MicroRNA-645 promotes cell metastasis and proliferation of renal clear cell carcinoma by targeting GK5.
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Objective To dissect the functioning mode of miR-645 on renal clear cell carcinoma cell metastasis and growth, and provide therapeutic targets for renal clear cell carcinoma. Patients and methods Quantitative Real-time PCR (qRT-PCR) assay was employed to detect miR-645 expression level. Wound healing assay and transwell assay were performed to investigate metastasis capacity of renal clear cell carcinoma cells. Cell Counting Kit 8 (CCK8) assay was incorporated to assess cell proliferation capacity. Flow cytometry was used to identify cell apoptosis and cell cycle distribution. Protein levels were assessed by Western blotting assay. The target gene was predicted and verified by bioinformatics analysis and luciferase assay. Results MiR-645 was upregulated in renal clear cell carcinoma tissues when compared with para-carcinoma tissues (n=32). Downregulated miR-645 could attenuate cell migration and invasion capacities, as well as inhibited cell proliferation capacity, promoted cell apoptosis and cell cycle arrest at G0/G1 phase. GK5 was chosen as the target gene of miR-645 by bioinformatics analysis and luciferase reporter assay. Moreover, silence of GK5 could rescue tumor suppression role of downregulated miR-645 on renal clear cell carcinoma metastasis. Conclusions Knockdown of miR-645 exerted tumor-suppressive effects on renal clear cell carcinoma metastasis and growth via targeting GK5 in vitro, which provided an innovative and candidate target for diagnose and treatment of renal clear cell carcinoma.Cite
The purpose of this study was to explore the functional role and mechanism of miR-155 in the development of renal cell carcinoma (RCC). miR-155 expression was quantified in renal cancers, matched adjacent non‑tumor tissues and renal cell lines using quantitative real-time PCR (RT-PCR). Cell proliferation, apoptosis and migratory activity were measured following suppression of miR-155 expression by antisense oligonucleotides. miR-155 targets were scanned using target prediction programs. Following the inhibition of miR-155, target gene expression was detected by western blotting. The expression of miR-155 was upregulated in clear cell RCC (ccRCC) tissue and renal cancer cell lines. The suppression of miR-155 inhibited cell proliferation and migratory activity and induced apoptosis in renal cancer cells. The suppressor gene suppressor of cytokine signaling (SOCS-1) and BACH1 were predicted as potential target genes by bioinformatics analysis. The suppression of miR-155 inhibited BACH1 protein expression. miR-155 may function as an oncogene by targeting BACH1. Thus, the inhibition of miR-155 may be an effective way to treat RCC.
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Pancreatic cancer (PC) is the most malignant tumor among all the tumors in the digestive system. MiR-217 has been reported to take a critical part in various malignant tumors. The aim of this study was to explore the function of MiR-217 in pancreatic cancer and its target genes.Twenty pairs of PC tissues and matched normal adjacent pancreatic tissues were collected. The expression of miR-217 in PC tissues and normal pancreatic tissues was detected by Real-time polymerase chain reaction (PCR). PC cells were transfected with miR-217 mimics, inhibitors and negative control, respectively. Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability. Cell apoptosis was checked via Annexin V-FITC/PI apoptosis kit. The protein expression of E2F3 was detected by Western blot. To detect repression by miR-217, HEK293T cells were co-transfected with the indicated E2F3 3'-UTR luciferase reporter.The expression of miR-217 was reduced in PC tissues comparing to normal pancreatic tissues. Meantime, the in-vitro study revealed that miR-217 suppressed PC cell growth, invasion but promoted apoptosis. Next, we proved that E2F3 was the target of miR-217 on PC cell function.miR-217 suppresses PC cell growth, invasion but promotes apoptosis in vitro through targeting E2F3. The miR-217-E2F3 axis may be used for PC therapy.
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Ovarian cancer is a common malignant cancer among women. Increasing studies have demonstrated that microRNAs function as important regulation factors in the progression of ovarian cancer.Human ovarian cancer cell lines HO8910 and OVCAR-3 were transfected with miR-934 inhibitor and corresponding negative control (inhibitor control). Cell proliferation and apoptosis were detected by cell counting kit-8 (CCK-8) and TUNEL assay, respectively. The expression levels of proliferation/apoptosis-related genes and BRMS1L were measured by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blotting. Furthermore, the association between miR-934 and BRMS1L was investigated through luciferase reporter assays.MiR-934 was significantly increased in ovarian cancer cell lines, whereas BRMS1L was significantly decreased. Downregulated miR-934 remarkably inhibited cell proliferation and induced cell apoptosis. Meanwhile, miR-934 could influence the expression levels of Ki67, Cyclin D1, Caspase3, and Bcl-2. In addition, BRMS1L was identified as a target gene of miR-934.Oncogene miR-934 promotes ovarian cancer cell proliferation and inhibits cell apoptosis through targeting BRMS1L. MiR-934 and BRMS1L may be novel biomarkers or therapeutic targets for ovarian cancer in the future.
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MicroRNA (miR)‑217 serves a pivotal role in the progression of colorectal cancer, renal cell carcinoma and glioma, however, the role of miR‑217 in cervical cancer (CC) remains unclear. In the present study, the mechanism of miR‑217 in cervical cancer was explored. The mRNA expression of miR‑217 and mitogen‑activated protein kinase 1 (MAPK1) were assessed using reverse transcription‑quantitative polymerase chain reaction analysis. Cell Counting‑Kit 8, wound‑healing and Transwell assays were performed to detect cell viability, migration and invasion, respectively. Apoptosis and cell cycle were determined by flow cytometry. TargetScan 7.2 and dual‑luciferase reporter assays were respectively used to determine miR‑217 target genes and their binding capacities. The protein expression levels of MAPK1, phosphorylated (p)‑extracellular signal‑regulated kinase 1/2 (ERK1/2)/ERK1/2, Bcl‑2, Bax and cleaved caspase‑3 were quantified by western blotting. It was found that miR‑217 was downregulated in patients with CC and in CC cells. The viability, migration and invasion of cells were suppressed by a miR‑217 mimic. It was also found that apoptosis was increased and cell cycle was inhibited by the miR‑217mimic, which was supported by changes in Bcl‑2, Bax and cleaved caspase‑3. MAPK1 was upregulated in patients with CC and was a target gene of miR‑217. MAPK1 reversed the inhibition of miR‑217 on cell viability, migration, invasion and apoptosis. The protein levels of MAPK1 and p‑ERK1/2, which were higher in the mimic MAPK1 group than those in the control or mimic groups, were ameliorated by PD98059. The results of the present study demonstrated that miR‑217 had an anti‑CC effect and may be effectively used in the treatment of CC.
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MicroRNAs (miRs) are critical regulators in cancer development and progression. The current study aimed to investigate the expression and potential function of miR-181a in thyroid cancer.A total of 15 paired thyroid cancer tissues and adjacent normal tissues were subjected to Real-time Polymerase Chain Reaction (PCR) to evaluate miR-181a expression. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, enzyme linked immunosorbent assay (ELISA) or flow cytometry was employed to assess the growth activity, apoptosis and cell cycle, respectively, upon modulation of the miR-181a expression in TPC-1 cells. Western blot was used to assess protein expression. The interaction between miR-181a and RB1 was tested by luciferase activity assay.The expression of miR-181a was significantly upregulated in thyroid cancer tissues compared with the adjacent tissues. Inhibition of miR-181a attenuated cell growth, which could be abrogated by miR-181a co-transfection. MiR-181a overexpression reduced apoptosis and promoted cell cycle progression; inhibition of miR-181a exerted opposite effects on both cell cycle and apoptosis. MiR-181a directly suppressed RB1 expression. RB1 expression in tumor tissues was downregulated and negatively correlated with miR-181a expression.miR-181a plays an oncogenic role in thyroid cancer; by targeting RB1, it promotes cell cycle progression and inhibits apoptosis.
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The downregulation of microRNA-15a has been reported in several human tumors. However, its expression and functional importance in renal cell carcinoma (RCC) remain unknown. The aim of the present study was to investigate its expression, biological functions and underlying mechanisms in RCC tumorigenesis. The expression levels of miR-15a were examined by qRT-PCR in 40 RCC specimens and adjacent‑paired normal tissues. Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry and Transwell assays were used to explore the potential influence of miR-15a transfection on RCC cell proliferation, the cell cycle, cell apoptosis, and cell invasion. Luciferase reporter assays were performed to confirm the potential target of miR-15a, in combination with qRT-PCR, western blotting and immunohistochemical assays. We found that miR-15a was significantly downregulated in most RCC specimens compared with adjacent normal tissues (P<0.01). Overexpression of miR-15a inhibited cellular growth, suppressed invasion and arrested cells at the G1/G0 phase, and induced cell apoptosis in RCC cells. Luciferase assays revealed that miR-15a directly targeted the binding site of the 3'-untranslated region (3'-UTR) of eIF4E, and inhibited its expression at both mRNA and protein levels. eIF4E expression was negatively associated with miR-15a expression in RCC tissues. eIF4E overexpression treatment partially abrogated the inhibitory effect of miR-15a on cell proliferation and invasion, as well as inactivated P13K/AKT/mTOR signaling in RCC cells. In conclusion, the present study indicated that miR-15a downregulation was associated with cell proliferation and invasion by directly targeting eIF4E during RCC progression. Thus, it may serve as a potential tumor suppressor and therapeutic target for the treatment of RCC.
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MicroRNA‑335 (miR‑335) was reported to suppress cell proliferation in prostate cancer (PC), a common malignancy in males. The expression of early growth response 3 (EGR3) was determined to be elevated in human PC tissues; however, the possible effects and underlying mechanism of miR‑335 on PC remains unknown. In the present study, miR‑335 mimics and miR‑335 inhibitors were respectively transfected into DU145 cells. Stable silencing of EGR3 was observed in DU145 cells following transfection with small interfering RNA. We also used Cell Counting Kit‑8 and in vitro angiogenesis assays to determine the viability and revascularization potential of DU145 cells. The expression levels of EGR and caspase‑3 activity were analyzed by immunohistochemistry and immunocytochemistry, respectively. We predicted the target of miR‑335 by bioinformatics analysis and a dual‑luciferase reporter gene assay. Western blot and quantitative real‑time polymerase chain reaction analyses were performed to determine the protein and mRNA expression of molecules. miR‑335 expression was downregulated in PC tissues and cell lines. Overexpression of miR‑335 significantly reduced the viability and the formation of regenerative tubes of DU145 cells, and inhibited the expression of inflammatory factors. EGR3 was proposed as a possible target of miR‑335, and was negatively regulated by miR‑335. Silencing EGR3 suppressed the viability and angiogenesis of DU145 cells, and reduced the activity of caspase‑3 and inflammatory factor expression. miR‑335 inhibition along with EGR3 silencing EGR3 inhibited the cell proliferation. Furthermore, miR‑335 inhibited the formation of a PC solid tumor xenograft in vivo. Thus, miR‑335 may exert an antitumor effect on DU145 cells by regulating the expression of EGR3. The findings of the present study may provide insight into a novel therapeutic strategy for the treatment of prostatic carcinoma.
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MicroRNA-204 (miR-204) has been reported to be frequently downregulated in various types of cancer, including renal, brain, ovary, hematological and colon cancer. The present study, investigated the effects of miR‑204 on renal cell carcinoma. Following transfection of miR‑204, an MTT assay, cell migration assay, cell invasion assay, western blot analysis and luciferase assay were performed in renal cell carcinoma cell lines. It was demonstrated that miR‑204 inhibits cell proliferation, migration and invasion in 786‑O and A498 cells. To the best of our knowledge, this study is the first to demonstrate that miR‑204 directly targets SOX4 in renal cell carcinoma. These results suggested that miR-204 may have value as a marker for the early detection of tumor metastasis and a therapeutic target preventing the invasion of renal cell carcinoma.
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Breast cancer causes significant mortality in women world over. The lack of efficient and reliable biomarkers and therapeutic targets impedes the treatment of breast cancer. Herein, the role and therapeutic potential of miR-155 was investigated in different breast cancer cell lines Methods: Cell viability was determined by WST-1 and colony formation assays. Transfections were performed by Lipofectamine 2000 reagent. Cell cycle analysis was carried out by flow cytometry and apoptosis was detected by AO (acridine orange)/EB (ethidium bromide) staining. Cell migration and cell invasion were determined by wound healing assay. RNA and protein expressions were determined by qRT-PCR and western blotting, respectively.miR-155 was significantly upregulated in all the breast cancer cells. Suppression of miR-155 in SK-BR-3 cells inhibited the growth and colony formation. The inhibition of SK-BR-3 cell proliferation was found to trigger apoptotic cell death and cell cycle arrest. Induction of apoptosis was also accompanied with enhancement of cytochrome c, Bax caspase 3, 8 and 9and inhibition of Bcl-2. Besides, suppression of miR-155 resulted in the decrease of cell migration and invasion. Bioinformatic analysis revealed MAPK7 to be the potential target of miR-155. The MAPK7 expression was also upregulated in all the breast cancer cells and suppression of miR-155 resulted in its downregulation.Taken together, miR-155 may prove essential in the management of breast cancer.
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Renal cell carcinoma (RCC) is one of the most common types of cancer of the urinary tract in the world. Long non‑coding RNA MALAT1 (lncR‑MALAT1) is upregulated in RCC and is associated with the proliferation and migration of RCC. The present study aimed to investigate the regulating role of lncR‑MALAT1 in RCC as well as the possible underlying mechanisms. The relative expression of MALAT1 and miR‑22‑3p in RCC tumor tissues and cell lines was detected by qRT‑PCR. CCK‑8 and wound healing assay were used to evaluate cell proliferation and migration ability. Western blot analysis was used to detect the expression of Ki‑67, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase‑3 (MMP‑3), migration and invasion inhibitory protein (MIIP), p‑PI3K and p‑Akt. The relationship between MALAT1 and miR‑22‑3p was examined by bioinformatic prediction analysis and luciferase reporter assay. Immunofluorescence was used to detect the activation of Akt. MALAT1 was highly expressed and the expression of miR‑22‑3p was suppressed in RCC tissues and cell lines. ShRNA‑mediated knockdown of MALAT1 significantly inhibited the viability and mobility of RCC cells in vitro and in vivo. Further experiments revealed that miR‑22‑3p was a target of MALAT1 and that miR‑22‑3p inhibitor abolished the effect of MALAT1 shRNA on cell proliferation, migration and inactivation of PI3K/AKT pathway. In conclusion, lncR‑MALAT1 affected the proliferation and migration of RCC cells by targeting miR‑22‑3p through the inactivation of the PI3K/Akt signaling pathway.
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