ABSTRACT
p21-activated kinase 5 (PAK5), a kind of the PAK family of serine / threonine kinases, modulates various cellular processes, including cytoskeletal remodeling, cell proliferation, migration and metastasis. Although studies have suggested that PAK5 is a key regulator of breast cancer progression, the link of PAK5 to senescence has not been reported yet. In this study, the CRISPR/ Cas9 lentivirus infection method was used to construct two PAK5 stably knockdown cell lines of human breast cancer (MDA-MB-231 and BT474). The effect of PAK5 knockdown on the proliferation of breast cancer cells was detected by CCK-8 and clone formation assays. The effect of PAK5 knockdown on cell cycle was detected by flow cytometry. The effect of PAK5 knockdown on cell senescence was observed by β-galactosidase staining. Western blotting was performed to detect the expression of senescence-related proteins including p53, p21 and p16 in breast cancer cells. Cells were treated by CHX and MG132 to explore the possible mechanism of PAK5 regulating p53 protein expression. The results showed that knockdown of PAK5 inhibited cell proliferation and blocked cells in the G
ABSTRACT
Objective: To investigate the effects of microRNA-129-5p (miR-129-5p) on the proliferation and apoptosis of osteosarcoma cells, and to explore the possible molecular mechanism. Methods: The expression of miR-129-5p in osteosarcoma MG63 cells and osteoblasts hFOB1.19 cells was determined by real-time fluorescent quantitative PCR (RFQ-PCR). After miR-129-5p mimics were transfected into osteosarcoma MG63 cells, the expression of miR-129-5p was detected by RFQ-PCR to verify the transfection efficiency. CCK-8 assay and plate clony formation experiment were used to detect the cell proliferative ability, while FCM was performed to analyze the cell apoptosis. Double luciferase reporter gene system was used to verify the interaction between miR-129-5p and the target gene p21-activated kinase 5 (PAK 5). The expressions of PAK5 mRNA and protein in MG63 cells with miR-129-5p overexpression were tested by RFQ-PCR and Western blotting, while the expression levels of phospho-glycogen synthase kinase-3β (p-GSK3β) and β-catenin were detected by Western blotting. After siRNA-PAK5, PAK5 overexpressed plasmids and miR-129-5p mimics＋ PAK5 overexpressed plasmids were separately transfected into osteosarcoma MG63 cells, the above methods were used to further verify the molecular mechanism of miR-129-5p regulating the proliferation and apoptosis of osteosarcoma cells. Results: The level of miR-129-5p in MG63 cells was lower than that in hFOB1.19 cells (P < 0.05). After the transfection with miR-129-5p mimics, the proliferation and clony formation abilities of MG63 cells were decreased (P < 0.001, P < 0.01), while the apoptosis rate was increased (P < 0.05). miR-129-5p regulated negatively the expression of PAK 5 gene (P < 0.05). After the transfection with miR-129-5p mimics or siRNA-PCK5, the expression levels of PAK5 mRNA and protein were decreased (all P < 0.01), while the expressions of p-GSK-3β and β-catenin proteins were also decreased (both P < 0.05, both P < 0.01). The overexpression of PAK5 could restore the effects of miR-129-5p on proliferation and apoptosis of osteosarcoma cells (both P < 0.05). Conclusion: miR-129-5p can inhibit the proliferation ability of osteosarcoma cells, and promote apoptosis by down-regulating the expression of PAK 5 gene. The mechanism may be associated with the expression regulation of p-GSK-3β and β-catenin proteins.
ABSTRACT
Objective The GATA1 mutant GATA1 S161A S187A (death type) and GATA1 S161D S187D (activated) eukaryotic expression vectors were constructed using the large primer method,and,to explore their biological function and potential tumor treatment targets,the expression and localization of the fusion protein in cells were confirmed. Methods S161A,S187A,S161D,and S187D mutants were amplified by GFP-GATA1 WT,which served as the template. The recombinant plasmid was cloned into a pEGFP-C1 expression vector and transfected into HEK293 cells by immunoblotting expression of the fusion protein. Results The eukaryotic expression vectors pEGFP-GATA1 S161A S187A and pEGFP-GATA1 S161D S187D were successfully constructed using the high primer PCR method,and expression of the fusion protein was verified. Confocal laser microscopy showed that the fusion protein was mainly located in the nuclei of HEK293 cells. Conclusion A eukaryotic expression vector of a GATA1 mutant was successfully constructed using the large primer method. This work lays the foundation for further studies on the structure and function of the mutant.
ABSTRACT
Background and purpose: p21-activated kinase 5 (PAK5) is a recently identified member of PAKs that regulate many intracellular processes such as cytoskeleton remodeling, cell proliferation, cell differentiation, gene transcription and cell apoptosis. Recently, studies found that PAK5 was overexpressed in some cancer such as gastric and colon cancer. However, the expression status and biological function of PAK5 in osteosarcoma are not clearly known. The objective of this study was to investigate the expression of PAK5 in osteosarcoma tissue and their relationships with the prognosis of osteosarcoma. Methods: The expression of PAK5 was detected by using immunohistochemical method in 92 specimens of human osteosarcoma tissues and 33 cases of osteoclastoma tissue, respectively. Results: The positive rate of PAK5 was 71.7% (66/92) in all the 92 cases of osteosarcoma. PAK5 expressions were not related to clinical variables such as gender, age, tumor location, tumor size, histological type and local recurrence, but signiifcantly related to Enneking grade, tumor cell necrosis rate and lung metastasis, and the high expression of PAK5 may reduce the efifciency of chemotherapy. Survival analysis indicated that high expression of PAK5 correlated with poor prognosis of patients with osteosarcoma. Univariate survival analysis showed that the signiifcant prognostic factors were tumor size, Enneking grade, local recurrence, lung metastasis and expression levels of PAK5. COX multivariate regression identified that the PAK5 expression levels (P=0.001) and lung metastasis (P=0.015) were independent prognostic factors of patients with osteosarcoma. Conclusion:The positive expressions of PAK5 closely correlate with Enneking grade, tumor cell necrosis rate and lung metastasis. Detection of PAK5 may be used as a molecular marker for prognosis of osteosarcoma. The high expression of PAK5 may reduce the efifciency of chemotherapy.