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@#Objective: To investigate the effect of long non-coding RNA CDKN2B antisense RNA 1 (CDKN2B-AS1) on malignant biological behaviors of melanoma B16-F10 cells by targeting miR-7-5p. Methods: Melanoma B16-F10 cells were chosen for this study. shRNA CDKN2B-AS1 vector was constructed and transfected into B16-F10 cells. The experimental cells were divided into control group, sh-CDKN2B-AS1 group, miR-7-5p mimic group and miR-7-5p inhibitor group. The expression level of CDKN2B-AS1 mRNA in the transfected B16-F10 cells was detected by RT-PCR; the number of clone formation and the proliferation ability of the cells were detected by Clone formation assay and MTT assay; and the migration and invasion ability of the cells were detected by Scratch-healing assay and Transwell assay. The targeting relationship between CDKN2B-AS1 and miR-7-5p was detected by Luciferase reporter gene assay. The mRNA expression of miR-7-5p and protein expressions of Ki67, cleaved caspase-3, E-cadherin, N-cadherin and Twist1 in B16-F10 cells after transfection with miR-7-5p mimics/inhibitor were detected by RT-PCR and Western blotting, respectively. Results: Compared with the control group, the expression level of CDKN2B-AS1 mRNA in B16-F10 cells of sh-CDKN2B-AS1 group was significantly decreased (P<0.01); the proliferation, migration and invasion ability of cells were significantly decreased (all P<0.01). Luciferase reporter gene assay showed that CDKN2B-AS1 directly targeted miR-7-5p. The mRNAexpression of miR-7-5p, and protein expressions of cleaved caspase-3 and E-cadherininsh-CDKN2B-AS1groupandmiR-7-5pmimic group were significantly up-regulated (all P<0.05), whiletheproteinexpressionsofKi67,N-cadherin,andTwist1weresignificantlydown-regulated (all P<0.05). Conclusion: CDKN2B-AS1 targets miR-7-5p to promote the development of melanoma, and interfering with CDKN2B-AS1 can inhibit the malignant biological behaviors of melanoma B16-F10 cells.
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@#Objective: To prepare the fusion protein mGM-CSF-GnRH3 (mGGn) of mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) combining with gonadotropin releasing hormone (GnRH) and the fusion protein mGM-CSF-GRP6 (mG6) of mGM-CSF combining with gastrin-releasing peptide (GRP), and to investigate the inhibitory effect of the above two fusion proteins on B16F10 melanoma in vitro as well as to preliminarily predict their isoelectric point, relative molecular weight, hydrophobicity, stability, subcellular localization, signal peptide, spatial structure and potential epitopes. Methods:After the successful preparation of mGGn and mG6, the effects of different concentrations of fusion proteins on tumor cell morphology, migration, proliferation and cell cycle were detected by microscopic observation, scratch test, CCK-8 method and flow cytometry, respectively. The protein online analysis systems EXPASY, GOR4, SWISS MODEL were used to predict the basic properties and secondary/tertiary structure of recombinant fusion proteins. The B cell epitopes were predicted by IEDB and ABCpred software, the CTL epitopes were comprehensively predicted by SYFPEITHI, BlMAS and NetCTL software, and the Th epitopes were predicted by NetMHCIIpan 3.1 Server and IEDB software. Results:Both mGGn and mG6 inhibited the migration and proliferation of tumor cells. mGGn could block B16F10 cell cycle at G1 phase while mG6 could block B16F10 cell cycle at S phase, all of which prevented cells entering into G2 phase to inhibit tumor cell growth. The mGGn and mG6 fusion proteins got diverse structures and had multiple potential B epitopes, CTL epitopes and Th epitopes. Conclusion: mGGn and mG6 have inhibitory effect on B16F10 melanoma in vitro, and bioinformatics predictions have laid a foundation for further study of the biological functions and immunological activities of these fusion proteins.
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Objective To evaluate the effect of fenretinide (4-HPR)-loaded liposomes (4-HPR-L) on the proliferation,apoptosis and migration of A375 and B16F10 melanoma cells.Methods A film-ultrasonic dispersion method was used to prepare 4-HPR-L.In vitro cultured A375 cells,as well as B16F10 melanoma cells,were divided into the following groups:blank control group treated with fresh culture medium alone,4-HPR groups treated with 4-HPR at concentrations of 0.1,1,15,30,50 and 70 mg/L separately,and 4-HPR-L groups treated with 4-HPR-L at concentrations of 0.1,1,15,30,50 and 70 mg/L separately.Cell counting kit-8 (CCK8) assay was conducted to detect cell proliferation,Hoechst33258 staining and flow cytometry to detect cell apoptosis,wound healing assay to evaluate cell migration ability,and laser scanning confocal microscopy to observe endocytic uptake of the liposomes.Statistical analysis was done by one-way analysis of variance (ANOVA) for intergroup comparison,and a two-sample t-test for comparisons between 2 groups with SPSS 20.0 software.Results Both 4-HPR and 4-HPR-L showed inhibitory effects on the proliferation of A375 and B16F10 melanoma cells.After 48-hour treatment,the survival rates of A375 cells in the 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR groups were (94.3 ± 1.4)％,(91.7 ± 2.5)％,(84.4 ± 2.5％),(78.8 ± 2.1)％,(59.0 ± 1.1)％ and (42.8 ± 2.0)％ respectively,and those in the 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR-L groups were (86.0 ± 0.2)％,(76.5 ± 0.6)％,(60.9 ± 1.5)％,(49.0 ± 0.5)％,(32.9 ± 0.2)％ and (18.9 ± 0.5)％ respectively.After 48-hour treatment,the survival rates of B16F10 cells in 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR groups were (95.4 ± 1.9)％,(90.5 ± 2.6)％,(77.0 ± 0.8％),(64.4 ± 3.5)％,(59.1 ± 2.9)％ and (49.9 ± 1.9)％ respectively,and those in the 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR-L groups were (88.4 ± 2.0)％,(80.9 ± 3.4)％,(60.9 ± 2.2)％,(51.5 ± 2.9)％,(41.1 ± 1.2)％ and (33.5 ± 2.4)％ respectively.The survival rates of A375 and B16F10 cells were significantly higher in the 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR groups than in the 4-HPR-L groups at the same concentrations (A375 cells:t =8.019,8.298,11.455,19.978,33.672,16.314 respectively,all P ＜ 0.01;B16F10 cells:t =3.573,3.153,9.953,4.019,8.097,7.53 respectively,all P ＜ 0.05).Hoechst33258 staining showed that the morphology of the melanoma cells in the control group and 4-HPR groups did not change obviously,but the cells.in the 4-HPR-L groups became smaller,with the cytoplasm concentrated,nuclei dissociated into fragments,and apoptotic bodies formed.Flow cytometry showed that the apoptosis rates of A375 and B16F10 cells were significantly higher in the 4-HPR-L groups than in the 4-HPR groups (all P ＜ 0.01).Cell wound healing assay showed that the inhibitory effect of 4-HPR-L on the migration of cells was stronger than that of 4-HPR,and 4-HPR-L could markedly decrease the degree of wound healing.Laser scanning confocal microscopy showed that C6 liposomes could be rapidly and successfully internalized into both A375 and B16F10 cells.Conclusion The 4-HPR-L can be internalized into A375 and B16F10 cells better than 4-HPR,effectively inhibit the proliferation and migration of A375 and B16F10 cells,and induce the apoptosis of these cells.
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Background: Melanoma is a metastatic type of skin cancer that is difficult to treat and the majority of efforts are directed to the design of new drugs. Medicinal Plants have been the primary source of medicines since life on earth; more than 50% of existing cancer treatments is derived from plants. Bauhinia variegata is well-known medicinal plant used from the ancient era to till date for their medicinal values. Scientific literatures have not documented any evidence of the antitumour potential of Bauhinia variegata against B16F10 melanoma tumor model in C57BL mice. The present investigation was undertaken to explore the antitumour activity of Leaf, stem bark and flower extract of Bauhinia variegata against B16F10 melanoma tumour model in C57BL mice. Methods: Hydro-methanolic extract prepared from the leaf, stem bark and flower of Bauhinia variegata were assessed for their antitumor activity. The extracts at doses of 500 and 750 mg/kg b.wt. were given orally along with cyclophosphamide (chemotherapeutic drug) for 40 days for exploring antitumor activity against melanoma tumor (B16F10) in C57BL mice. Inhibition of tumor growth, increase in survival time of animal with treatment, histopathological studies and antioxidant parameter were determined. Results: The Present investigation showed significant effect of the B. variegata L. in preventing melanoma tumor by B16F10 cell line in C57BL/6 mice. As compared with the tumour control group, the remarkable results especially in the group which received B. variegata extract and cyclophosphamide together were obtained for all of the measured parameters. Dose dependent response was observed in tumor volume, inhibition rate, life span time and antioxidant parameter of extracts. Combination treatment of cyclophosphamide and B. variegata extracts showed more pronounced effect. Conclusions: These findings suggest that B. variegata hydromethanolic extract may contain bioactive compounds of potential therapeutic significance which are relatively safe from toxic effects, and can compromise the medicinal use of this plant in folk medicine (US)