Objective To investigate the effects of nucleus localization signal linked nucleic kinase substrate short peptide (NNS) conjugated chitosan (CS) (NNSCS) mediated the transfection of microRNA-140 (miR-140) in rabbit articular chondrocytes in vitro. Methods Recombinant plasmid GV268-miR-140 and empty plasmid GV268 were combined with NNSCS to form NNSCS/pDNA complexes, respectively. Chondrocytes were isolated and cultured through trypsin and collagenase digestion from articular cartilage of newborn New Zealand white rabbits. The second generation chondrocytes were divided into 3 intervention groups: normal cell control group (group A), NNSCS/GV268 empty plasmid transfection group (group B), and NNSCS/GV268-miR-140 transfection group (group C). NNSCS/GV268 and NNSCS/GV268-miR- 140 complexes were transiently transfected into cells of groups B and C. After transfection, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expressions of exogenous miR-140; Annexin Ⅴ-FITC/PI double staining and MTT assay were used to detect the effect of exogenous miR-140 on apoptosis and proliferation of transfected chondrocytes; the expressions of Sox9, Aggrecan, and histone deacetylase 4 (Hdac4) were detected by RT-qPCR. Results RT-qPCR showed that the expression of miR-140 in group C was significantly higher than that in groups A and B (P<0.05). Compared with groups A and B, the apoptosis rate in group C was decreased and the proliferation activity was improved, Sox9 and Aggrecan gene expressions were significantly up-regulated, and Hdac4 gene expression was significantly down-regulated (P<0.05). There was no significant difference in above indexes between groups A and B (P>0.05). Conclusion Exogenous gene can be carried into the chondrocytes by NNSCS and expressed efficiently, the high expression of miR-140 can improve the biological activity of chondrocytes cultured in vitro, which provides important experimental basis for the treatment of cartilage damage diseases.
ObjectiveTo detect the expressions of signal peptide-CUB-EGF-like domain containing protein 3 (SCUBE3) and specificity protein 1 (SP1) in breast cancer tissues, and explore relations between their protein expressions and clinicopathologic features or prognosis.MethodsFrom February 2013 to October 2015, the breast cancer tissues and the corresponding adjacent normal breast tissues of 80 women patients with breast cancer in the Mianyang Central Hospital were selected, and the expressions of SCUBE3 and SP1 proteins in the tissues were detected by immunohistochemistry. The relations between the expressions of SCUBE3 and SP1 and clinicopathologic parameters of breast cancer were analyzed, the correlation between the SCUBE3 and SP1 was analyzed by Spearman rank correlation analysis. Kaplan-Meier method was used to analyze the survival of patients with breast cancer; and Cox proportional hazards regression model was used to analyze the risk factors of overall survival of patients with breast cancer.ResultsThe positive rates of SCUBE3 and SP1 proteins expressions in the breast cancer tissues were higher than those in the corresponding adjacent normal breast tissues (P<0.05). The positive rates of SCUBE3 and SP1 protein expressions were higher in the breast cancer tissues with lymph node metastasis and molecular subtypes of Luminal A or B (P<0.05), and the positive rates of SCUBE3 protein expression were higher in the breast cancer tissues with TNM stage Ⅱ–Ⅳ and high Ki67 (P<0.05). The retsult of Spearman rank correlation analysis showed that the positive rates of SCUBE3 and SP1 proteins expressions in the breast cancer tissues was positive correlation (χ2=7.979, rs=0.316, P=0.005). Kaplan-Meier curve showed that the overall survival of the patients with positive expression of SCUBE3 or SP1 protein was worse than that of the patients with negative expression (χ2=4.042, P=0.044; χ2=10.676, P=0.001). The Cox proportional hazards regression model multivariate analysis showed that the positive SCUBE3 (HR=6.020, P=0.016), positive SP1 (HR=4.077, P=0.018), lymph node metastasis (HR=3.518, P=0.017), and higher Ki67 expression (HR=7.989, P<0.001) were the independent risk factors of overall survival for the patients with breast cancer.ConclusionPositive rates of SCUBE3 and SP1 proteins expressions in breast cancer tissues are higher and there is a positive correlation between them, which are closely related to clinicopathologic parameters such as lymph node metastasis and molecular subtypes and prognosis of patients with breast cancer.