MiR-326 regulates EphB3 to inhibit invasion and metastasis of breast cancer cells / 中国药理学通报

;Aim To investigate the molecular mechanism of miR-326 inhibiting breast cancer invasion and metastasis by regulating EphB3 expression. Methods RTFQ-PCR was used to examine the expression of miR-326 in normal breast epithelial cells and breast cancer cells and the transfection efficiency of miR-326 overexpression plasmid. EdU cell proliferation assay and Transwell assay were used to examine the changes in proliferation, migration and invasion ability of different subgroups of cells. Dual luciferase assay was used to verify the presence of binding sites for miR-326 and EphB3. Western blot was used to detect the expression of EphB3 in breast cancer cells after overexpression of miR-326. Results RTFQ-PCR results showed that miR-326 was lowly expressed in breast cancer cells and successfully transfected (P < 0. 05). EdU proliferation assay and Transwell assay results showed that overexpression of miR-326 in breast cancer cells inhibited proliferation, migration and invasive ability (P < 0. 05). The results of dual luciferase assay showed that miR-326 could interact with the 3'-UTR of EphB3 (P < 0. 05). Western blot and Transwell assays showed that miR-326 could negatively regulate EphB3 to inhibit invasive metastasis of breast cancer cells (P < 0. 05). Conclusions MiR-326 acts as a cancer suppressor genes in the development of breast cancer and suppresses the invasion and metastasis of breast cancer cells by regulating the expression of EphB3.

Targeted inhibition of lncRNA CTB-191K22.5 reduces the proliferation and invasion of colorectal cancer SW480 cells and its mechanism / 国际外科学杂志

Objective:To explore the effect of down-regulation of long non-coding RNA (lncRNA) CTB-191K22.5 on the proliferation and invasion of colorectal cancer SW480 cells and the molecular mechanism.Methods:The TCGA database was used to analyze the expression differences of CTB-191K22.5 in colorectal cancer tissues and normal tissues. The CTB-191K22.5 inhibitor (Anti-CTB-191K22.5) and negative inhibitor (Control) were transfected into colorectal cancer SW480 cells, denoted as Observation group and Control group, real-time quantitative polymerase chain reaction (qRT) -PCR) was used to evaluate the inhibitory effect. MTT method and Transwell chamber method were used to evaluate the proliferation and invasion of SW480 cells. Western blot was used to evaluate the protein levels of PI 3K/AKT/mTOR signaling pathway in SW480 cells. The bioinformatics software starBase v2.0 was used to predict the target genes of CTB-191K22.5. qRT-PCR was used to evaluate the expression of CTB-191K22.5 target gene in SW480 cells. Measurement data were expressed as Mean±SD, and t-test was used for comparison between two groups. Results:Compared with normal tissues, the expression of CTB-191K22.5 in colorectal cancer tissues was significantly increased ( P<0.01). The expression of CTB-191K22.5 in SW480 cells of the Control group and Observation group were 6.60±0.85 and 1.08±0.21, respectively. The expression level of CTB-191K22.5 decreased after transfection with Anti-CTB-191K22.5 ( P<0.01). Compared with the Control group, the SW480 cell proliferation ability of the Observation group decreased ( P<0.01). The invasion numbers of SW480 cells in the Control group and Observation group were (135.4 ± 16.29) and (42.24±14.59), respectively. The invasion ability of SW480 cells decreased after transfection with Anti-CTB-191K22.5 ( P<0.01). Compared with the Control group, the expression levels of PI 3K/AKT/mTOR signaling pathway protein in SW480 cells in the Observation group decreased. miR-326 may be the target gene of CTB-191K22.5. Compared with the Control group, transfection with Anti-CTB-191K22.5 significantly increased the expression level of miR-326 in SW480 cells ( P<0.01). Conclusion:CTB-191K22.5 is highly expressed in colorectal cancer tissues, and down-regulation of CTB-191K22.5 may inhibit the proliferation and invasion of colorectal cancer SW480 cells by targeting miR-326.

Oncogenic circDHTKD1 promotes tumor growth and metastasis of oral squamous cell carcinoma in vitro and in vivo via upregulating miR-326-mediated GAB1

Circular RNAs (circRNAs) have been extensively elucidated with regard to their significant implications in oral squamous cell carcinoma (OSCC). This study performed the functional investigation of circRNA dehydrogenase E1 and transketolase domain containing 1 (circDHTKD1) in OSCC. RNA expression levels of different molecules were measured via quantitative real-time polymerase chain reaction (qRT-PCR). Cellular behaviors were detected by 3-(4, 5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) for cell viability, colony formation assay for clonal capacity, flow cytometry for cell apoptosis, wound healing assay for migration, and transwell assay for migration/invasion. Western blot was used for analyzing protein expression. RNA pull-down and dual-luciferase reporter assays were applied to assess the binding between targets. A xenograft tumor model was established in nude mice for in vivo experiments. Our expression analysis revealed that circDHTKD1 was upregulated in OSCC tissues and cells. circDHTKD1 knockdown was shown to impede OSCC cell growth and metastasis but motivate apoptosis. Additionally, circDHTKD1 served as a microRNA-326 (miR-326) sponge and the function of circDHTKD1 was achieved by sponging miR-326 in OSCC cells. Also, miR-326 inhibited OSCC development via targeting GRB2-associated-binding protein 1 (GAB1). circDHTKD1 could sponge miR-326 to alter GAB1 expression. Furthermore, circDHTKD1 contributed to OSCC progression in vivo via the miR-326/GAB1 axis. These data disclosed a specific circDHTKD1/miR-326/GAB1 signal axis in governing the malignant progression of OSCC, showing the considerable possibility of circDHTKD1 as a predictive and therapeutic target for clinical diagnosis and treatment of OSCC.

PINK1 Deficiency Decreases Expression Levels of mir-326, mir-330, and mir-3099 during Brain Development and Neural Stem Cell Differentiation

PTEN-induced putative kinase 1 (PINK1) is a Parkinson's disease (PD) gene. We examined miRNAs regulated by PINK1 during brain development and neural stem cell (NSC) differentiation, and found that lvels of miRNAs related to tumors and inflammation were different between 1-day-old-wild type (WT) and PINK1-knockout (KO) mouse brains. Notably, levels of miR-326, miR-330 and miR-3099, which are related to astroglioma, increased during brain development and NSC differentiation, and were significantly reduced in the absence of PINK1. Interestingly, in the presence of ciliary neurotrophic factor (CNTF), which pushes differentiation of NSCs into astrocytes, miR-326, miR-330, and miR-3099 levels in KO NSCs were also lower than those in WT NSCs. Furthermore, mimics of all three miRNAs increased expression of the astrocytic marker glial fibrillary acidic protein (GFAP) during differentiation of KO NSCs, but inhibitors of these miRNAs decreased GFAP expression in WT NSCs. Moreover, these miRNAs increased the translational efficacy of GFAP through the 3'-UTR of GFAP mRNA. Taken together, these results suggest that PINK1 deficiency reduce expression levels of miR-326, miR-330 and miR-3099, which may regulate GFAP expression during NSC differentiation and brain development.