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Objective:To explore the effect of miR-1249-5p on the proliferation, metastasis and cell cycle of PC-3 cell in prostate cancer.Methods:The relationship between the expression level of miR-1249-5p and the overall survival of prostate cancer patients was analyzed using OncoMir Cancer Database (OMCD). The human prostate cancer cell line PC-3 was divided into two groups: miR-1249-5p group and negative control group. Mediated by Lipofectamine 2000, miR-1249-5p mimics liposome complex or negative miRNA liposome complex were transfected into PC-3 cell at logarithmic growth stage. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-1249-5p in PC-3 cell of two groups. Colony formation assay was used to detect the changes of the proliferation ability of PC-3 cell in the two groups. Transwell experiment was used to detect the changes of PC-3 cell invasion in the two groups, and the cell cycle changes of the two groups of PC-3 were detected by flow cytometry. The miRNA prediction software miRGator was used to predict the target gene of miR-1249-5p. RT-qPCR and Western blotting were used to detect the target gene expression of miR-1249-5p. Measurement data were expressed as mean±standard deviation ( ± s), and t-test was used for comparison between two groups. Results:Compared with prostate cancer patients with low miR-1249-5p expression, prostate cancer patients with higher miR-1249-5p expression had longer overall survival, and the difference was statistically significant ( P<0.01). The expression level of miR-1249-5p in the miR-1249-5p group (10.74±1.19) was significantly higher than that of the negative control group (1.56±0.27), the difference was statistically significant ( P<0.01). The number of colonies formed in the miR-1249-5p group (35.86±6.94) was significantly less than that in the negative control group (88.94±11.66), and the difference was statistically significant ( P<0.01). The number of transmembrane cells [(25.01±6.83)/high power field of view] in the miR-1249-5p group was significantly less than that of the negative control group [(82.76±8.35)/high power field of view], and the difference was statistically significant ( P<0.01). The proportion of cells in the G 0-G 1 phase in the miR-1249-5p group [(50.79±6.61)%] was significantly higher than that in the negative control group [(27.09±2.30)%], the difference was statistically significant ( P<0.01), and PC-3 cell were inhibited in the G 0-G 1 phase. Neural precursor cell expressed developmentally down-regulated 9 ( NEDD9) may be the target gene of miR-1249-5p. Compared with the negative control group, the NEDD9 gene expression in the miR-1249-5p group was significantly lower than that of the negative control group, the difference was statistically significant ( P<0.01). Conclusion:miR-1249-5p can inhibit the proliferation, metastasis and cell cycle of PC-3 cell in prostate cancer, which may be achieved by negatively regulating the expression of proto-oncogene NEDD9.
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Objective:To explore the expression level of miR-769-3p in bladder cancer tissues, and observe the effect of silencing miR-769-3p on the migration ability and cell cycle of J82 cells by down-regulating the expression level of miR-769-3p in bladder cancer J82 cells.Methods:The OncomiR database was used to analyze the expression differences of miR-769-3p in bladder cancer tissues and adjacent tissues. J82 cells were transfected with Lipofectamine 2000 transfection reagent and divided into si-miR-769-3p group (transfected with miR-769-3p small molecule interference fragments) and control group (transfected with meaningless sequences). quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative expression level of miR-769-3p after transfection. The cell scratch test and flow cytometry were used to compare the migration ability and cell cycle differences between the two groups of J82 cells. The bioinformatics software MicroRNAdb was used to predict the target gene of miR-769-3p. The dual-luciferase reporter gene assay was used to verify the complementary binding of miR-769-3p to the target gene. qRT-PCR and Western blotting were used to detect the expression levels of miR-769-3p target gene. Measurement data were expressed as mean ± standard deviation ( ± s), and t-test was used for comparison between two groups. Results:The expression of miR-769-3p was significantly increased in bladder cancer tissues compared with adjacent tissues, the difference was statistically significant ( P<0.01). The relative expression of miR-769-3p in the si-miR-769-3p group (1.02 ± 0.16) was significantly lower than that of the control group (4.50 ± 0.60), the difference was statistically significant ( P<0.01). The cell migration rate of the si-miR-769-3p group [(26.67±3.98)%] was significantly lower than that of the control group [(61.86±4.70)%], the difference was statistically significant ( P<0.01). The proportion of cells in the G 0-G 1 phase in the si-miR-769-3p group [(57.66±5.74)%] was significantly higher than that in the control group [(31.26±3.24)%], the difference was statistically significant ( P<0.01). Dual-luciferase reporter gene assay confirmed that endothelin 3 ( EDN3) was the target gene of miR-769-3p. The relative expression of EDN3 mRNA in J82 cells in control group and si-miR-769-3p group was 1.99 ± 0.66 and 6.98 ± 0.76, compared with the control group, the EDN3 mRNA relative expression level of the si-miR-769-3p group was significantly higher than that of the control group, the difference was statistically significant ( P<0.01). Conclusion:Low expression of miR-769-3p can inhibit the migration of bladder cancer J82 cells and block the J82 cell cycle by promoting the expression of EDN3 gene.
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Objective:To investigate the effects of astragalin on the cell proliferation and cell cycle of prostate cancer cell line C4-2B through up-regulating the expression of miRNA-513 (miR-513).Methods:Prostate cancer cell line C4-2B cells were taken and treated with 125 μg/L of astragalin for 48 h (astragalin group), and untreated C4-2B cells were set as the control group. The methyl thiazolyl tetrazolium (MTT) method was used to detect the proliferation ability of C4-2B cells in the two groups, and cell cycle was detected by using flow cytometry. The miRNAMap prediction software was used to predict that the targeted gene of miR-513 was the forkhead box protein R2 (FOXR2), and the dual luciferase gene reporter assay was used to verify it. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the relative expression levels of miR-513 and FOXR2 mRNA in the two groups of cells. Western blotting was used to detect the expressions of FOXR2, cyclin-dependent kinase 7 (CDK7), β-actin and cyclin H in the two groups of C4-2B cells.Results:Compared with the control group, the proliferation activity of C4-2B cells in the astragalin group was decreased from day 2 to day 5 (all P < 0.05). The proportions of S-phase cells in the control group and the astragalin group were (48.1±3.2)% and (36.0±2.1)%, respectively. The proportion of S-phase cells in the astragalin group was decreased ( t = 3.12, P = 0.021); the proportions of G 2-phase cells were (24.9±3.3)% and (11.8±2.4)%, respectively. The proportion of G 2-phase cells in the astragalin group was decreased ( t = 3.18, P = 0.019). The relative expression levels of miR-513 in C4-2B cells of the control group and the astragalin group were 1.01±0.22 and 6.55±0.61, respectively. The relative expression levels of miR-513 in C4-2B cells in the astragalin group was increased ( t = 7.70, P < 0.01). The dual luciferase reporter gene assay verified that FOXR2 was the targeted gene of miR-513. The relative expression level of FOXR2 mRNA in C4-2B cells of the control group and the astragalin group was 1.04±0.14 and 0.19±0.06, respectively, and the difference was statistically significant ( t = 5.53, P = 0.002), suggesting that after astragalin promoted the expression of miR-513, the FOXR2 mRNA expression was decreased. The relative expression levels of FOXR2, CDK7 and cyclin H protein in C4-2B cells in the astragalin group were all decreased compared with those in the control group. Conclusions:Astragalin inhibits the proliferation of prostate cancer C4-2B cells and induces cell cycle arrest by up-regulating the expression of miR-513.
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Objective:To investigate the mechanism of physcion affecting the cell cycle and proliferation of prostate cancer DU145 cell line by regulating the expression of miR-380-3p.Methods:Prostate cancer DU145 cells were treated with 50 μg/mL physcion as physcion group, and normal cultured DU145 cells without any treatment were used as control group. Flow cytometry was used to detect DU145 cell cycle changes. MTT proliferation test was used to detect the proliferation of DU145 cells. quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-380-3p in DU145 cells. The bioinformatics software RNAhybrid was used to predict the target genes of miR-380-3p. qRT-PCR and Western blotting methods were used to detect the expression of miR-380-3p target gene. Measurement data were expressed as mean ± standard deviation ( ± s), t-test was used for comparison between two groups. Results:Compared with the control group, DU145 cells in the physcion group were blocked in the G 0/G 1 phase ( P<0.01), and the proliferation ability of DU145 cells was significantly inhibited ( P<0.05). The expression of miR-380-3p in DU145 cells in the control group and physcion group was 8.36 ± 1.42 and 1.08 ± 0.39, respectively. Physcion could promote the expression of miR-380-3p ( t=4.96, P<0.01). The functional target gene of miR-380-3p may be UHRF1. The relative expression levels of UHRF1 mRNA in DU145 cells in the physcion group and control group were 0.23±0.06 and 1.04±0.15, respectively. Compared with the control group, the expression of UHRF1 gene in DU145 cells in the physcion group was decreased ( t=4.55, P<0.01). Conclusion:Physcion can inhibit the proliferation of prostate cancer DU145 cells and induce G 0/G 1 block in DU145 cells, which may be closely related to the regulation of miR-380-3p.
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Objective:To explore the expression of microRNA (miRNA)-6516-5p in renal cancer cell lines and the molecular mechanisms regulating the proliferation and migration of renal cancer cells.Methods:quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-6516-5p in renal cancer cell lines and normal proximal renal tubular epithelial cell lines. The liposome method was used to transiently transfect miR-6516-5p mimic and nonsense sequence (NC) into renal cancer cells with the lowest expression of miR-6516-5p, namely miR-6516-5p group and NC group. qRT-PCR was used to detect the expression of miR-6516-5p in transfected cells. CCK-8 and Transwell migration experiment were used to detect the proliferation and migration of transfected cells. Bioinformatics software and dual luciferase gene report experiment were used to predict and verify the regulation of miR-6516-5p on target gene, respectively. qRT-PCR and Western blotting were used to detect the expression of target gene in transfected cells. Measurement data were expressed as mean±standard deviation ( ± s), t-test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. Results:The expression of miR-6516-5p in renal cancer cell lines was significantly lower than that of normal proximal tubular epithelial cells ( P<0.01), and the expression of miR-6516-5p in 786-O cells was the lowest ( F=27.69, P<0.01). The expression of miR-6516-5p in 786-O cells in NC group and miR-6516-5p group was 1.01±0.08 and 9.91±1.16, respectively. Compared with the NC group, the expression of miR-6516-5p in 786-O cells in the miR-6516-5p group was significantly increased ( t=7.63, P<0.01). Up-regulation of miR-6516-5p can significantly inhibit the proliferation of 786-O cells ( P<0.05). The migration numbers of NC group and miR-6516-5p group were 85.65±8.77 and 28.05±6.20, respectively. Overexpression of miR-6516-5p could inhibit the migration of 786-O cells ( t=5.36, P< 0.01). The target gene of miR-6516-5p may be ornithine decarboxylase 1 ( ODC1), miR-6516-5p can significantly inhibit the luciferase activity of wild-type ODC1-3′UTR ( t=9.83, P<0.01). Up-regulation of miR-6516-5p can reduce the expression of ODC1 mRNA and protein in 786-O cells ( P<0.01). Conclusion:The expression of miR-6516-5p is reduced in renal cancer cell lines, miR-6516-5p inhibits the proliferation and migration of renal cancer 786-O cells by targeting ODC1, miR-6516-5p may become a potential molecular target of renal cancer.
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Objective:To investigate the expression of long non-coding RNA (lncRNA) BDNF-AS in kidney cancer tissues, and its effect on the proliferation and migration ability of kidney cancer cells and the molecular mechanism.Methods:Real-time reverse quantitative polymerase chain reaction (rRT-PCR) was used to detect the expression levels of BDNF-AS gene in renal cancer tissues, tumor-adjacent tissues of 67 renal cancer patients and normal renal tubular epithelial cells HK-2 and renal cancer cell lines A498, ACHN, OS-RC-2, Caki-1, 786-O in Huangshi Central Hospital of Edong Medical Group from May 2017 to July 2018. The kidney cancer cell line with the lowest expression of BDNF-AS was taken as the research object. Transient transfection with BDNF-AS overexpression plasmid was treated as the experiment group or a plasmid carrying meaningless sequences was treated as the control group. rRT-PCR was used to detect transfection efficiency. After the transfection with Caki-1 for 24 h, methythiazolyl tetrazolium (MTT) method was used to detect the proliferation of cells in both groups, Transwell migration assay was applied to detect the cell migration ability, rRT-PCR was used to detect the expression level of protein tyrosine phosphatase receptor type G (PTPRG) mRNA and Western blot was used to detect the expression level of PI3K-AKT pathway related-proteins.Results:The relative expression level of BDNF-AS in kidney cancer tissues was lower than that in tumor-adjacent tissues (0.96±0.24 vs. 4.62±0.84, t = 41.76, P < 0.01). The relative expression of BDNF-AS in kidney cancer cell lines was lower than that in normal renal tubular epithelial cells HK-2 (all P < 0.05), and the relative expression in Caki-1 cells was the lowest (0.10±0.01). The relative expression of BDNF-AS in the experimental group was higher than that in the control group ( P < 0.01). From the second day of transfection, the proliferation ability of Caki-1 cells in the experimental group was lower than that in the control group (all P < 0.05). The number of Caki-1 migrated cells in the experimental group was lower than that in the control group after migration for 15 h of Caki-1 cells transfected for 24 h [(51±8) vs. (192±25), t = 5.31, P < 0.01]. After 48 h transfection, the relative expression of PTPRG mRNA in Caki-1 cells ( P < 0.01) and protein expression of the experimental group were higher than those of the control group, the expression levels of PI3K-AKT signaling pathway related-proteins p-PI3K, p-AKT, p-Tpl2 in Caki-1 cells of the experimental group were lower than those of the control group. Conclusions:The expression of BDNF-AS is down-regulated in kidney cancer tissues and cell lines. Overexpression of BDNF-AS can inhibit the proliferation and migration ability of kidney cancer Caki-1 cells. The molecular mechanism may be related to the transduction that BDNF-AS promotes PTPRG gene expression and interferes with PI3K-AKT signaling pathway.
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Objective:To explore the molecular mechanism of microRNA (miRNA, miR)-1914-3p regulating the expression of ARL4C and affecting the invasion and proliferation of renal cancer cells.Methods:Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of miR-1914-3p in tumor tissues and adjacent tissues of 53 renal cancer patients, 4 types of renal cancer cell lines (ACHN, OS-RC-2, 786-O, A498) and normal proximal renal tubular epithelial cell line (HK-2). The nonsense sequence (NC) and miR-1914-3p mimic were transiently transfected into renal cancer cells with the lowest miR-1914-3p expression by liposome method, namely the NC group and miR-1914-3p group. qRT-PCR was used to detect the expression level of miR-1914-3p in transfected cells. Transwell invasion test and cell counting kit-8 (CCK-8) were used to detect the invasion and proliferation ability of each group of cells. Bioinformatics software and dual luciferase gene report experiment were used to predict and test the targeted regulation mechanism of miR-1914-3p on target genes. qRT-PCR and Western blot was conducted to analyze the target gene expression level in cells of each group.Results:The expression level of miR-1914-3p in renal cancer tissue was significantly lower than that in adjacent tissues ( P<0.01). The expression level of miR-1914-3p in renal cancer cell lines was significantly lower than that in HK-2 cell lines ( P<0.01), and the expression of miR-1914-3p in OS-RC-2 cells was the lowest ( P<0.01). The expression of miR-1914-3p in the NC group and the miR-1914-3p group were (1.04±0.17) and (11.40±0.91), respectively. The expression level of miR-1914-3p in the miR-1914-3p group was significantly increased ( P<0.01), indicating that the transfection was successful. Overexpression of miR-1914-3p can significantly inhibit the invasion ( P<0.01) and proliferation ( P<0.05) of renal cancer OS-RC-2 cells. Dual luciferase gene report experiment indicated that the target gene of miR-1914-3p may be ADP-ribosylation factor-like 4C (ARL4C); miR-1914-3p can significantly inhibit the luciferase activity of wild-type ARL4C-3′UTR ( P<0.01). Overexpression of miR-1914-3p decreased the expression of ARL4C mRNA and protein in OS-RC-2 cells ( P<0.01), and decreased the expression of cell invasion phenotype proteins (Snail, Slug) and cell proliferation phenotype proteins (Mcm2, Mcm7) ( P<0.01). Conclusions:miR-1914-3p is low-expressed in renal cell carcinoma. It inhibits the invasion and proliferation of renal cell carcinoma OS-RC-2 cells through targeted interference with the expression of the oncogene ARL4C, and participates in the occurrence and development of renal cell carcinoma.
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Objective:To observe the expression of long non-coding RNA (lncRNA) PEBP1P2 in renal cell carcinoma (RCC) tissues and its effect on the proliferation and migration of RCC cells.Methods:The expression of PEBP1P2 in 51 RCC tissues and RCC cell lines was detected by real-time quantitative polymerase chain reaction (qPCR). The A498 cells with the lowest expression of PEBP1P2 were transfected, and the cells transfected with PEBP1P2 plasmid were used as the PEBP1P2 group, and the cells transfected with the negative control plasmid were used as the NC group. qPCR was used to detect the expression of PEBP1P2 in the two groups of cells. MTT assay and Transwell migration assay were used to detect the proliferation and migration ability of RCC cells. qPCR and Western blotting were used to detect the expression of caspase recruitment domain family member 10 ( CARD10) gene and NF-κB pathway protein, respectively. Measurement data were expressed as mean±standard deviation ( Mean± SD), and LSD- t test was used for comparison between groups. Results:The expression of PEBP1P2 in RCC tissues was lower than that in adjacent tissues ( t=4.89, P<0.01). The expression of PEBP1P2 in RCC cells was lower than that in normal renal tubular epithelial cells ( P<0.01). The expression of PEBP1P2 in A498 cells of the PEBP1P2 group and NC group was (11.01±1.26) and (1.06±0.19), respectively, and the PEBP1P2 group was significantly higher than that in the NC group ( t=7.81, P<0.01). Overexpression of PEBP1P2 significantly inhibited the proliferation of RCC cells ( P<0.05) and migration ability ( t=3.65, P<0.05). Overexpression of PEBP1P2 significantly suppressed the expression of CARD10 gene in RCC A498 cells ( t=6.83, P<0.01) and inhibited the transduction of NF-κB signaling pathway proteins. Conclusions:PEBP1P2 expression was significantly decreased in RCC tissues. Overexpression of PEBP1P2 significantly inhibited the proliferation and migration of RCC A498 cells. Its molecular mechanism is that PEBP1P2 down-regulates CARD10 gene expression and inhibits NF-κB signaling pathway.
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Objective:To explore the effect of long non-coding RNA (lncRNA) AC068768.1 on the cycle and proliferation of renal cancer cells and its molecular mechanism.Methods:Real-time quantitative polymerase chain reaction (qPCR) was used to detect the expression of AC068768.1 in renal cancer cell lines. The OS-RC-2 cells with the lowest expression of AC068768.1 were used as the transfection objects, OS-RC-2 transfected with the negative control plasmid was set as the control group, and the cells transfected with the AC068768.1 plasmid were set as the AC068768.1 group. qPCR was used to detect the expression of AC068768.1 in transfected OS-RC-2 cells. The effects of AC068768.1 on the cell cycle and proliferation of OS-RC-2 were detected by flow cytometry and tetramethylazazole blue colorimetric (MTT) proliferation experiments. Using bioinformatics methods to predict the microRNA (miRNA) that AC068768.1 may bind. qPCR was used to detect the expression of miRNA and downstream gene mRNA, and Western blot was used to detect the expression of downstream gene protein.The measurement data were expressed as mean±standard deviation ( Mean± SD), the comparison between the two groups adopts the t-test, and the comparison among multiple groups adopts the One-way analysis of variance. Results:Compared with normal renal tubular epithelial cells, the expression of AC068768.1 in renal cancer cell lines was significantly reduced, the difference was statistically significant ( P<0.01). The expression of AC068768.1 in OS-RC-2 cells in the AC068768.1 group was significantly higher than that in the control group, the difference was statistically significant ( P<0.01). Up-regulating the expression of AC068768.1 can inhibit the cycle ( P<0.05) and proliferating ability ( P<0.05) of renal cancer cells. miR-21-5p may be the functional target gene of AC068768.1. Up-regulation of AC068768.1 can significantly inhibit the expression of miR-21-5p ( P<0.01) and promote the expression of tissue inhibitor of metalloproteinase 3 (TIMP3) ( P<0.01). Conclusion:AC068768.1 promotes the expression of TIMP3 gene by regulating the expression of miR-21-5p, thereby inhibiting the cell cycle and proliferation of renal cancer OS-RC-2 cells.
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Objective:To explore the expression of long non-coding RNA LINC00630 in bladder cancer cell lines, and to explore the effect of interference with its expression in vitro on the proliferation and migration of bladder cancer cells.Methods:Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of LINC00630 in bladder cancer cell lines 5637, BIU-87, T24, J82 and normal bladder epithelial cell line SV-HUC-1. The bladder cancer cell line with the highest LINC00630 expression was selected for follow-up experiments, then the cell line infected with the control lentivirus was used as the control group, and the cell line infected with the lentivirus that could interfere with the expression of LINC00630 was used as the experimental group. qRT-PCR was used to detect the expression of LINC00630 in the two groups of cells. MTS method and cell scratch test were used to detect the proliferation and migration abilities of cells in the two groups. qRT-PCR was used to detect the expression of neuregulin 1 (NRG1) mRNA in the two groups of cells, and Western blot was used to detect the expressions of NRG1 protein, cell proliferation-related proteins (cyclin D3 and CDK2) and cell migration-related proteins (Vimentin and N-cadherin) in the two groups of cells.Results:Compared with SV-HUC-1 cells (1.05±0.17), the expression of LINC00630 was significantly increased in all bladder cancer cell lines (all P < 0.01), and the expression was highest in J82 cells (relative expression 5.83±0.42). Compared with J82 cells of the control group, the expression of LINC00630 in J82 cells of the experimental group decreased (0.18±0.02 vs. 1.00±0.05, t=14.36, P < 0.01); from day 2 of transfection, the cell proliferation activity of the experimental group was lower than that of the control group (all P < 0.05). The cell scratch closure rate of the experimental group was lower than that of the control group [(27.4±7.1)% vs. (66.0±5.4)%, t = 4.31, P < 0.01]. Therelative expression of NRG1 mRNA in the experimental group was lower than that in the control group (0.34±0.03 vs. 1.07±0.24, t = 2.99, P < 0.05). Compared with the control group, the expressions of NRG1 protein, cell proliferation-related proteins and cell migration-related proteins in the experimental group were reduced. Conclusions:LINC00630 is up-regulated in bladder cancer cell lines, and interference with LINC00630 may inhibit the proliferation and migration of J82 cells by down-regulating the expression of NRG1 gene. LINC00630 may be a new molecular target for the treatment of bladder cancer.
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Objective:To observe the expression of microRNA (miRNA, miR) -7850 in renal cancer tissues, and to explore the effect of miR-7850 on the proliferation and migration of renal cancer cells and on the regulation of serine proteinase inhibitor B3 (SERPINB3) gene expression.Methods:Real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-7850 in renal cancer tissues and renal cancer cell lines. The renal cell carcinoma cell line with the lowest expression of miR-7850 was selected, and the negative control sequence (miR-NC) and miR-7850 mimics were transfected into renal cell carcinoma cells by Lipofectamine 2000 transfection reagent, respectively, which were defined as miR-NC group and miR-7850 group. qRT-PCR was used to detect the expression of miR-7850 in transfected renal cancer cells. The cell proliferation and migration ability after transfection were detected by cell counting kit-8 (CCK-8) method and transwell experiment. Bioinformatics prediction and dual luciferase reporter gene experiments were used to verify the target gene of miR-7850. qRT-PCR and Western blot were used to detect the expression of target genes in renal cancer cells after transfection.Results:Compared with adjacent tissues (5.95±0.44), the expression of miR-7850 in kidney cancer tissues (1.19±0.33) was lower ( P<0.01). Compared with immortalized proximal renal tubular epithelial cells (1.01±0.07), the expression of miR-7850 was lower in renal cancer cell lines ( P<0.05), and the lowest in A498 cells (0.13±0.01) ( P<0.01). The expression of miR-7850 in the miR-7850 group (7.46±0.93) was significantly higher than that in the miR-NC group (1.01±0.08) ( P<0.01), indicating successful transfection. Compared with the miR-NC group, the cell proliferation ability of the miR-7850 group was significantly reduced ( P<0.05). The number of migrating cells in miR-NC group and miR-7850 group were (139.50±12.31) and (75.09±16.05) cells, respectively, and the cell migration ability in miR-7850 group decreased significantly ( P<0.01). Bioinformatics technology shows that the target gene of miR-7850 was SERPINB3. The dual luciferase reporter gene experiment confirmed that miR-7850 can target the SERPINB3 gene ( P<0.05). Compared with the miR-NC group, the expression of SERPINB3 in cells of miR-7850 group was significantly reduced ( P<0.05), as well as the CDK4, CyclinD, Snail and Vimentin. Conclusions:miR-7850 is lowly expressed in renal cancer tissues and cell lines. miR-7850 can inhibit the proliferation and migration of renal cancer A498 cells, which may be related to its inhibition of SERPINB3 gene expression.
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Objective:To analyze the expression of long non-coding RNA (lncRNA) ZFPM2-AS1 in bladder cancer tissues and cell lines, and to observe the effect of down-regulating ZFPM2-AS1 on the migration and proliferation of bladder cancer cells and explore its molecular mechanism.Methods:Real-time quantitative fluorescent polymerase chain reaction (qRT-PCR) was used to detect the expression of ZFPM2-AS1 in 51 pairs of bladder cancer tissues and adjacent tissues, bladder cancer cell lines (J82, 5637, BIU-87, T24) and human normal bladder epithelial cells SV-HUC-1. The bladder cancer cells with the highest ZFPM2-AS1 expression were selected and transfected with the small interfering siRNA-ZFPM2-AS1 plasmid and the negative control plasmid, respectively, and defined as the experimental group and the control group. qRT-PCR was used to detect the expression of ZFPM2-AS1 in two groups of cells. Transwell migration test and tetramethylazozole blue (MTT) method were used to detect the cell migration ability and proliferation ability of the two groups. qRT-PCR was used to detect the expression of Up-frameshift mutant 1 (UPF1) mRNA in two groups of cells. Western blot was used to detect the expression of UPF1 and mTOR signaling pathway proteins in the two groups of cells.Results:The expression of ZFPM2-AS1 in bladder cancer tissues was significantly higher than that in adjacent tissues ( P<0.01). The expression of ZFPM2-AS1 in bladder cancer cell lines was significantly higher than that in human normal bladder epithelial cells ( P<0.01), and ZFPM2-AS1 had the highest expression in BIU-87 cells ( P<0.01). Compared with the control group, the expression of ZFPM2-AS1 in BIU-87 cells in the experimental group was significantly reduced [(1.01±0.06) vs (0.16±0.04), t=12.28, P<0.01]. Compared with the control group, the migration ability of BIU-87 cells in the experimental group was decreased ( P<0.05), and the proliferation ability of BIU-87 cells was significantly decreased from the second day ( P<0.05). Compared with the control group, UPF1 mRNA expression in BIU-87 cells in the experimental group was significantly decreased [(1.00±0.02) vs (0.28±0.04), t=15.49, P<0.01]. Western blot results showed that UPF1 protein expression and mammalian rapamycin target protein (mTOR), GRB2, IRS1 and p-PI3K signal pathway protein expression were decreased in BIU-87 cells. Conclusions:ZFPM2-AS1 is highly expressed in bladder cancer tissues and cell lines. Down-regulating ZFPM2-AS1 can inhibit the migration and proliferation of BIU-87 cells. The molecular mechanism may be related to the inhibition of UPF1 gene expression.
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Objective:To investigate the expression of long non-coding RNA (lncRNA) COX10-AS1 in renal cell carcinoma tissues and cell lines and its effect on proliferation and migration of renal cancer cells.Methods:Fluorescence real-time quantitative PCR (qRT-PCR) was used to detect the expression of COX10-AS1 in surgical specimens that have been diagnosed as renal cancer tissues and adjacent tissues by pathology, renal cancer cell lines (786-O, CaKi-1, A498, ACHN) and normal renal tubular epithelium cell line (HK-2). The ACHN cells with the lowest expression were divided into a control group (transfected with a negative control plasmid carrying nonsense sequences) and an experimental group (transfected with a plasmid carrying COX10-AS1 sequences). The expression level of COX10-AS1 was detected by qRT-PCR in two groups of cells. The proliferation and migration ability of ACHN cells were detected by MTS assay and cell scratch assay. The expression of MFN2 mRNA was detected by qRT-PCR. The expressions of MFN2 and Ras-NF-κB signaling pathway proteins were detected by Western blotting. The measurement data were expressed as mean±standard deviation ( Mean± SD), the comparison between the two groups used the t-test, and the comparison among multiple groups adopts the one-way analysis of variance. Results:The expression of COX10-AS1 in renal cell carcinoma was significantly lower than that in adjacent tissues ( P<0.01), The expression of COX10-AS1 in renal cell carcinoma cells was significantly lower than that in renal tubular epithelial cells ( P<0.05), the expression of COX10-AS1 was the lowest in ACHN cells( P<0.01), the above differences were statistically significant compared with the control group, the expression of COX10-AS1 in ACHN cells of experimental group was significantly increased ( P<0.01), the above differences were statistically significant compared with the control cells, the proliferation of ACHN cells in the experimental group was significantly decreased ( P<0.05), and the cell migration ability was significantly decreased ( P<0.01). Compared with the control cells, the expression of MFN2 mRNA in ACHN cells of experimental group was significantly increased ( P<0.01). The expression levels of MFN2 were significantly up-regulated ( P<0.01), and Ras-NF-κB signaling pathway proteins were significantly down-regulated ( P<0.05), the above differences were statistically significant. Conclusions:The expression of COX10-AS1 is decreased in renal cell carcinoma tissues and cell lines. COX10-AS1 may inhibit the proliferation and migration of ACHN cells by promoting the expression of MFN2 gene.
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Objective To observe the effect of mitofusion 2 (MFN2) on the proliferation of prostate cancer cells and its molecular mechanism. Methods Lentivirus containing the MFN2 coding sequence (Lenti-MFN2) were used to infect the prostate cancer cell lines DU-145 and LNCaP, and the lentivirus containing the green fluorescent protein gene (Lenti-GFP) were defined as the control. Real-time quantitative PCR (qRT-PCR) and Western blot were used to detect the expression of MFN2 mRNA and protein in the infected cells. MTT assay and colony formation assay were used to detect the cell proliferation. Cell cycle distribution was measured by flow cytometry.Western blot was used to detect the expression of Ras,p-Raf and p-Erk1/2 proteins in infected cells. Results The expressions of MFN2 mRNA in DU-145 and LNCaP cells of Lenti-MFN2 group were 2.79±0.91 and 3.87±1.06, which were higher than those in Lenti-GFP group (1.02± 0.27 and 1.13±0.59),the differences were statistically significant(t=3.726,P=0.010;t=5.209,P =0.002). Compared with Lenti-GFP group, the expression of MFN2 protein in Lenti-MFN2 group was increased. The number of colonies formed in DU-145 and LNCaP cells of Lenti-MFN2 group was 147.42±32.91 and 130.26± 62.47, respectively, which was lower than that of the Lenti-GFP group (255.46±50.91 and 238.10±49.77), the differences were statistically significant (t =3.565, P=0.012; t =2.700, P=0.036). The cell cycle was arrested at G0/G1phase,and the expressions of Ras, p-Raf and p-Erk1/2 proteins were significantly decreased. Conclusion MFN2 can inhibit the proliferation of prostate cancer cells,and its mechanism may be related to the inhibition of activation of Ras-Raf1-Erk1/2 signaling pathway.
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Objective To investigate the activation effect of microRNA-1280 (miR-1280) on the expression of p21 gene in bladder cancer cell line BIU-87 and its effect on cell cycle and proliferation of bladder cancer cell line.Methods Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expressions of miR-1280 in bladder cancer cell lines T24,5637,J82,BIU-87 and normal bladder epithelial cells SV-HUC-1.miR-1280 mimics (experimental group) and miR-NC (control group) were transfected into the bladder cancer cells with the lowest expression of miR-1280.The expressions of miR-1280 and p21 mRNA were detected by qRT-PCR.Chromatin immunoprecipitation (ChIP) was used to verify the targeting effect of miR-1280 and p21 gene promoter.Western blotting was used to detect the expressions of p21,cell cycle-dependent kinase 1 (CDK1),Cyclin A2 mRNA and protein in the two groups.Cell cycle was detected by flow cytometry,and cell proliferation was detected by methyl thiazolyl tetrazclium (MTT) assay.Results The results of qRT-PCR indicated that the expression levels of miR-1280 in bladder cancer cell lines T24,5637,J82 and BIU-87 and normal urothelium cell line SV-HUC-1 were 0.503 ±0.094,0.611 ±0.054,0.567 ± 0.077,0.257 ± 0.032 and 1.014 ± 0.090 respectively,with a significant difference (F =1.880,P ＜0.001).Compared with bladder cancer cell lines T24,5637 and J82 cells,the expression of miR-1280 in BIU-87 cell was the lowest (P =0.026,P =0.003,P =0.008).Compared with the control group,the expression of miR-1280 in BIU-87 cell was significantly increased (1 041.000 ± 157.500 vs.1.023 ± 0.118,t =6.606,P ＜0.001),and the expression of p21 mRNA was also significantly increased (5.280 ± 0.660 vs.1.007 ± 0.070,t =6.440,P ＜ 0.001).Western blotting showed that p21 protein expression was up-regulated,CDK1 and Cyclin A2 protein expressions were down-regulated.ChIP experiments showed that compared with the miR-NC transfection group,the concentration of biotin modified miR-1280 in the p21 gene promoter region was significantly increased (1.246 ±0.171 vs.0.519 ± 0.087,t =3.787,P =0.009).The proportion of G0-G1 cells in the experimental group BIU-87 cells was significantly higher than that in the control group (68.360％ ±3.064％ vs.46.970％ ±3.971％,t =4.263,P =0.005).The results of MTT showed that compared with the control group,the cell proliferation ability of BIU-87 cells after being transfected miR-1280 was significantly decreased starting from day 3 (0.826 ± 0.099 vs.1.224 ± 0.057,t =3.505,P =0.013).Conclusion miR-1280 can activate the expression of p21 gene in bladder cancer cell line BIU-87 by binding the promoter region of p21 gene,blocking the progression of cell cycle and inhibiting cell proliferation,which provides a new direction for bladder cancer targeted therapy theory.
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Objective To investigate the effects of microRNA-1291 (miR-1291) on the expression of Zinc finger protein 8 (PHF8) gene in renal cell carcinoma and its effect on cell cycle and proliferation of renal cell carcinoma.Methods Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-1291 in renal cell carcinoma cell lines OS-RC-2,ACHN,A498,786-O and human proximal tubular epithelial cells HK-2.miR-1291 (miR-1291 group) and miR-NC (miR-NC group) were transfected into the renal cell lines with the lowest expression of miR-1291.qRT-PCR was used to detect the expression of miR-1291 and PHF8 mRNA in the transfected cells.The expression levels of PHF8,Cyclin-dependent kinase 6 (CDK6) and Cyclin D1 were detected by Western blotting.The effect of miR-1291 on the transcriptional activity of PHF8 was detected by double luciferase reporter gene system.Flow cytometry was used to detect cell cycle distribution.Methyl thiazolyl tetrazolium (MTT) assay and colony formation assay were used to detect cell viability and proliferation.Results The expressions of miR-1291 in renal carcinoma cell lines OS-RC-2,ACHN,A498,786-O and human proximal renal tubular epithelial cell HK-2 were 0.64 ± 0.17,0.60 ±0.15,0.29 ±0.08,0.63 ±0.08 and 1.01 ±0.17 respectively,with a significant difference (F=13.790,P ＜ 0.001).Compared with renal carcinoma cell lines OS-RC-2,ACHN and 786-O,the expression level of miR-1291 in A498 cell line was the lowest (P =0.002,P =0.006,P =0.003).The expression levels of miR-1291 in A498 cell lines of miR-NC group and miR-1291 group were 1.00 ± 0.03 and 775.25 ± 329.91 respectively,with a significant difference (t =4.694,P =0.003);and the expression levels of PHF8 mRNA were 1.00 ±0.11 and 0.57 ±0.18 respectively,with a significant difference (t =4.122,P =0.006).The results of Western blotting were consistent with the results of qRT-PCR,and the expressions of CDK6 and Cyclin D1 were significantly decreased.The double luciferase reporter gene showed that miR-1291could directly inhibit the activity of luciferase in the 3'un-translated region of target gene PHF8.Compared with miR-NC group,the proportion of renal carcinoma cells in S phase (23.40 ± 4.29 vs.32.19 ± 2.64;t =3.491,P =0.013) and G2-M phase (14.38 ± 4.05 vs.25.59 ± 6.01;t =3.095,P =0.021) decreased;and the proportion of cells in G0-G1 phase increased (62.22 ± 7.56 vs.42.22 ± 5.23,t =4.351,P =0.005).MTT assay showed that the cell viability of miR-1291 was significantly decreased.Colony formation experiments showed that the numbers of colonies formed by A498 cells in miR-NC group and miR-1291 group were 246.64 ± 39.94 and 87.34 ± 21.93 respectively,with a significant difference (t =6.993,P ＜ 0.001).Conclusion The expression of miR-1291 is significantly decreased in renal cancer cell lines.miR-1291 can significantly inhibit the proliferation of renal cell carcinoma cells by targeting interfering PHF8 gene expression,which may contribute to the development of new renal cancer target.
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Objective To investigate the effect of miR-103b on the expression of P21 protein in renal cell carcinoma cell line 769-P and ACHN cells,and its effect on the growth of renal cell carcinoma.Methods Renal cancer cells were divided into two groups according to the transfected RNA,miR-103b (experimental group) and dsControl (control group),respectively.Real-time PCR and Western blotting were used to detect the expression of P21,cell cycle-dependent kinase 6,Cyclin D1 mRNA and protein expression.Flow cytometry was used to detect the cell cycle distribution.MTT assay was used to detect cell viability and colony formation assay was used to detect cell proliferation.Measurement data were represented as x ± s.Comparison between groups was analyed using t test.Results Real-time PCR results showed that the relative expression levels of P21,cell cycle-dependent kinase 6 and Cyclin D1 mRNA in 769-P and ACHN which belong to control group cells were 1.00 ±0.10 and 1.02 ±0.27,1.00 ±0.08 and 1.01 ±0.17,1.01 ±0.19 and 1.00 ±0.02.The experimental group was 2.36 ±0.51 and 2.03 ± 0.49,0.33 ± 0.20 and 0.58 ± 0.22,0.48 ± 0.11 and 0.60 ± 0.23,respectively,and the difference was statistically significant (P ＜ 0.05).Western blotting results were consistent with Real-time PCR results.Flow cytometry results showed that compared with the control group,the proportion of cells located in G0/G1 phase in the experimental group increased (P ＜ 0.05),suggesting that the cells were arrested in G0/G1 phase.MTT assay showed that the viability of 769-P and ACHN cells in the experimental group was significantly lower than that in the control group.Colony formation experiments showed that the number of colony formation in the experimental group was significantly less,suggesting that the cell proliferation capacity decreased.Conclusion miR-103b can inhibit the growth of renal cell carcinoma cells by activating the expression of P21 protein and blocking the progression of the renal cell cycle,which provides a theoretical basis for the molecular targeted therapy of renal cell carcinoma.
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Objective To investigate the regulatory effect of miRNA-370(miR-370)on the expression of tumor suppressor gene p21 in renal cell carcinoma cell lines ACHN and 786-O and its effect on cell growth. Methods RCC cells were transfected with dsRNA known lack homology to human genes (control group) and miR-370 (experimental group) by Lipofectamine 3000 respectively. Real-time fluorescence quantitative polynucleotide chain reaction (RT-qPCR) and Western blot were used to detect the expression of p21 mRNA and protein. The cell cycle distribution was identified by flow cytometry (FCM). Cell viability and proliferation ability were measured by cell viability assay (MTS) and colony culture assay. Results The expression of p21 mRNA in ACTN and 786-O cells in control group was 1.04±0.33, 1.04±0.31, respectively. The expression of p21 mRNA in experimental group was significantly increased by 3.68±0.62 (t=7.535, P<0.001), 3.15±0.29 (t=9.975, P<0.001). Western blot further demonstrated that the increased expression of p21 protein in both renal cell lines was consistent with the upregulation of p21 mRNA level. FCM results showed that the cell cycle of more cells was blocked in G0-G1phase after transfection of miR-370.MTS results showed that after transfection of miR-370,the number of colonies formed by ACHN and 786-O cells in the control group was 113±30 and 106±27 respectively. The number of colonies formed by experimental group was significantly reduced by 53±17 (t=2.982, P=0.041) and 50±16 (t=3.089, P=0.037). Conclusion miR-370 can significantly up-regulate the expression of tumor suppressor gene p21 in renal cell carcinoma and inhibit the growth of renal cell carcinoma.
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Objective To investigate the effect of dsP21-555 transfection on the expression of tumor suppressor gene p21 in renal clear cell carcinoma cell lines ACHN and 786-O.Methods Renal clear cell carcinoma cells were transfected with dsControl and dsP21-555 with Lipofectamine 3000 respectively.Real-time quantitative PCR (RT-qPCR) and Western blotting were used to detect the expression of p21 mRNA and protein.Cell cycle distribution was detected by flow cytometry (FCM).Cell viability and proliferation were analyzed by cell viability assay (MTS method) and colony culture assay.Results In ACHN and 786-O cells, the expressions of p21 mRNA in dsP21-555 group (2.86±0.33, 1.96±0.35) were significantly higher than those in dsControl group (1.05±0.34, 1.01±0.14), which were increased to 2.72 times (t=7.640, P<0.001) and 1.95 times (t=5.058, P=0.002).Western blotting showed that the expressions of P21 protein were up-regulated in both renal cell lines, which was consistent with p21 mRNA up-regulation.The result of FCM showed that the cell cycle was blocked in G0-G1 phase (57.08%±5.66% vs.46.06%±4.60%, t=3.023, P=0.023;61.58%±6.23% vs.42.25%±6.08%, t=4.444, P=0.004) after transfection of dsP21-555 in renal clear cell carcinoma cells.MTS result showed that the vitality of both cell lines after transfection of dsP21-555 decreased compared with dsControl group, their absorbance values were 0.85±0.20 vs.1.27±0.13, t=3.410, P=0.014;1.04±0.25 vs.1.55±0.10, t=3.758, P=0.009.Colony culture experiments showed that the numbers of colonies formed by ACHN and 786-O in the dsControl group were 110.91±26.21 and 129.99±22.87 respectively, and the numbers of colonies formed in the dsP21-555 group were 59.37±14.23 (t=3.456, P=0.014) and 71.26±21.38 (t=3.745, P=0.010), indicating that the proliferation of cells in the dsP21-555 group was significantly reduced.Conclusion dsP21-555 can up-regulate the expression of p21 gene in renal clear cell carcinoma cells and inhibit the growth of carcinoma cells, suggesting that dsP21-555 may become a new gene therapy tool.
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Objective To investigate the effect of microRN-206 (miR-206) on the expression of Cyclin-dependent kinase 4 (CDK4) and Cyclin G-associated protein kinase (GAK), and the growth of prostate cancer cells.Methods Prostate cancer cell lines DU-145 and PC-3 were transfected with miR-NC (the control group) or miR-206 (the experimental group).The expressions of CDK4 and GAK mRNA were detected by real-time quantitative PCR (qRT-PCR).The expressions of CDK4 and GAK protein were detected by Western blotting.Cell cycle distribution was detected by flow cytometry.EdU proliferation assay and colony forming assay were used to analyze the cell proliferation ability.Results In DU-145 and PC-3 cells, the expressions of CDK4 mRNA in miR-NC group were 1.00±0.09, 1.00±0.10, the expressions of GAK mRNA were 1.00±0.05, 1.00±0.06.The expressions of CDK4 mRNA in miR-206 group were significantly decreased in DU-145 (0.36±0.18;t=6.572, P=0.001) and PC-3 cell lines (0.43±0.17;t=5.794, P=0.001).The expressions of GAK mRNA were also significantly decreased in DU-145 (0.23±0.04;t=22.420, P<0.001) and PC-3 cell lines (0.32±0.08;t=14.500, P<0.001).Western blotting results were consistent with qRT-PCR results.The results of flow cytometry showed that compared with the miR-NC group of DU-145 and PC-3 cell lines, the percentage of cells in S phase (23.60%±5.68% vs.32.53%±4.52%, t=2.462, P=0.049;22.09%±4.35% vs.30.96%±4.86%, t=2.720, P=0.035) and G2-M phase (16.28%±7.12% vs.26.63%±4.33%, t=2.484, P=0.048;14.60%±1.62% vs.24.68%±7.13%, t=2.758, P=0.033) decreased after transfection of miR-206, and the percentage of cells in G0-G1 phase (60.13%±5.82% vs.40.84%±5.37%, t=4.872, P=0.003;63.31%±3.27% vs.44.36%±3.82%, t=7.533, P<0.001) increased.The results of EdU proliferation assay showed that the proliferation abilities were significantly attenuated after transfection of miR-206 (22.56±3.81 vs.38.90±8.51, t=3.503, P=0.013;25.12±6.42 vs.48.45±8.92, t=4.244, P=0.005).The results of colony formation experiments showed that the numbers of colonies formed by DU-145 and PC-3 in miR-NC group were 218.66±44.59 and 177.35±24.49, respectively.The numbers of colonies formed in miR-206 group were 125.38±32.80 (t=3.370, P=0.015) and 82.65±14.05 (t=6.708, P=0.001), suggesting that cell proliferation ability in miR-206 group was reduced.Conclusion miR-206 significantly inhibits the growth of prostate cancer cells by interfering with the expressions of CDK4 and GAK, suggesting that miR-206 may be a molecular targeted therapy tool for prostate cancer.