ABSTRACT
Objective:To investigate the effect and molecular mechanism of circ_0001955 on the radiosensitivity of prostate cancer DU145 cells.Methods:The si-con, si-circ_0001955, miR-con and miR-149 were transfected into DU145 cells and recorded as the si-con group, si-circ_0001955 group, miR-con group, miR-149 group. The miR-149 and pc-circ_0001955 were co-transfected into DU145 cells and recorded as the miR-149+ pc-circ_0001955 group. Untreated cells were used as the blank control (NC) group. Real-time quantitative PCR was employed to detect the expression levels of circ_0001955 and miR-149. MTT assay was performed to detect cell viability. Flow cytometry was carried out to detect cell apoptosis. Transwell chamber assay was conducted to observe cell migration and invasion. Western blot was performed to detect the expression levels of MMP-2, MMP-9, Cleaved caspase-3, Cleaved caspase-9 and γ-H 2AX proteins. Colony formation assay was employed to determine the cell radiosensitivity. Dual-luciferase reporter assay was conducted to verify the targeting relationship between circ_0001955 and miR-149. Results:The circ_0001955 was highly expressed, whereas the miR-149 was lowly expressed in prostate cancer DU145 cells. Silencing circ_0001955 or over-expressing miR-149 could decrease the cell viability, migration and invasion, down-regulate the expression levels of MMP-2 and MMP-9, up-regulate the expression levels of Cleaved caspase-3 and Cleaved caspase-9, and increase the apoptosis rate (all P<0.05). After 4 Gy dose irradiation, the expression level of γ-H 2AX was up-regulated, the cell survival fraction was decreased, and the sensitivity ratio was 1.38. circ_0001955 could targetedly regulate the expression level of miR-149. After simultaneous overexpression of circ_0001955 and miR-149, cell proliferation activity and the number of migrating and invading cells were increased, cell apoptosis rate was decreased, and cell survival fraction was increased, and the sensitivity ratio was calculated as 0.72. Conclusion:Silencing circ_0001955 can targetedly up-regulate the expression level of miR-149, which inhibits the proliferation, migration, and invasion, induces cell cycle arrest, induces cell apoptosis and increases the radiosensitivity of prostate cancer DU145 cells.
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@#Objective: To explore the effects of miR-149-3p on the proliferation, apoptosis, invasion and migration of cervical cancer HeLa cells and the possible mechanisms. Methods: HeLa cells were randomly divided into five groups, including untransfected (HeLa) group, mimic-scramble group (the negative control of miR-149 mimic), miR-149 mimic group, FOXP3 over-expression (pc-FOXP3) group, and co-transfection (mimic+pc-FOXP3) group. The targeted relationship of miR-149-3p and FOXP3 was verified by luciferase assay. The expressions of miR-149-3p and FOXP3 mRNA were tested by quantitative real-time reverse transcription PCR (qRT-PCR). The protein levels of FOXP3 were measured by Western blotting. The proliferation was detected by CCK-8; the apoptosis was tested by flow cytometry, the cell invasion was measured by transwell invasion assay and cell migration was detected by scratch assay. Results: The luciferase assay showed that miR-149-3p could target combine with FOXP3. Compared with untransfected group, the expression of miR-149-3p was increased while mRNA level of FOXP3 was decreased in miR-149 mimic group (all P<0.01). Moreover, the protein level of FOXP3 in miR-149 mimic group was lower than that in untransfected group (P<0.01), while the protein level of FOXP3 in pcFOXP3 group was higher than that in untransfected group (P<0.01); Compared with pc-FOXP3 group, the protein levels of FOXP3 in mimic+pc-FOXP3 group were reduced (P<0.01). The proliferation in miR-149 mimic group was lower than that in untransfected group (P<0.01), while the proliferation in pc-FOXP3 was higher than that in untransfected group (P<0.01); compared with pc-FOXP3 group, the proliferation in mimic+pc-FOXP3 group was decreased (P<0.01). The apoptosis rate of HeLa cells in miR-149 mimic group was higher than that in untransfected group (P<0.01), while the apoptosis rate in pc-FOXP3 was lower than that in untransfected group (P< 0.01); compared with pc-FOXP3 group, the apoptosis in mimic+pc-FOXP3 group was elevated (P<0.01). The number of invasive cells per field and wound healing rate in miR-149 mimic group was lower than those in untransfeccted group (P<0.01) while the invasive cells and wound healing rate in pc-FOXP3 group was higher than those in untransfeceted group (P<0.01); compared with pc-FOXP3 group, the number of invasive cells per field and wound healing rate in mimic+pc-FOXP3 group was reduced (P<0.01). Conclusion: miR-149-3p inhibits proliferation, invasion and migration and promotes apoptosis of cervical cancer HeLa cells via targeting FOXP3.
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Purpose To investigate the expression and significance of USP10 protein and mRNA in normal colorectal mucosa and colorectal adenocarcinoma,and to analyze the cause of the disorder.Methods 99 cases of colorectal adenocarcinoma and 83 cases of normal intestinal mucosa tissue were selected.Using tissue microarray and immunohistochemistry the expression of USP10 protein was detected,and the relationship was analyzed between USP10 protein and clinical pathological parameters or prognosis survival time.The expression of USP10 mRNA was analyzed by GEO datesets.Some miRNAs that down-regulate the expression of USP10 protein were screened by bioinformatics methods.The expression of USP10 protein and miR-149 in colorectal cancer cell lines were detected by Western blot and real-time quantitative PCR.Results The positive rate of USP10 protein in normal intestinal mucosa tissues was 71.08％(59/83),which was significantly higher than that in colorectal adenocarcinoma tissues (53.54％,53/99,P =0.015).No correlation were proved between USP10 protein expression and clinical pathological parameters or survival time (P ＞ 0.05).The expression level of USP10 mRNA in colorectal adenocarcinoma was 1.07 ～ 1.45 times that were higher than that of normal intestinal mucosa,which showed that the down-regulation of USP10 protein was at the post-transcriptional level.The program predicted a putative highly-conserved binding site in the USP10 mRNA 3'UTR for miR-149 which was up regulated in colorectal adenocarcinoma tissues.In addition,the expression of miR-149 was negatively correlated with the expression of USP10 protein in colorectal cancer cell lines.Conclusion The down-regulation of USP10 protein which occurs at the post-transcriptional level is closely related to the pathogenesis of colorectal adenocarcinoma.The high expression of miR-149 may be one of the factors that negatively regulate the expression of USP10 protein.
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Objective:To study the frequencies of allele and genotype distribution of miR-146a C>G(rs2910164) and miR-149 T>C( rs2292832) gene, and to analyze the statistical differences between different racial and nationalities.Methods:The Polymerase Chain Reaction-Single Base Extension ( PCR-SBE) technique and DNA sequencing methods were used for the determination of the SNP in miR-146a C>G and miR-149 T>C gene,and compared with the European, African, Japanese and People in Beijing from the Human Genome Project (HapMap).Results:There were no statistical differences of allele and genotype distribution in miR-146a C>G,miR-149 T>C between female and male group (P>0.05).There were significant difference frequencies of allele and genotype distribution of miR-146a C>G and miR-149 T>C gene by compared with the European, African and People in Beijing( PG and miR-149 T>C in Guangxi populations, and there were significant differences by compared with other ethnic populations, which may play an important role in the human inherited disease research.
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Objective:To investigate the effects of the miR-149 on the growth and invasion of prostate carcinoma cells. Meth-ods:Real-time fluorescence quantitative polymerase chain reaction was performed to detect the expression of miR-149 on prostate car-cinoma tissues and paraneoplastic tissues. The PC3 and DU145 cells were transfected with miR-149 mimics and negative controls. The cell growth and invasion abilities were tested in terms of colony formation and via Transwell invasion assay. The cells were transfected with the siRNA of the target gene FOXM1 and siRNA control. Western blot was used to detect the expression of FOXM1. The cell colo-ny formation and invasion ability were also detected. Results:Compared with the paraneoplastic tissues, miR-149 was down-regulated in the prostate carcinoma tissues (P<0.01), and the FOXM1 mRNA was highly expressed (P<0.01). PC3 and DU145 cells with miR-149 mimics had only a few colonies and invading cells (P<0.01). Moreover, PC3 (P<0.01) and DU145 (P<0.05) with miR-149 mimics had a low protein level of FOXM1. The FOXM1 expression was knocked down by the siRNA of FOXM1 in the PC3 and the DU145 cells (P<0.01). The knocking down of FOXM1 resulted in an inhibition of the cell colony formation and invasion abilities (P<0.01). Conclusion:The miR-149 inhibits prostate carcinoma cell growth and invasion by suppressing the FOXM1. Our data suggest that miR-149 may function as an effective tool for the molecular treatment of prostate cancer.
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Objective To investigate the function and mechanism of miR-149 in nasopharyngeal carcinoma (NPC).Methods The expression of miR-149 was examined by real-time PCR and calculated by 2-△△Ct method. The cell proliferation was analyzed by MTT assay. The cell migration and invasion were shown by the wound healing assay and transwell migration assay, and the expression of E-cadherin was detected by Western blot. Results The expression of miR-149 was higher in NPC cell lines 5-8F and 6-10B than that in normal immortalized nasopharyngeal epithelial NP69. MTT assay showed that miR-149 promoted the proliferation of NPC cell lines. The wound healing assay showed miR-149 promoted the mobility and invasion of NPC cell lines. Inhibition of miR-149 reduced the ability of NPC cell lines to proliferate and invade. miR-149 downregulated the expression of E-cadherin, whereas antagomir which mediated knockdown of miR-149 significantly upregulated the expression of E-cadherin. Conclusion miR-149 might be involved in the invasion and metastasis of NPC through regulation of epithelial-mesenchymal transition (EMT).