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Objective To explore the expression of miR-101-3p in gastric cancer and its mechanism on the invasion, metastasis, and angiogenesis of gastric cancer cells by targeting the STC-1 gene to regulate the PI3K/AKT signaling pathway. Methods qRT-PCR was used to detect the expression of miR-101-3p and STC-1 mRNA in gastric cancer tissues and BGC-823 cell and analyze the relationship between miR-101-3p expression and patients' clinical pathological factors. The cells were transfected with miRNA mimics and plasmids separately or in combination with LipofectamineTM 2000. TargetScanHuman prediction and dual-luciferase assay were used to verify the targeted regulation of miR-101-3p on STC-1. The effect and possible mechanism of miR-101-3p targeting the STC-1 gene on the invasion, metastasis, and angiogenesis of cancer cells were verified by scratch test, Transwell chamber test, Matrigel in vitro tube forming test, and Western blot assay. The development of the transplanted tumor was detected by nude mouse tumorigenicity test. Results The expression of STC-1 in gastric cancer tissues was higher than that in normal tissues. Compared with normal gastric tissues and GES-1 cells, miR-101-3p was down-regulated, and STC-1 mRNA was up-regulated in gastric cancer tissues and BGC-823 cell. The level of miR-101-3p was negatively correlated with the level of STC-1, and significantly correlated with the degree of tumor differentiation, TNM stage, and lymph node metastasis (P < 0.05). miR-101-3p directly targeted STC-1. The overexpression of miR-101-3p inhibited STC-1 expression and downregulated the expression of p-PI3K/PI3K, p-AKT/AKT, MMP-2, MMP-9, VEGF, and Ang2, consequently, inhibited tumor cell invasion, metastasis, and angiogenesis and reduced the size and weight of the transplanted tumors (P < 0.05). Conclusion miR-101-3p is down-regulated in gastric cancer and can target the STC-1 gene to regulate the PI3K/AKT signaling pathway and inhibit the invasion, metastasis, and angiogenesis of BGC-823 gastric cancer cells and the development of transplanted tumors in vivo.
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Patients with osteosarcoma (OS) usually have poor overall survival because of frequent metastasis. Long non-coding RNAs (lncRNAs) have been reported to be associated with tumorigenesis and metastasis. In this study, we investigated the expression and roles of lncRNA human histocompatibility leukocyte antigen (HLA) complex P5 (HCP5) in OS, aiming to provide a novel molecular mechanism for OS. HCP5 was up-regulated both in OS tissues and cell lines and high expression of HCP5 was associated to low survival in OS patients. Down-regulation of HCP5 inhibited cell proliferation, migration, and invasion, suggesting its carcinogenic role in OS. miR-101 was targeted by HCP5 and its expression was decreased in OS. The inhibitor of miR-101 reversed the impact of HCP5 down-regulation on cell proliferation, apoptosis, and metastasis in OS. Ephrin receptor 7 (EPHA7) was proved to be a target of miR-101 and had ability to recover the effects of miR-101 inhibitor in OS. In conclusion, lncRNA HCP5 knockdown suppressed cell proliferation, migration, and invasion, and induced apoptosis through depleting the expression of EPHA7 by binding to miR-101, providing a potential therapeutic strategy of HCP5 in OS.
Subject(s)
Humans , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Osteosarcoma/genetics , Osteosarcoma/pathology , MicroRNAs/metabolism , RNA, Long Noncoding/genetics , Down-Regulation , Gene Expression Regulation, Neoplastic , Cell Movement , Receptor, EphA7/metabolism , Cell Line, Tumor , Cell Proliferation , Neoplasm InvasivenessABSTRACT
@#[Abstract] Objective: To investigate the molecular mechanism of microRNA-101 (miR-101) inhibiting the migration and invasion of non-small cell lung cancer (NSCLC) via targeting fibroblast growth factor 2 (FGF2). Methods: qPCR was used to detect the expression levels of miR-101 and FGF2 in human normal lung epithelial BEAS-2B cells and NSCLC cell lines (A549, H661 and SK-MES-1) as well as A549 cells after transfection. MiR-NC, miR-101 mimics, miR-IN-NC, miR-101 inhibitor or pcDNA-3.1 empty plasmid, pcDNA-FGF2 were respectively transfected into A549 cells. Wound healing assay and Transwell assay were used to examine the effects of overexpression of miR-101 and FGF2 on the migration and invasion of A549 cells. Western blotting(WB) was used to detect the expression levels of FGF2, E-cadherin, N-cadherin, Vimentin, ERK1/2 and p-ERK1/2 in A549 cells in each group. Results: The expression level of miR-101 in NSCLC cell lines were significantly lower than that in normal lung epithelial cells (all P<0.05), while the expression level in A549 cells was the lowest. Overexpression of miR-101 significantly inhibited the migration (P<0.05) and invasion (P<0.01) of A549 cells, increased the expression level of E-cadherin but decreased the expression level of Vimentin (P<0.05),N-cadherin (P<0.01) and p-ERK1/2 (P<0.05). Inhibition of miR-101 significantly enhanced the invasion and migration of A549 cells (all P<0.05), decreased the expression level of E-cadherin but increased the expression levels of Vimentin, N-cadherin and p-ERK1/2 (all P<0.05). The results of WB and Dual-luciferase reporter gene assay verified that FGF2 is a direct target gene of miR-101, and over‐expression of FGF2 significantly enhanced the invasion and migration of A549 cells (all P<0.01), decreased the expression of E-cadherin (P<0.01) but increased the expressions of Vimentin (P<0.01), N-cadherin (P<0.05) and p-ERK1/2 (P<0.01). Compared with the FGF2 overexpression alone group, co-overexpression of miR-101 and FGF2 significantly reduced the invasion and migration of A549 cells (all P<0.01), increased the expression of E-cadherin (P<0.01), and decreased the expressions of Vimentin (P<0.01), N-cadherin (P<0.05) and p-ERK1/2 (P<0.01). Conclusion: By targeting FGF2, miR-101 inhibits the invasion and migration of NSCLC cells through suppressing the epithelial-mesenchymal transition (EMT) and ERK signaling pathway.
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Objective To investigate the effects of miR-101 expression on retinoblastoma cell proliferation and invasion.Methods A total of 31 cases of retinoblastoma tissues and 7 cases of normal retinal tissues were collected.Human normal retinal vascular endothehal cell line ACBRI-181 and retinoblastoma cell line HXO-Rb44 were cultured.HXO-Rb44 cells were transfected with Lipofectamine 2000 and grouped as follows:negative control (NC) group 1,in which cells were transfected with miRNA-101 negative controls,miR-101 expression group,cells transfected with miRNA-101 mimics;NC group 2,in which cells were transfected with histone-lysine N-methyltransferase (EZH2) negative controls,siRNA-EZH2 group,cells transfected with EZH2 siRNA,siRNA-EZH2 + mimics group,cells transfected with EZH2 siRNA and miRNA-101 mimics,and EZH2 + mimics group,cells transfected with EZH2 expression vector and miRNA-101 mimics.Normal HXO-Rb44 cells were served as blank group,miR-101 and EZH2 mRNA expression were detected by qRT-PCR,and EZH2 protein expression was measured by Western blot.Cell proliferation and invasion ability were determined by MTT and Transwell assays,respectively.Luciferase reporter assay was used to assess the targeting relationship between miR-101 and EZH2.Xenograft in nude mice was performed to detect cell proliferation ability in vivo.Results Compared with normal retinal tissues and ACBRI-181 cells,the relative expression of miR-101 in retinoblastoma tissues and HXO-Rb44 cells was significantly down-regulated (both P ＜ 0.05).The relative expression of EZH2 mRNA and protein in the miR-101 expression group was significantly lower than that in the blank group and NC group 1 (all P ＜ 0.05).At 72-96 h,the A values of the miR-101 expression group was significantly lower than those of the blank group and NC group 1 (both P ＜0.01).The number of invasive cells in the miR-101-expressing group (51 ± 6) was significantly lower than that of the blank group (97 ± 11)and NC group 1 (92 ± 8) (both P ＜ 0.01).EZH2 was the target gene of miR-101.At 48-96 h,the A values of the siRNA-EZH2 group and siRNA-EZH2 + mimics group were significantly lower than those of the blank group and NC group 2 (all P ＜0.01).At 72-96 h,the A values of the siRNA-EZH2 + mimics group were obviously lower than those of the siRNA-EZH2 group (both P ＜ 0.05).At 24-96 h,the A values of EZH2 + mimics group were not statistically different from the blank group or NC group 2 (all P ＞ 0.05).The number of invasive cells in the siRNA-EZH2 group (48 ± 4) and siRNA-EZH2 +mimics group (38 ±3) was significantly lower than that in the blank group (95 ± 10) and NC group 2 (90 ±6) (all P ＜0.01),and siRNA-EZH2 + mimics group was significantly lower than siRNA-EZH2 group (P ＜0.05).The number of invading cells of the EZH2 +mimics group (101 ± 11) was not statistically different from the blank group and NC group 2 (both P ＞ 0.05).At 5-7 weeks,the tumor volumes of siRNA-EZH2 group and siRNA-EZH2 + mimics group were significantly lower than those of the blank group and NC group 2 (all P ＜ 0.01),and siRNA-EZH2 + mimics group was significantly lower than the siRNA-EZH2 group (P ＜ 0.05).There was no significant difference in the tumor volume between the EZH2 + mimics group and the blank group or the NC group 2 (all P ＞ 0.05).Conclusion Up-regulation of miR-101 expression can inhibit the proliferation and invasion of HXO-Rb44 cells,which might be achieved by inhibiting the expression of EZH2.
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Emerging evidence has indicated that circular RNAs (circRNAs) play pivotal roles in the regulation of cellular processes and are found to be aberrantly expressed in a variety of tumors.However,the clinical role of circRNAs in bladder cancer (BC) and the molecular mechanisms have yet to be fully understood.In this study,the clinical specimens were obtained and the expression level of a circRNA BCRC4 was detected by real-time PCR in both BC tissues and cell line.The circular RNA over-expression plasmid was constructed and transfected into BC cells and related cell line.The cell cycles and apoptosis were observed using inverted microscope and flow cytometry.Western blotting was used to compare the relative protein expression of groups with different treatments.It was found that circRNA BCRC4 expression was lower in BC tissues than in adjacent normal tissues.Furthermore,consequences of fomed-expression of BCRC4 promoted apoptosis and inhibited viability of T24T and UMUC3 cells,and up-regulated BCRC4-inereased miR-101 level,which suppressed EZH2 expression in both RNA and protein levels.In addition,gambogic acid (GA) is a promising natural anticancer compound for BC therapy,and GA treatment increased the BCRC4 expression in T24T and UMUC3 cells in a dose-dependent manner.Altogether,our findings suggest that BCRC4 functions as a tumor suppressor in BC,and mediates anticancer function,at least in part,by up-regulating the expression of miR-101.Targeting this newly identified circRNA may help us develop a novel strategy for treating human BC.
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Objective To investigate the effect and underlying mechanism of micro ribonucleic acid (miR)-101 on the epithelial-mesenchymal transition in human hepatocellular carcinoma MHCC97H cells. Methods MHCC97H cell lines were transfected with miR-101 mimics (M101 group) and negative control mimic (NCM group), and the cell lines without transfection were used as the control group. The expression levels of miR-101 in cells of 3 groups were quantitatively measured using reverse transcription polymerase chain reaction (RT-PCR). Transwell assay was performed to evaluate the cell migration and invasion ability of 3 groups. The expression levels of vimentin, α-catenin, E-cadherin and USP22 proteins in cells of 3 groups were measured by Western blot. The relationship between miR-101 and USP22 was analyzed by dual-luciferase assay. Results In the M101 group, the expression level of miR-101 was significantly up-regulated, which was approximately 761 times of that in the control group (P<0.05). In the M101 group, the quantity of migrating cells was 15.7±1.6, significantly lower than 94.1± 1.8 in the control group (P<0.05). In the M101 group, the quantity of invasive cells was 9.1±0.4, significantly lower compared with 51.6±0.9 in the control group (P<0.05). In the M101 group, the expression levels of vimentin and USP22 protein were significantly down-regulated, whereas the expression levels of α-catenin and E-cadherin protein were significantly up-regulated. Dual-luciferase assay revealed that USP22 was the target gene of the downstream miR-101 signaling pathway. Conclusions miR-101 regulates the expression of epithelial-mesenchymal transition-related proteins and suppresses the epithelial-mesenchymal transition of hepatocellular carcinoma MHCC97H cells probably through down-regulating the expression level of USP22.
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Objective To study the effects of microRNAl01 (miR-101)on radiosensitization of human uterine cervix cancer HeLa cells and underlying mechanism.Methods HeLa cells were divided into three groups including blank control,miRNA negative control and miR-101 transfection group.The cells were irradiated by 160 kVp X-ray generated from a linear accelerator at a dose rate of 1.15 Gy/min.Real-time quantitative PCR (qRT-PCR) was used to detect the expression of miR-101.The clonogenic survival assay was applied to evaluate the effect of miR-101 on radiosensitization of HeLa cells.γ-H2AX immunofluorescence and Western blot assays were performed to observe DNA double-strand breaks and the protein expressions of ATM and DNA-PKcs of HeLa cells,respectively.Results Compared with the negative control group,the expression of miR-101 was significantly increased in the HeLa cells at 48 h after transfection with miR-101 mimic,and the survival of HeLa cells over expression of miR-101 was significantly reduced(t =10.75,P ＜ 0.05).The miR-101 had remarkable radiosensitive effect on HeLa cells(F =7.72,P ＜0.05) with a SERD0 of 1.29.Moreover,over-expression of miR-101 could inhibit the repair of DNA damage induced by irradiation.Compared with the control group,the protein expressions of ATM and DNA-PKcs were significantly decreased in the HeLa cells over expression of miR-101.Conclusions Over-expressions of miR-101 could inhibit cell growth and enhance radiosensitivity of HeLa cells by inhibiting the repair of radiation-induced DNA damage.
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Objective: To explore the effect of microRNA-101 on apoptosis of condylar cartilage cells and the specific mechanism of molecular biology. Methods: IL-1 was used to stimulate and establish the model of apoptosis of condylar cartilage cells. The expression change of miR-101 in control group was compared with that in IL-1 stimulation group by qRT-PCR. Overexpression and down-regulation models of miR-101 were established by transfecting Mimics and Inhibitor and verified by qRT-PCR. Flow cytometry was used to detect the effect of miR-101 overexpression and down-regulation on apoptosis. Target gene of miR-101 was analyzed and calculated through bioinformatics. Western blot and Luciferase report assay were used to detect whether SOX9 could become the target gene of miR-101. Results: qRT-PCR results showed that IL-1 stimulation could cause the increase of miR-101 expression. After the transfection of rabbit condylar cartilage cells by Mimics and Inhibitor, qRT-PCR results confirmed the significant effect of miR-101 overexpression and down-regulation. It was confirmed by flow cytometry that overexpression of miR-101 could promote the apoptosis of condylar cartilage cells, and down-regulation of miR-101 could reduce the apoptosis. It was confirmed by Western blot and Luciferase report assay that SOX9 was the target gene of miR-101, and miR-101 inhibited SOX9 expression through complementary pairing with 3'UTR of SOX9 mRNA. Conclusions: miR-101 can promote the apoptosis of condylar cartilage cells through inhibiting the protein level of target gene SOX9.
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Objective:To explore the effect of microRNA-101 on apoptosis of condylar cartilage cells and the specific mechanism of molecular biology. Methods: IL-1 was used to stimulate and establish the model of apoptosis of condylar cartilage cells. The expression change of miR-101 in control group was compared with that in IL-1 stimulation group by qRT-PCR. Overexpression and down-regulation models of miR-101 were established by transfecting Mimics and Inhibitor and verified by qRT-PCR. Flow cytometry was used to detect the effect of miR-101 overexpression and down-regulation on apoptosis. Target gene of miR-101 was analyzed and calculated through bioinformatics. Western blot and Luciferase report assay were used to detect whether Sox9 could become the target gene of miR-101. Results:qRT-PCR results showed that IL-1 stimulation could cause the increase of miR-101 expression. After the transfection of rabbit condylar cartilage cells by Mimics and Inhibitor, qRT-PCR results confirmed the significant effect of miR-101 overexpression and down-regulation. It was confirmed by flow cytometry that overexpression of miR-101 could promote the apoptosis of condylar cartilage cells, and down-regulation of miR-101 could reduce the apoptosis. It was confirmed by Western blot and Luciferase report assay that Sox9 was the target gene of miR-101, and miR-101 inhibited SOX9 expression through complementary pairing with 3’UTR of Sox9 mRNA. Conclusions:miR-101 can promote the apoptosis of condylar cartilage cells through inhibiting the protein level of target gene SOX9.
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Background and purpose:miR-101 has been reported to be down-regulated in gastric cancer, colorectal cancer, breast cancer as well as prostate cancer acting as a tumor suppressor gene. However, its function in ovarian cancer is still unknown. The aim of this study was to investigate whether miR-101 can suppress cell growth and invasion of ovarian cancer cells by targeting DNMT3A, so as to reveal molecular mechanism to inhibit ovarian cancer. Methods:Quantitative real-time palymerase chain reaction (qRT-PCR) method was employed to detect the expression of miR-101 in ovarian cancer and cancer adjacent normal ovarian tissues. SKOV3 cells were transfected with miR-101 mimics, and DNMT3A siRNA was transfected as a positive control. Then Western blot was used to detect the expres-sion of DNMT3A protein regulated by miR-101 in SKOV3 cells. The growth and invasion ability of SKOV3 cells were evaluated by MTT and Transwell invasion assays.Results:qRT-PCR showed that miR-101 was down-regulated in ovarian cancer tissues. Western blot showed that the level of DNMT3A protein was inhibited by restored miR-101 or knock-down of DNMT3A in SKOV3 cells. Following transfection of miR-101 mimics or knock-down of DNMT3A for 48, 72 and 96 h respectively, MTT assay showed that theD values were signiifcantly lower than the control group, (P<0.05). After transfection of miR-101 mimics or knock-down of DNMT3A for 36 h, Transwell invasion assay showed that the numbers of cells through the basement membrane was (105±7) and (107±13), respectively, which are signiifcantly different from the control group (213±11), indicating invasion of SKOV3 cells signiifcantly slowed down (P<0.05).Conclusion:miR-101 suppresses cell growth and invasion by targeting DNMT3A in ovarian cancer.
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OBJECTIVE@#To explore the effect of microRNA-101 on apoptosis of condylar cartilage cells and the specific mechanism of molecular biology.@*METHODS@#IL-1 was used to stimulate and establish the model of apoptosis of condylar cartilage cells. The expression change of miR-101 in control group was compared with that in IL-1 stimulation group by qRT-PCR. Overexpression and down-regulation models of miR-101 were established by transfecting Mimics and Inhibitor and verified by qRT-PCR. Flow cytometry was used to detect the effect of miR-101 overexpression and down-regulation on apoptosis. Target gene of miR-101 was analyzed and calculated through bioinformatics. Western blot and Luciferase report assay were used to detect whether SOX9 could become the target gene of miR-101.@*RESULTS@#qRT-PCR results showed that IL-1 stimulation could cause the increase of miR-101 expression. After the transfection of rabbit condylar cartilage cells by Mimics and Inhibitor, qRT-PCR results confirmed the significant effect of miR-101 overexpression and down-regulation. It was confirmed by flow cytometry that overexpression of miR-101 could promote the apoptosis of condylar cartilage cells, and down-regulation of miR-101 could reduce the apoptosis. It was confirmed by Western blot and Luciferase report assay that SOX9 was the target gene of miR-101, and miR-101 inhibited SOX9 expression through complementary pairing with 3'UTR of SOX9 mRNA.@*CONCLUSIONS@#miR-101 can promote the apoptosis of condylar cartilage cells through inhibiting the protein level of target gene SOX9.
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Objective:This study was conducted to investigate Epstein-Barr virus (EBV) infection in gastric cancer in Wuwei ar-ea of Gansu province (China) and the roles of miR-101, EZH2, and COX-2 in EBV-associated gastric carcinomas (EBVaGC). Meth-ods:Tissue microarray technique, immunohistochemistry, and in situ hybridization were performed to detect the expression of EBV-en-coded small RNAs (EBERs), miR-101, EZH2, and COX-2, in gastric cancer tissues (n=120) and the corresponding adjacent tissues (n=120). Results:The positive rate of EBV was 10.0% in 120 cases of gastric cancer tissues. EBVaGC was not significantly associated with lymph node metastasis and developed most often in the cardia and body (P<0.05). The differences in the positive rates of miR-101, EZH2, and COX-2 in 120 cases of gastric cancer tissues and corresponding adjacent tissues were significant (P<0.05). The differences in the positive rates of miR-101, EZH2, and COX-2 in 12 cases of EBVaGC and in 108 cases of EBV-negative gastric can-cer (EBVnGC) tissues were significant (P<0.05). The infection of EBV in gastric cancer tissues was positively related to miR-101 ex-pression. By contrast, miR-101 expression was negatively related to lymph node metastasis and expression of COX-2 and EZH2 in EB-VaGC tissues (P<0.05). Conclusion:EBV infection was related to gastric cancer in Wuwei area of Gansu province. EBVaGC and EB-VnGC have significant differences in lymph node metastasis and in the location of cancer. MiR-101, EZH2, and COX-2 were related to the development of EBVaGC.
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Objective To investigate the expression of miR-101 in pancreatic cancer and the effect of down-regulation miR-101 on proliferation of pancreatic cancer cell line ASPC1. Methods Real-time PCR was used to determine the expression of miR-101 in pancreatic cancer, adjacent tissues and pancreatic cancer cell line ASPC-1. The miR-101 over-expression vector (peGFPc1-miR-101) was constructed and was transfected into ASPC-1 cell. Transfection efficiency was measured by fluorescence microscope. The expression of miR101in the transfected cells was detected by real-time PCR. Cell viability analysis was performed by MTT. The targeted genes of miR-101 in pancreatic cancer were scanned by the online targeted gene prediction software (target Scan). Results The expression of miR-101 was in pancreatic cancer tissues, adjacent tissues and ASPC-1 cell line, respectively. The expressions in pancreatic cancer tissues and ASPC-1 cells were significantly lower than that in adjacent tissues ( P ＜ 0.01 ). The expression of miR 101 in transfected cells increased to 19.8 folds as much as that in the control group (P ＜0.01 ). Proliferation rate of transfected cells was significantly decreased, which was only 26% of primary cells ( P ＜ 0.01 ). EZH2 was the potential targeted gene of miR-101 in pancreatic cancer. Conclusions miR-101 was weakly expressed and it may affect the proliferation of pancreatic cancer cell by inhibiting the EZH2 expression.