Long non-coding RNA taurine up regulated 1 promotes osteosarcoma cell proliferation and invasion through upregulating Ezrin expression as a competing endogenous RNA of micro RNA-377-3p.

Osteosarcoma (OS) is the most common primary malignant tumor of bone mainly occurring in children and young people, which has a high rate of recurrence and metastasis. Long non-coding RNAs (lncRNAs) have capabilities in regulating target gene expression in various tumors served as competing endogenous RNAs (ceRNAs) to sponge microRNAs (miRNAs). In addition, Ezrin (EZR) is a member of ERM (ezrin/Radixin/moesin) protein family that contributes to the progression of multiple tumors. Previous studies have correlated lncRNA taurine upregulated 1 (TUG1) or Ezrin with OS. However, the correlation between lncRNA TUG1 and Ezrin in OS remains unclear. The expressions of lncRNA TUG1 and Ezrin were upregulated in OS tissues and cells determined by quantitative reverse transcription-PCR (qRT-PCR) and Western blot (WB), respectively. In addition, both lncRNA TUG1 and Ezrin promoted OS cell proliferation identified by Cell Counting Kit-8 (CCK-8) assay and clone formation assay, and enhanced OS cell invasion detected using Transwell assay for cell invasion. Moreover, lncRNA TUG1 upregulated Ezrin expression through sponging miR-377-3p determined by dual-luciferase reporter gene assay and WB. In conclusion, our work revealed that lncRNA TUG1 promoted OS cell proliferation and invasion through upregulating Ezrin expression as a ceRNA of miR-377-3p, which might provide novel therapeutic targets for OS therapy.

MicroRNA miR-425 promotes tumor progression by inhibiting Dickkopf-related protein-3 in gastric cancer.

Gastric cancer is a prevalent yet heterogeneous disease which ranks as the fifth most common cancer in the world. Dietary habit, genetic background, Helicobacter Pylori infections were the risk factors of gastric cancer. MicroRNA miR-425 is highly expressed in gastric cancer, but little attention has been devoted to the mechanism of miR-425 in tumorigenesis. This study aim to investigate the role of miR-425 in gastric cancer.The expression of miR-425 and Dickkopf-related protein-3(DKK-3) were analyzed by qRT-PCR. Gastric cell line BGC-823 and SGC-7901 were transfected miR-425 inhibitors or NC. Then, cell viability was determined by CCK-8, cell apoptosis and cell cycle were assessed by flow cytometer. Cell migration and cell invasion were analyzed by wound healing and trans-well assays. Luciferase reporter assay was conducted to assess the correlation between miR-425 and DKK-3. Downstream regulators, such as p-ASK1 and p-JNK, were analysis by western blot.Compared with normal gastric epithelium cell line, miR-425 was obviously upregulated in gastric cancer cell lines. MiR-425 inhibitor suppressed the cell viability, cell migration and cell invasion. The Luciferase assay data identified that DKK-3 is a target of miR-425. While miR-425 could lower the expression of DKK-3 which mediate tumorigenesis in a certain way.

The E2F3/miR-125a/DKK3 regulatory axis promotes the development and progression of gastric cancer.

BACKGROUND: Gastric cancer (GC) is one of the most common malignant tumours with high mortality and metastasis rates. E2F3, miR-125a and DKK3 have been reported to be involved in various cancer types, but their detailed roles in GC have not been fully understood. METHODS: A QRT-PCR assay was used to examine the expression of E2F3, miR-125a and DKK3 in metastatic and nonmetastatic GC tissues. DKK3 plasmids, DKK3 shRNA, miR-125a mimic and miR-125a inhibitor were transfected into BGC823 cells to evaluate the biological functions of DKK3 and miR-125a. A scratch wound healing assay and Transwell assay were utilized to determine the migratory and invasive ability of BGC823 cells transfected with the DKK3 plasmids, DKK3 shRNA, miR-125a mimic and miR-125a inhibitor. Moreover, qRT-PCR and WB analysis were used to analyse the mRNA and protein expression levels of metastasis-related genes after proper transfection. The target relationship between miR-125a and the DKK3 mRNA 3'UTR was determined by a dual luciferase reporter assay, while the interaction between E2F3 and miR-125a was analysed by a ChIP assay. RESULTS: The clinical data showed that the DKK3 expression level in metastatic GC samples was significantly less than that in nonmetastatic GC samples, whereas the E2F3 and miR-125a expression levels in metastatic GC samples were notably greater than those in nonmetastatic GC samples. Moreover, knockdown of DKK3 and overexpression of miR-125a markedly promoted the migratory and invasive abilities of GC cells. Additionally, the protein and mRNA expression levels of metastasis-related genes, including N-cadherin, Vimentin, MMP2 and MMP9, were markedly decreased in the DKK3 and miR-125a inhibitor groups compared to their control groups and markedly increased in the DKK3 shRNA and miR-125a groups compared with the control group. Finally, a dual luciferase reporter assay and ChIP assay showed that E2F3 binds to the miR-125a promoter and that the DKK3 mRNA 3'UTR is a direct target of miR-125a. Furthermore, analysis of Kaplan-Meier curves also confirmed the regulatory role of E2F3 on miR-125a. Additionally, BGC823 cells transfected with E2F3 plasmids and shRNA downregulated and upregulated the expression of DKK3, respectively. CONCLUSION: Our results suggested that E2F3 might play a tumour-promoting role in the metastasis and progression of GC by regulating the miR-125a/DKK3 axis.

microRNA-211 regulates cell proliferation, apoptosis and migration/invasion in human osteosarcoma via targeting EZRIN.

BACKGROUND: In recent years, microRNA-211 (miR211) has been considered as a tumor suppressor in multiple malignancies. However, the function of miR211 in human osteosarcoma has not been explored intensively so far. In this study, the relationship between miR211 and EZRIN was analyzed in human osteosarcoma. METHODS: The expression levels of miR211 and EZRIN were measured in both human osteosarcoma cells and tissues. The direct regulatory relationship between miR211 and EZRIN was evaluated using dual-luciferase assay. The effect of miR211 and EZRIN overexpression on cell proliferation, migration/invasion, and apoptosis was detected. RESULTS: The expression of miR211 was obviously lower in osteosarcoma tissues than paracancerous tissues. EZRIN was identified as the direct target of miR211, and up-regulation of miR211 increased the percentage of cell apoptosis, and suppressed cell proliferation as well as cell migration/invasion via directly regulating EZRIN. CONCLUSIONS: Our study indicated that miR211 has an important role in the development and progress of osteosarcoma, and it might become a novel target in the diagnosis and treatment of human osteosarcoma.

microRNA-96 acts as a tumor suppressor gene in human osteosarcoma via target regulation of EZRIN.

AIMS: microRNA-96 (miR-96) is considered as a tumor suppressor in multiple malignancies. Some studies have indicated that EZRIN could be regulated by the miR-96 in several kinds of cancer cells. However, the function of miR-96 in human osteosarcoma has not been investigated intensively so far. In this study, we mainly explored the relationship between miR-96 and the expression of EZRIN in human osteosarcoma cells. MAIN METHODS: The levels of miR-96 and EZRIN in osteosarcoma tissues and paracancerous tissues of patients were evaluated with qPCR. The targeted regulation between miR-96 and EZRIN was further confirmed with Dual luciferase assay. Overexpression of miR-96 and EZRIN was induced in human osteosarcoma cell line, and the changing of cell viability was analyzed to determine the role of miR-96 in human osteosarcoma in vitro and in vivo. KEY FINDINGS: Our results showed that the expression level of miR-96 was much lower in human osteosarcoma tissues compared with human paracancerous tissues. Dual luciferase assay indicated that EZRIN as the direct target of miR-96 in osteosarcoma cells. Besides, the up-regulation of miR-96 could inhibit cell proliferation, cell migration and invasion, tumor formation ability, and increase the percentage of cell apoptosis in osteosarcoma cells through inhibiting EZRIN. SIGNIFICANCE: Therefore, our study indicated that miR-96 hold an important role for the development and progress of human osteosarcoma, and this miRNA might become a novel target in the diagnosis and treatment of human osteosarcoma and even other cancers.

Influence of ezrin-shRNA in combination with HSP70 on the apoptosis and proliferation of osteosarcoma cells.

Ezrin and heat shock protein (HSP)70 have been reported to regulate cell apoptosis and tumor development of osteosarcoma. However, there has not been reported the synergy effect of knocking down ezrin and overexpressing HSP70. In the present study, two vectors, pGFP-V-RS-shRNA and pGFP-V-RS-shRNA-HSP70, were constructed and transfected into LM8 cells [denoted as small hairpin (sh)RNA group and dual group, respectively]. The apoptosis rates in these two transfected groups were significantly higher than those in the control group (empty vector) (P=0.036), while significantly lower proliferation rates were observed in these two groups (P=0.023). The cytotoxic T lymphocyte activity on target LM8 tumor cells in the dual group was significantly higher than in other groups, with cytotoxicity as high as 55.56±2.10%. Further studies revealed that the transfection of ezrin-shRNA/HSP70 also suppressed tumor formation in vivo in nude mice. A lower cluster of differentiation (CD)4/CD8 ratio was detected in the tumor formed by injecting cells in the dual group (P=0.006). Furthermore, the serum level of interleukin-4 in the dual group was significantly decreased, while the serum level of interferon-γ was significantly increased, compared with the other two groups (P=0.004). Simultaneously knocking down ezrin and overexpressing HSP70 promotes cellular apoptosis and suppresses the proliferation of osteosarcoma cells in vitro, and enhances the tumor killing effects of HSP70-induced immune killing.

GATA4 promotes hepatoblastoma cell proliferation by altering expression of miR125b and DKK3.

GATA4 is a zinc finger DNA-binding protein that plays an important role in mammalian liver development. However, the effects of GATA4 in hepatoblastoma (HB), a common liver cancer in pediatric patients, remain largely unknown. In this study, we demonstrate that GATA4 promotes growth and survival in the Huh6 human hepatoblastoma cell line. GATA4 expression was high in Huh6 cells, and its knockdown decreased expression of Dickkopf-related protein 3 (DKK3), a gene that may contribute to premature or undifferentiated phenotypes in HB. GATA4 also directly bound to the promoter regions of the miRNA miR125b and inhibited its expression in Huh6 cells. DKK3 was a direct target of miR125b in Huh6 cells. Inhibition of miR125b or overexpression of DKK3 promoted proliferation, survival, migration, and invasion in Huh6 cells. This is the first report to demonstrate that GATA4 promotes oncogenesis by inhibiting miR125b-dependent suppression of DKK3 expression. This GATA4/miR125b/DKK3 axis may be a major regulator of growth, migration, invasion, and survival in hepatoma cells, and is therefore a potential therapeutic target or biomarker for progression in HB patients.

Effects of Ezrin and Heat Shock Protein 70 on Apoptosis and Proliferation of Human Osteosarcoma Cells.

OBJECTIVE: To investigate the influence of knocking down ezrin expression in combination with heat shock protein (HSP)-induced immune killing on the apoptosis and proliferation of mouse osteosarcoma cells. METHODS: The HSP70 and ezrin-shRNA DNA fragments cloned into the expression vector pGFP-V-RS and the expression vectors pGFP-V-RS-shRNA and pGFP-V-RS-shRNA-HSP70 constructed and transfected into MG63 cell line, where their status was observed by fluorescent microscopy. Expression of ezrin and HSP70 was determined by RT-PCR and western blot. Changes in cell apoptosis and proliferation were assessed by flow cytometry and MTS and changes in expression of apoptosis and cell cycle-related proteins by western blot. Specific cytotoxic T lymphocytes (CTLs) were induced by HSP70 and its lethal effect on target MG63 tumor cells analyzed by MTS assay. RESULTS: The specific vector simultaneously downregulated ezrin and upregulated HSP70. Compared with ezrin knockdown alone, simultaneous HSP70 overexpression partially recovered the promoted cellular apoptosis and proliferation suppression by induced by ezrin knockdown; however, the apoptosis rate of MG63 cells was significantly greater than that of a negative control. In addition, ezrin-shRNA and ezrin-shRNA/HSP70 promoted expression of Bax. However, expression of these agents reduces Bcl-2 and Cyclin D1. The cytotoxic effects of CTLs on target MG63 tumor cells were significantly greater in the CTL + IL-2 + HSP70 group than the CTL + IL-2 group. CONCLUSIONS: Simultaneously knocking down ezrin and overexpressing HSP70 promotes apoptosis and inhibits proliferation of osteosarcoma cells and HSP70 induces CTL, enhancing the lethal effect on tumor cells.

[Differential proteomic analysis and function study of human prostate carcinoma cells with different osseous metastatic tendency].

OBJECTIVE: To investigate the differential protein expression profiles of human prostatic carcinoma cells with different metastatic tendency and to screen the osseous metastasis associated proteins and investigate their function. METHODS: Proteomics and Western blotting were applied to screen and identify the differentially expressed proteins in the prostatic carcinoma cells of different lines: line T3B with high osseous metastasis potential, line P2-4 with high lymphatic metastasis potential, and their common parent cell line PC-3. The eukaryotic expression vector carrying human Pgenesil-1/HMGB1 siRNA was constructed and transfected into T3B cells by Lipofectamine 2000 and the positive clones was screened by G418. Pgenesil-1/HMGB1 siRNA/T3B, Pgenesil-1/T3B, and T3B cells were inoculated into the left ventricles of nude mice. Twelve weeks later the mice were killed. The number of osseous metastatic nodules and osseous metastasis inhibition rate were calculated. The mice metastatic tumor cells were identified by immunohistochemistry. RESULT: Six differential expressed proteins, correlated with cytoskeleton, transcriptional control, cellular metabolism, and phosphorylation were identified by proteomics and Western blotting. The recombinant plasmid Pgenesil-1/HMGB1 siRNA was successfully constructed. The HMGB1 expression of the T3B cells transfected with Pgenesil-1/HMGB1 siRNA was significantly lower than those of the other 2 groups (both P <0.05). The number of osseous metastatic nodules of the mice inoculated with Pgenesil-1/HMGB1 siRNA/T3B was significantly less than those of the other 2 groups (both P < 0.05). The metastatic osseous tumor cells were identified as the human prostatic carcinoma cells. CONCLUSION: Osseous metastasis associated proteins exist in prostatic carcinoma cells of the line with high osseous metastasis potential. HMGB1 is closely related to the osseous metastasis of human prostatic carcinoma cells. siRNA targeting HMGB1 specifically suppresses the expression of HMGB1 gene in the human prostatic carcinoma cells with high osseous metastasis potential and effectively inhibits the osseous metastasis.