of miR-17-92 gene cluster on the biological characteristics of prostate cancer cells.@*Methods@#DU145 cells were transfected with miR-17-92 gene expression plasmid and clones with stable ectopic miR-17-92 overexpression were established. The cell viabilities of DU145-17-92 and DU145-control cells were monitored by xCELLigence system. Cell proliferation and apoptosis were analyzed by Ki-67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Cell cycle was detected by flow cytometry. Expression levels of proteins involved in apoptosis and Akt pathway were determined by western blotting.@*Results@#xCELLigence RTCA array data showed that the growth rate of DU145-17-92 cells was significantly higher than that of DU145-control cells after 24 h of seeding (P<0.01). The Ki-67-positive rates of the DU145-control group at 24, 48 and 72 hours were (56.57±1.68)%, (85.48±0.26)% and (90.85±2.08)%, respectively. While the Ki-67 positive rates of the DU145-17-92 group at the desired time points were (73.64±0.68)%, (93.43±1.23)% and (97.36±0.86)%, respectively, with a statistically significant difference at 24 hours (P<0.01). The percentages of apoptotic cells of the DU145-control group at 24, 48 and 72 hours were (6.76±0.09)%, (14.51±0.86)% and (20.73±1.64)%, respectively, while the apoptotic percentages of the DU145-17-92 group were (1.86±0.15)%, (7.90±0.40)% and (4.92±0.48)%, respectively. The percentages of apoptotic cells of the DU145-control group at different time were significantly higher than those of DU145-17-92 group (P<0.01 for all). The result of western blotting showed that the protein expression levels of Bcl-2 interacting mediator of cell death (BIM) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) in DU145-control cells were 0.83±0.00 and 0.91±0.00, respectively, significantly higher than 0.16±0.00 and 0.13±0.00 of DU145-17-92 cells (both P<0.01). Overexpression of miR17-92 induced the phosphorylation of protein kinase B (Akt) at Ser473 while no appreciable effect on the phosphorylation of Akt at Thr308. The phosphorylated level of extracellular regulated protein kinases (ERK) in DU145-control cells was 0.21±0.01, significantly lower than 0.72±0.01 of DU145-17-92 cells (P<0.01).@*Conclusions@#Overexpression of miR-17-92 gene plays a pivotal role in growth, proliferation, apoptosis and cell cycle of DU145 cells through down-regulation of apoptotic protein BIM and tumor suppressor PTEN and activation of Akt and ERK signaling pathway.
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BACKGROUND: This study aimed to evaluate the expression of miR‑17‑92 host gene (MIR17HG), in gastric cancer and paired normal adjacent tissues for the 1st time. METHODS: Using quantitative real‑time‑polymerase chain reaction, the MIR17HG expression was assessed in 30 paired tumoral and nontumoral gastric tissue samples. RESULTS: Our results showed that this transcript was significantly underexpressed in gastric tumors compared with normal ones. Furthermore, there was an association between the expression levels of MIR17HG and gastric cancer grades and stages. Moreover, the expression level of MIR17HG was conversely associated with the size of tumoral specimens in early stages (stages I and II). We also observed an association between the presence of metastasis and lower expression of MIR17HG. CONCLUSION: Our results suggest that MIR17HG gene expression is dysregulated in gastric cancer in which it may indicate a tumor suppressive function of this miRNA cluster host gene in gastric cancer.
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Objective:To determine the expression of miR-17-92 cluster in osteosarcoma tissue samples and explore its associa-tion with clinical significance. Methods: Quantitative polymerase chain reactiom analysis was used to examine the expression of miR-17-92 cluster in osteosarcoma tissues. Normal bone tissues from 63 patients were matched, and the relationships between the ex-pression of miR-17-92 cluster and the clinicopathological features and prognosis of osteosarcoma were explored. Results:The relative expression of miR-17-92 cluster in osteosarcoma tissues was significantly higher than those in adjacent normal tissues (P<0.05). The high expression of miR-17-92 had a significant correlation with reduced survival (P=0.027). Conclusion:The expression of miR-17-92 cluster closely correlates with the occurrence and progress of osteosarcoma and may be used as an indicator for osteosarcoma prognosis.
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MicroRNAs (miRNAs) have gradually been recognized as regulators of embryonic development; however, relatively few miRNAs have been identified that regulate cardiac development. A series of recent papers have established an essential role for the miRNA-17-92 (miR-17-92) cluster of miRNAs in the development of the heart. Previous research has shown that the Friend of Gata-2 (FOG-2) is critical for cardiac development. To investigate the possibility that the miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation in mouse embryonic cardiomyocytes we initially used bioinformatics to analyze 3’ untranslated regions (3’UTR) of FOG-2 to predict the potential of miR-17-92 to target it. We used luciferase assays to demonstrate that miR-17-5p and miR-20a of miR-17-92 interact with the predicted target sites in the 3’UTR of FOG-2. Furthermore, RT-PCR and Western blot were used to demonstrate the post-transcriptional regulation of FOG-2 by miR-17-92 in embryonic cardiomyocytes from E12.5-day pregnant C57BL/6J mice. Finally, EdU cell assays together with the FOG-2 rescue strategy were employed to evaluate the effect of proliferation on embryonic cardiomyocytes. We first found that the miR-17-5p and miR-20a of miR-17-92 directly target the 3’UTR of FOG-2 and post-transcriptionally repress the expression of FOG-2. Moreover, our findings demonstrated that over-expression of miR-17-92 may inhibit cell proliferation via post-transcriptional repression of FOG-2 in embryonic cardiomyocytes. These results indicate that the miR-17-92 cluster regulates the expression of FOG-2 protein and suggest that the miR-17-92 cluster might play an important role in heart development.