MiR-491 functions as a tumor suppressor through Wnt3a/ß-catenin signaling in the development of glioma.

OBJECTIVE: Glioma is the most frequent brain tumor that has high invasion and usually disperses to the whole brain through blood and basement membranes. MicroRNA-491 (miR-491) has been reported to have low expression and act as a tumor suppressor in several cancers. The Wnt/ß-catenin signaling is a classic signaling pathway that participated in several biological processes. Our purpose was to detect the molecular mechanism of miR-491 in regulating the growth and metastasis of glioma. MATERIALS AND METHODS: Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was applied to calculate the mRNA level of miR-491 and target gene. The protein expression of special genes was assessed by Western blot. The proliferation and invasive abilities were measured by the Cell Counting Kit-8 (CCK-8) and transwell assays. The Kaplan-Meier method was conducted to evaluate the association between the expressions of miR-491 with the overall survival of glioma patients. RESULTS: We discovered that miR-491 was lowly expressed in glioma and downregulation of miR-491 predicted poor outcome of glioma patients. Similarly, a high expression of miR-491 suppressed the growth and metastasis in glioma cell line LN229. MiR-491 high expression inhibited the growth of glioma in a mouse xenograft model. Moreover, Wnt3a was a target gene of miR-491 and miR-491 mediated the invasion-mediated epithelial-mesenchymal transition (EMT) by regulating the expression of Wnt3a. Additionally, miR-491 regulated the proliferation through the Wnt/ß-Catenin pathway by targeting Wnt3a. CONCLUSIONS: MiR-491 overexpression inhibited the proliferation through the Wnt3a/ß-catenin pathway and invasion-mediated EMT in glioma. The newly identified miR-491/Wnt3a/ß-catenin axis provides novel insight into the pathogenesis of glioma.

[Synthesis and cloning of V gamma 3 cDNA of monoclonal antibody].

With the guanidinium isothiocyanate method, total RNA was isolated from hybridoma cells that secrete monoclonal antibody against Brucella melitenses. Poly (A)+ RNA was obtained by oligo (dT)-cellulose affinity chromatography. Reverse transcriptase reaction was performed with a primer 3'A-T-A-G-G-T-G-A-C-C 5' that is complement to the codons of No. 122-125 amino acid residues in 5' terminus of constant region. The size of synthesized ds-cDNA is about 300bp, that is consistent with the length of variable region genes of heavy chain. The ds-cDNA was inserted into plasmid pUC19 with dC: dG tailing method, and the inserted plasmid was used to transform E. coli HB101. It has been proved that the insert was a variable region gene of heavy chain by clone hybridization in situ, size of insert and Southern blot.