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OBJECTIVES: Malignant melanoma (MM) is an invasive tumor that poses a threat to patient health. Circular RNAs (circRNAs) are important regulators of MM carcinogenesis. In this study, we investigated the expression characteristics and biological functions of, and mechanism underlying, circ_0119872 expression in MM. METHODS: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was employed to examine the circ_0119872, microRNA (miR)-582-3p, and E2F transcription factor 3 (E2F3) mRNA expression levels in MM tissues and cell lines. Western blotting was performed to quantify E2F3 protein expression. MM cells with circ_0119872 knockdown were established, and cell counting kit 8 (CCK-8) and transwell assays were utilized to examine the function of circ_0119872 and its effects on the malignant characteristics of MM cells. The MiRDB and TargetScan databases were used to predict the target genes of miR-582-3p. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was used to explore the biological functions of the target genes of miR-582-3p. Additionally, a dual-luciferase reporter gene experiment was performed to verify the targeting relationship between circ_0119872 and miR-582-3p as well as that between miR-582-3p and E2F3. RESULTS: Circ_0119872 was remarkably upregulated in MM tissues and cell lines. Circ_0119872 knockdown suppressed the cell proliferation and metastasis In addition, miR-582-3p was identified as a downstream target of circ_0119872. The target genes of miR-193a-3p are involved in melanogenesis and cancer-related signaling pathways. Mechanistically, circ_0119872 facilitated MM progression by adsorbing miR-582-3p and upregulating E2F3 expression. CONCLUSION: Circ_0119872 is an oncogenic circRNA that participates in the promotion of MM progression by regulating the miR-582-3p/E2F3 axis.
Subject(s)
Humans , MicroRNAs/genetics , Melanoma/genetics , Gene Expression Regulation, Neoplastic , Cell Line, Tumor , E2F Transcription FactorsABSTRACT
@#[Abstract] Objective: To investigate the expression of miR144-3p in bladder cancer tissues and cells and its effect on the proliferation and invasion of T24 cells. Methods: A total of 36 cases of bladder cancer tissue specimens and 10 cases of normal bladder epithelial tissue specimens were collected from Tangdu Hospital of Air Force Medical University during February 2018 and December 2018. In addition, bladder cancer T24 cell line and normal urothelial cell line SV-HUC-1 were also collected for this study. The levels of miR144-3p in bladder cancer tissues and cells were detected by qPCR methods. The miR-144-3p mimics and miR-NC were transfected into T24 cells by LipofectamineTM 2000, respectively. The proliferation, cell cycle distribution and invasion abilities were detected by MTT, Flow cytometry and Transwell chamber methods, respectively. TargetScan software was used to predict the binding site between miR-144-3p and E2F3 (E2F transcription factor 3); Dual luciferase reporter gene assay was used to verify the relationship between miR-144-3p and E2F3; and WB was used to detect the expression levels of miR-144-3p and E2F3 in cells. Results: The expression of miR-144-3p was downregulated in bladder cancer tissues and cells (all P<0.01). In addition, the expression level of miR-144-3p in muscular invasive bladder cancer tissues was significantly lower than that in non-muscular invasive bladder cancer tissues (P<0.05). Dual luciferase reporter gene assay confirmed that there was a targeted relationship between miR-144-3p and E2F3. Overexpression of miR-144-3p inhibited the proliferation and invasion of T24 cells (all P<0.01) and downregulated the expression of E2F3 (P<0.01); upregulation of E2F3 could reverse the inhibitory effect of miR-144-3p overexpression on proliferation and invasion of T24 cells. Conclusion: miR-144-3p has low expression level in bladder cancer tissues. It inhibits proliferation and invasion of bladder cancer cells by downregulating E2F3.
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Diabetic nephropathy (DN) has been identified as the major cause of end-stage renal disease (ESRD) in most developed countries. MicroRNA-770-5p depletion could repress high glucose (HG)-triggered apoptosis in podocytes, and downregulation of E2F transcription factor 3 (E2F3) could facilitate podocyte injury. Nevertheless, whether E2F3 is involved in miR-770-5p knockdown-mediated improvement of DN is still unclear. The expression levels of miR-770-5p and E2F3 were detected in HG-treated podocytes by RT-qPCR. The expression levels of E2F3, apoptosis-related proteins Bcl-2 related X protein (Bax), B-cell lymphoma-2 (Bcl-2), Bad, apoptotic peptidase activating factor 1 (APAF1), C-caspase3, C-caspase7, and C-caspase9 were detected by western blot assay. The effects of miR-770-5p and E2F3 on HG-treated podocytes proliferation and apoptosis were detected by CCK-8 and flow cytometry assays. The interaction between miR-770-5p and E2F3 was predicted by Targetscan, and then verified by the dual-luciferase reporter assay. MiR-770-5p was upregulated and E2F3 was downregulated in HG-treated podocytes. MiR-770-5p inhibited proliferation and promoted apoptosis and E2F3 promoted proliferation and suppressed apoptosis in HG-treated podocytes. E2F3 is a target gene of miR-770-5p and it partially abolished the effect of miR-770-5p in HG-triggered proliferation and apoptosis of podocytes. MiR-770-5p deficiency blocked HG-induced APAF1/caspase9 pathway via targeting E2F3 in podocytes. We firstly confirmed that E2F3 was a target of miR-770-5p in podocytes. These findings suggested that miR-770-5p expedited podocyte injury by targeting E2F3, and the miR-770-5p/E2F3 axis might represent a pathological mechanism of DN progression.
Subject(s)
Humans , MicroRNAs , Diabetes Mellitus , Diabetic Nephropathies , Podocytes , Apoptosis , E2F3 Transcription Factor , GlucoseABSTRACT
Cataract, an eye disease that threatens the health of millions of people, brings about severe economic burden for patients and society. MicroRNA (miR)-378a-5p and miR-630 were recognized as essential regulators in multiple cancers. However, the exact functions of miR-378a-5p and miR-630 in cataract are still unclear. The expression of miR-378a-5p, miR-630, and E2F transcription factor 3 (E2F3) in tissues and cells was measured by quantitative real-time polymerase chain reaction. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was used to evaluate cell viability. Flow cytometry was conducted to analyze cell apoptosis. The interaction between E2F3 and miR-378a-5p or miR-630 was confirmed by dual-luciferase reporter assay. The expression of proteins E2F3, B cell lymphoma (Bcl-2), Bcl-2 associated X (Bax), and cleaved caspase 3 was detected by western blot assay. The expression of miR-378a-5p and miR-630 was up-regulated whereas E2F3 was down-regulated in human cataract lens tissues compared with normal lens tissues. Depletion of miR-378a-5p or miR-630 enhanced proliferation and reduced apoptosis of human lens epithelial cells. Interestingly, up-regulation of E2F3 exhibited the same trend. Next, dual-luciferase reporter assay validated the interaction between E2F3 and miR-378a-5p or miR-630. The rescue experiments further revealed that E2F3 knockdown could recover miR-378a-5p, and miR-630 inhibitor induced promotion of cell proliferation and inhibition of apoptosis in cataract. miR-378a-5p and miR-630 repressed proliferation and induced apoptosis of lens epithelial cells by targeting E2F3 in cataract, representing a prospective alternative therapy for cataract.
Subject(s)
Humans , Male , Female , Middle Aged , Aged , Cataract/metabolism , Apoptosis , MicroRNAs/metabolism , Epithelial Cells/metabolism , E2F3 Transcription Factor/metabolism , Down-Regulation , Blotting, Western , Disease Progression , Real-Time Polymerase Chain Reaction , Flow CytometryABSTRACT
Objective To investigate the effect of interfering with E2F3 gene expression on the invasion and migration of prostate cancer cells and its mechanism. Methods The expression of E2F3 gene in human prostate cancer Du145 cells was knocked down by siRNA. The cells were divided into three groups: control group, Du145 NC group (siRNA-NC)and Du145-siRNA group(siRNA-E2F3). Cell migration and invasion were detected by Transwell invasion and wound healing assay. The expressions of E2F3, E-cadherin and MMP-9 proteins were detected by western blotting. Results After transfection, the expression of E2F3 protein in the Du145-siRNA group was significantly lower than the control group(P< 0.01). The number of invasive cells and wound healing rate of Du145 cells in the Du145-siRNA group were significantly lower than the control group(P < 0.01). Furthermore, the protein expression of E-cadherin was significantly increased(P< 0.01)while MMP-9 decreased(P< 0.01)in the Du145-siRNA group. Conclusions E2F3 silencing can inhibit the invasion and migration ability of prostate cancer Du145 cells, and this might be accomplished by regulating E-cadherin and MMP-9 protein.
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Objective To investigate the role of combined analysis of E2F3a and CASP8AP2 expression in prognosis evaluation in pediatric acute lymphoblastic leukemia (ALL). Methods The study included 141 newly diag-nosed pediatric ALL patients enrolled at the Hematology Center,Beijing Children′s Hospital,Capital Medical Universi-ty between March 2008 and July 2010,including 97 boys and 44 girls(aged 1. 2 - 15. 5 years,median 5. 2 years). E2F3a and CASP8AP2 expressions were quantified in 141 children with ALL by adopting real - time quantitative poly-merase chain reaction (qPCR). Receiver operating characteristic (ROC)curve was used to find the best cut - off point to divide the patients into E2F3a or CASP8AP2 low - and high - expression groups,and the treatment outcome between the groups was compared. Cox regression was used to analyze the prognostic significance of the combined expression of E2F3a and CASP8AP2. Results The estimated 5 - year relapse free survival(RFS)rate,event free survival(EFS) rate and overall survival (OS)rate of patients with low - E2F3a and low - CASP8AP2 expression were (58. 9 ± 10. 0)％,(56. 0 ± 9. 9)％ and (72. 0 ± 9. 0)％,respectively. They were significantly lower than those of patients with high - E2F3a and high - CASP8AP2 expression,whose RFS,EFS and OS were (94. 9 ± 2. 5)％,(93. 7 ± 2. 7)％ and (96. 2 ± 2. 2)％,and the differences were all statistically significant(all P < 0. 05),respectively. Compared with other patients,the one with low expression of both E2F3a and CASP8AP2 had a poorer prognosis. In addition to MLL rear-rangements and minimal residual disease level at the end of remission induction,low expression of both E2F3a and CASP8AP2 remained as independent prognostic factors. Conclusion Low expressions of E2F3a and CASP8AP2 pre-dict poor prognosis in pediatric ALL. Combined assessment of E2F3a and CASP8AP2 expression could predict poor prognosis and relapse more accurately.
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Objective To explore the effect of inhibition of miR-214 expression on the proliferation of hepatocellular carcinoma cells via regulation of E2F3 expression.Methods The expression of miR-214 in SMMC-7721, HepG2, SK-Hep-1 and Huh 7 cells was examined by RT-PCR.Hepatocellular carcinoma cells were transfected with miR-214 NC and miR-214 mimics with liposomes.The expression of miR-214 was detected by RT-PCR.The cell viability was detected by MTT assay.Cell apoptosis was detected by Hoechst staining.Cell cycle was detected by flow cytometry.Western blot, RT-PCR and dual luciferase reporter gene assay were used to detect whether E2F3 was a downstream target gene of miR-214.Results The expression of miR-214 in SMMC-7721, HepG2, SK-Hep-1 and Huh 7 cells was 0.83±0.08, 0.32±0.03, 0.33±0.03, and 0.08±0.01, respectively.The expression of miR-214 in the HepG2 cells was the lowest, so HepG2 cells were selected as the subsequent experimental cell line.Compared with the miR-214 NC group, the expression of miR-214 (0.65±0.06 vs.0.14±0.01) was up-regulated, the cell viability (0.35±0.03 vs.0.69±0.06) was decreased, cell apoptosis rate [(36.37±3.43)% vs.(3.74±0.34)%] was increased, the G1 phase of the cell cycle (57.79±5.78 vs.45.319±4.53) was prolonged, the expression of E2F3 protein (0.23±0.02 vs.0.98±0.09) and mRNA (0.24±0.02 vs.0.99±0.10) was significantly down-regulated in the miR-214 mimics group (P<0.01).Conclusion miR-214 mimics suppress the HepG2 cell proliferation via targeted down-regulation of E2F3 expression.
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Objective To explore the correlation and role of E2F3 gene,miR-17-5p and miR-20a in the cell lines of transitional cell carcinoma of bladder. Methods The plasmids of pcDNA3.1-HA-E2F3 and pAAV-siRNA-E2F3 were used to overexpress and knockdown E2F3.The mimics of miR-17-5p,miR-20a and their anti-miRNA oligonucleotides were used to overexpress and screen miR-17-5p and miR-20a.The expression levels of E2F3 gene,miR-17-5p and miR-20a were detected by quantitative real-time PCR,and E2F3 protein were detected by Western blot. Results When E2F3 was overexpressed,the 2- △△Ct of miR-17-5p and miR-20a were 2.26 ± 0.30 and 4.04 ± 0.51,it was statistically significant to compared with control (P ＜ 0.05) ; when E2F3 was knockdown,the 2 △△Ct of miR-17-5p and miR-20a were 0.49 ± 0.02and 0.65 ± 0.04 (P ＜ 0.05) ; when miR-17-5p and miR-20a were overexpressed simultaneously,the level of E2F3 mRNA was significantly decreased,the average E2F3 protein gray scale was 55.31 ± 7.89,the control was 103.67 ± 13.61 (P ＜ 0.05 ) ; when miR-17-5p and miR-20a were knockdown simultaneously,the E2F3 mRNA was significantly increased,the E2F3 protein gray scale was 295.68 ± 19.25,the control was 103.67 ± 13.61 ( P ＜ 0.05 ). Conclusions miR-17-5p and miR-20a could be up-regulated by E2F3 gene,and the E2F3 gene could be down-regulated by miR-17-5p and miR-20a.The regulatory feedback loop of E2F3 gene,miR-17-5p and miR-20a exists in transitional cell carcinoma of bladder. The loop maybe plays a key role in the development of bladder cancer.
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PURPOSE: E2F3 is important for cell cycle regulation and DNA replication. Recent studies have reported that members of the E2F family can play specific and diverse roles in the tumorigenesis of human malignancies, and the E2F3 expression appears to provide a growth advantage to tumor cells by activating cell proliferation in bladder tumors. We studied the prognostic significance of E2F3 expression in bladder cancer. MATERIALS AND METHODS: We examined the expression of E2F3 with using immunohistochemical staining in the tumor samples from 109 patients suffering with bladder cancer, and we analyzed the prognostic significance of E2F3 according to the grade, stage, recurrence and progression of bladder cancer. RESULTS: We found positive staining for E2F3 in 23 cases (21.1%). The E2F3 expression was correlated with the tumor stage (superficial vs. invasive, p<0.001) and the tumor grade (p=0.001). The E2F3 expression was not correlated with the recurrence and progression of superficial bladder cancer. CONCLUSIONS: In this study, our results showed that the E2F3 expression was observed in a portion of the bladder cancer specimens. These results suggest that E2F3 may contribute to the development of bladder cancer, but it may not play a role as a prognostic factor of bladder cancer.
Subject(s)
Humans , Carcinogenesis , Cell Cycle , Cell Proliferation , DNA Replication , E2F3 Transcription Factor , Recurrence , Urinary Bladder Neoplasms , Urinary BladderABSTRACT
Objective To study the expression of E2F-3 and pRb in primary prostate cancer(PCa) and its clinical significance.Methods The expression of E2F-3 protein and pRb was detected in 49 PCa,20 benign prostatic hyperplasia(BPH),10 normal prostate tissues(NP) by EliVision~(TM) plus immunohistochemical staining.Results The positive expression rate of E2F-3 in PCa,BPH,NP was 63.27%(31/49),20%((4/20)) and 10%(1/10),respectively.The expression level of E2F-3 in PCa was significantly higher than that in BPH(P
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Objective:To investigate the expression of E2F3 mRNA in bladder urothelial carcinoma and normal bladder urothelial tussue to explore its relationship with the genesis and progression of bladder urothelial carcinoma.Methods:The expression of E2F3 mRNA in 34 cases of bladder urothelial carcinoma and 19 cases of normal bladder urothelial tussue was examined by RT-PCR.Result:The positivity rates of E2F3 mRNA in bladder urothelial carcinomas and normal bladder urothelial tussues were 47.1%(16/34) and 0%(0/19) respectively.The expression level of E2F3 mRNA in bladder urothelial carcinomas was significantly higher than that in normal bladder urothelial tussues(P