Effect of LncRNA GHET1 on Proliferation, Apoptosis, and Metastasis of Gallbladder Cancer Cells by Targeting miR-27b / 肿瘤防治研究

Objective To investigate the effect of GHET1 on the biological behavior of gallbladder cancer cells and the regulatory mechanism of GHET1 on miR-27b. Methods The expression of GHET1 and miR-27b in 50 samples of gallbladder cancer was detected by real-time quantitative PCR. The si-NC vector, si-GHET1 vector, miR-27b inhibitor, and si-GHET1 vector+miR-27b inhibitor were transfected into SGC-996 cells and set as the control group, GHET1 interference group, miR-27b interference group, and GHET1+miR-27b interference group. Cell proliferation, apoptosis, and metastasis in each group were detected by MTT, flow cytometry, and Transwell assays. The regulatory effect of GHET1 on miR-27b was validated by luciferase reporter gene assay. Results GHET1 expression was higher in cancer tissues than that in paracancerous ones. miR-27b expression was lower in cancer tissues than that in paracancerous tissues. GHET1 was negatively correlated with miR-27b expression (P<0.05), and GHET1 expression was associated with TNM staging and lymph node metastasis (P<0.05). High GHET1 expression was associated with poor prognosis of patients with gallbladder cancer (P<0.05). Compared with the control group, the GHET1 interference group showed decreased cell-proliferation ability, increased apoptosis rate, and reduced number of cell metastasis. The miR-27b interference group showed increased cell-proliferation ability, decreased apoptosis rate, and increased number of cell metastasis (P<0.05). Compared with the GHET1 interference group, the GHET1+miR-27b interference group showed increased cell-proliferation ability, decreased apoptosis rate, and increased number of cell metastasis (P<0.05). GHET1 inhibited miR-27b expression by acting as a sponge of miR-27b. Conclusion GHET1 promotes the proliferation and metastasis and inhibits the apoptosis of gallbladder cancer cells by targeting miR-27b, suggesting that GHET1/miR-27b axis plays a role in gallbladder cancer progression.

Elevated miR-27b-3p enhances the radioresistance by targeting PLK2 in breast cancer / 中华放射医学与防护杂志

Objective:To investigate the effect of miR-27b-3p on radiation resistance of breast cancer cells.Methods:The relative expression levels of miR-27b-3p in normal tissues and breast cancer tissues were analyzed through GEO database and verified by the qRT-PCR assay. Cloning formation, immunofluorescence, and EDU assay were used to assess the functions of miR-27b-3p on radioresistance of breast cancer cells. The luciferase reporter assay was used to verify whether miR-27b-3p directly targeted PLK2 mRNA. A rescue experiment was performed to identify the influence of PLK2 overexpression on miR-27b-3p regulated radioresistance.Results:The expression level of miR-27b-3p in both breast cancer tissues ( t=2.99, P<0.01) and breast cancer cells ( t=21.21, 32.88, P<0.05) was significantly higher than those in normal breast tissues and cells, especially, it was elevated in radioresistant MCF-7R cells ( t=25.63, P<0.05). Overexpression of miR-27b-3p enhanced the cloning efficiency of MCF-7 cells ( t=10.32, P<0.05), and had a protective effect on the proliferation of irradiated MCF-7 cells ( t=8.77, 8.26, 8.03, P<0.05). But interference of miR-27b-3p reduced the cloning efficiency and proliferation of MCF-7R cells ( t=40.00, P<0.05) after irradiation with different doses( t=8.54, 8.32, 8.23, P<0.05). Moreover, PLK2 was verified to be a direct target of miR-27b-3p, and overexpression of PLK2 inhibited miR-27b-3p-mediated radioresistance of breast cancer cells (MCF-7: t=9.66, P<0.05; MCF-7R: t=6.42, P<0.05). Conclusions:miR-27b-3p contributes to the radioresistance of breast cancer cells by targeting PLK2.

Upregulation of miR-27b Facilitates Apoptosis of TNF-α-Stimulated Fibroblast-Like Synoviocytes

PURPOSE: The aim of this study was to explore the function of microRNA-27b (miR-27b) in fibroblast-like synoviocytes (FLSs) stimulated by tumor necrosis factor α (TNF-α). MATERIALS AND METHODS: mRNA expression of miR-27b in FLS cells (MH7A) treated with or without TNF-α was determined by q-PCR. MiR-27b mimics was transfected into MH7A cells to upregulate miR-27b expression. MTT assay and flow cytometry analysis were performed to investigate the effect of miR-27b on MH7A cell viability and apoptosis. The targets of miR-27b were predicted by TargetScan. The direct regulation of miR-27b on IL-1β expression was verified by luciferase assay. The protein expression levels of apoptosis-related proteins, IL-1β, and NF-κB signaling-related proteins were detected by Western blot. RESULTS: We discovered that miR-27b expression was decreased in MH7A cells stimulated by TNF-α. Upregulation of miR-27b by miR-27b mimics significantly inhibited the proliferation and promoted the apoptosis of TNF-α-stimulated MH7A cells. Consistently, upregulation of miR-27 decreased the level of Bcl-2 and increased Bax and caspase-3 expression in MH7A cells stimulated by TNF-α. Luciferase assay revealed that IL-1β was indeed a target of miR-27b. By quantitative real-time PCR and Western blot, we found that the expression of IL-1β is negatively regulated by miR-27b. Moreover, the NF-κB signaling pathway was significantly inhibited by miR-27b. CONCLUSION: Taken together, our results illustrated that enhanced miR-27b expression results in the suppression of proliferation and the promotion of apoptosis in FLSs stimulated by TNF-α, partially by regulating IL-1β expression and NF-κB signaling.

Effects of TET2-targeting miR-27b on oxidized low-density lipoprotein-induced inflammatory responses and apoptosis in endothelial cells / 中华微生物学和免疫学杂志

Objective@#To investigate the effects of miR-27b targeting ten-eleven translocation methylcytosine dioxygenase 2 (TET2) on oxidized low-density lipoprotein (ox-LDL) induced inflammatory responses and apoptosis in endothelial cells.@*Methods@#Double luciferase reporter gene analysis verified the targeting effect of miR-27b on TET2. Human umbilical vein endothelial cells (HUVECs) were induced by ox-LDL in vitro. Eight groups were set up including control group, ox-LDL group, ox-LDL+ anti-miR-con group, ox-LDL+ anti-miR-27b group, ox-LDL+ pcDNA group, ox-LDL+ pcDNA-TET2 group, anti-miR-27b+ si-con group and anti-miR-27b+ si-TET2 group. qRT-PCR was used to detect the expression of miR-27b and TET2 at mRNA level. Cell viability was detected by MTT assay. Cell apoptosis rate was detected by flow cytometry. Western blot was used to detect the expression of TET2, Cyclin D1 and caspase-3 at protein level.@*Results@#TET2 was the target gene of miR-27b. TET2 expression could be negatively regulated by miR-27b. ox-LDL increased the expression of miR-27b and reduced the expression of TET2 in HUVECs. The secretion of inflammatory factors and apoptosis rates of HUVECs in the control, ox-LDL+ anti-miR-27b, ox-LDL+ pcDNA-TET2 and anti-miR-27b+ si-con groups were significantly lower than those in the ox-LDL, ox-LDL+ anti-miR-con, ox-LDL+ pcDNA and anti-miR-27b+ si-TET2 groups, respectively (P<0.05).@*Conclusions@#miR-27b promoted ox-LDL-induced inflammatory responses and apoptosis in endothelial cells through down-regulating the expression of TET2.

miR-27b inhibits gastric cancer metastasis by targeting NR2F2

Increasing attention is focused on the down-regulation of miRNAs in cancer process. Nuclear receptor subfamily 2 (NR2F2, also known as COUP-TFII) is involved in the development of many types of cancers, but its role in gastric cancer remains elusive. In this experiment, oncomine and Kaplan-meier database revealed that NR2F2 was up-regulated in gastric cancer and that the high NR2F2 expression contributed to poor survival. MicroRNA-27b was targeted and down-regulated by NR2F2 in human gastric cancer tissues and cells. The ectopic expression of miR-27b inhibited gastric cancer cell proliferation and tumor growth in vitro and in vivo. Assays suggested that the overexpression of miR-27b could promote MGC-803 cells' migration and invasion and retard their metastasis to the liver. In addition, down-regulation of miR-27b enhanced GES-1 cells' proliferation and metastasis in vitro. These findings reveal that miR-27b is a tumor suppressor in gastric cancer and a biomarker for improving patients' survival.

Feasibility study on plasmatic microRNA-27b-3p as a potential biomarker for early diagnosis of gastric cancer / 肿瘤

Objective: To investigate the potential role of plasmatic microRNA-27b-3p (miR-27b-3p) as a biomarker in early diagnosis of gastric cancer (GC). Methods: The expressions of miR-27b-3p in plasma from 46 GC patients and 40 age- and gender-matched healthy controls were detected by real-time fluorescent quantitative-PCR (RFQ-PCR). The expression of miR-27b-3p in peripheral blood cells from 12 GC patients and 12 healthy controls were also detected by RFQ-PCR. The association of plasmatic miR-27b-3p level with the clinicopathological features of GC was statistically analyzed. The sensitivity and specificity of miR-27b-3p for diagnosis of GC were evaluated by receiver operating characteristic (ROC) curve and area under the curve (AUC). Results: The level of plasmatic miR-27b-3p in GC patients was significantly lower than that in the healthy controls (P 0.05). The downregulation of plasmatic miR-27b-3p level was correlated to the differentiation of GC (P 0.05). The ROC curve analysis showed that AUC value was 0.724, and the sensitivity and specificity of plasmatic miR-27b-3p used for differential diagnosis of GC were 60.0% and 76.1%, respectively. Conclusion: Downregulation of plasmatic miR-27b-3p level may indicate the occurrence of GC. MiR-27b-3p may be a potential biomarker for early diagnosis of GC.

Effects of interleukin-1βon MMP-13 expression in rat chondrocytes and its regulation of miR-27b / 天津医药

Objective To observe the effect of interleukin-1β(IL-1β) on expression of Matrix Metalloproteinases 13 (MMP-13) in rat chondrocytes and its regulation of miR-27b. Methods Chondrocytes were extracted from 7 Wistar male rats. Expression of MMP-13 were examined by Western blot at 0 h, 24 h, 48 h after IL-1βstimulation. Differential miRNAs expression profiles were examined by miRNAs microarray. The most obviously down-regulated miRNAs were confirmed by quantitative Real-time PCR. Targeted regulation relationship between miR-27b and MMP-13 was set up by Luciferase re?porter gene experiments. Results Expression of MMP-13 in rat chondrocytes was increased at a timely dependent manner upon IL-1βstimulation(P<0.05);Microarray revealed 36 miRNAs whose expression changed, among which 6(miR-27b, miR-31, miR-26a, miR-26b, miR-23, miR-204)were especially obvious. Real-time PCR confirmed that miR-27b was the one whose expression level were most down-regulated. Transient co-transfection of miR-27b mimics with luciferase expres?sion plasmids resulted in significant repression of luciferase activity in rat chondrocytes (P < 0.05). Conclusion IL-1βstimulation result in down-regulation of miR-27b and up-regulation of MMP-13 expression. MiR-27b and MMP-13 show targeted regulation relationship.