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Objective To investigate the effects of lncRNA SBF2-AS1 on the proliferation and invasion of hepatoma cells by regulating the miR-372-3p/CDK6 pathway. Methods Bel7402 and SK-hep1 cells were selected as research objects. The expression levels of SBF2-AS1, miR-372-3p, and CDK6 were up- or down-regulated according to different experimental stages, while the expression levels of miR-372-3p and CDK6 in cells were detected by real-time fluorescence quantitative PCR and Western blot. Dual luciferase reporter assay verified the targeting relationships between SBF2-AS1 and miR-372-3p as well as miR-372-3p and CDK6, respectively. CCK-8, colony formation assay, Transwell, cell cycle assay, and flow cytometry were used to analyze cell proliferation, colony formation, migration/invasion ability, cell cycle activity, and apoptosis. Results SBF2-AS1 was highly expressed in hepatocellular carcinoma cells (P<0.05). SBF2-AS1 knockdown resulted in decreased proliferation and invasion of Bel7402 and SK-hep1 cells (P<0.05). After miR-372-3p knockdown, the proliferation capacity and invasion number of Bel7402 cells were significantly increased. However, the above results were reversed after SBF2-AS1 knockdown (P<0.05). In addition, miR-372-3p targeted CDK6 and inhibited its expression, although over-expressing SFB2-AS1 could reverse the above results (P<0.05). Over-expressing CDK6 could reverse the inhibition of over-expressing miR-372-3p on the proliferation and invasion of Bel7402 cells. Conclusion LncRNA SBF2-AS1 can positively regulate the expression of CDK6 through miR-372-3p. It can also influence the distribution of cell cycle and affect the proliferation and invasion abilities of hepatocellular carcinoma cells.
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ABSTRACT Purpose: This study aimed to optimize the effective doses of mitomycin C, 5-fluorouracil, and their combination on cultivated basal cell carcinoma. Methods: Cultivated basal cell carcinoma and fibroblastic cells were treated with different concentrations of mitomycin C, 5-fluorouracil, and their combination. Cell viability, cell cycle, apoptosis, and expression levels of TP53, CDKN1A, and CDK6 were investigated. The most effective drug with its optimum dosage was administered via multiple intralesional injections to a 65-year-old woman with advanced periorbital nodulo-ulcerative BCC. Results: The concentrations of 0.00312 and 0.312 mg/mL were considered optimum for mitomycin C and 5-fluorouracil, respectively. The mean viabilities of basal cell carcinoma treated with mitomycin C alone and its combination with 5-fluorouracil were significantly less than those of the controls (p=0.002 and p=0.04, respectively). The cell cycle of all the treated basal cell carcinoma groups was arrested in the S phase. The apoptotic rates (p=0.002) of mitomycin C treated basal cell carcinoma were higher than those of the other treated cells, and their TP53 was significantly upregulated (p=0.0001). Moreover, CDKN1A was upregulated, whereas CDK6 was downregulated in basal cell carcinoma treated with either 5-fluorouracil (p=0.0001 and p=0.01, respectively) or the combination of 5-fluorouracil and mitomycin C (p=0.007 and p=0.001, respectively). Basal cell carcinoma lesions were significantly alleviated following mitomycin C injections in the reported patient. Conclusion: Our in vitro results revealed that the effective doses of mitomycin C and 5-fluorouracil on cultivated basal cell carcinoma were optimized. Mitomycin C was more effective in inducing the apoptosis of basal cell carcinoma than 5-fluorouracil and their combination. The intralesional injections of the optimum dose of mitomycin C could be proposed for the nonsurgical treatment of advanced eyelid basal cell carcinoma.
RESUMO Objetivo: Otimizar a dose efetiva de mitomicina C, 5fluorouracil e da combinação de ambos em culturas de células de carcinoma basocelular (CBC). Métodos: Culturas de células de células de carcinoma basocelular e de fibroblastos foram tratadas com diferentes concentrações de mitomicina C, 5fluorouracil e combinação de ambos. Além disto, foram investigados a viabilidade celular, o ciclo celular, a apoptose e a expressão dos genes TP53, CDKN1A e CDK6. O medicamento mais eficaz, em sua dosagem otimizada, foi administrado em últiplas injeções intralesionais em uma mulher de 65 anos com carcinoma basocelular nódulo-ulcerativo periorbital avançado. Resultados: A concentração de 0,00312 mg/mL de mitomicina C e a de 0,312 mg/mL de 5fluorouracil foram consideradas as ideias. A viabilidade média das células de carcinoma basocelular tratadas com mitomicina C isoladamente e em combinação foi significativamente menor que nas células de controle (respectivamente, p=0,002 e p=0,04). Todos os grupos de carcinoma basocelular tratados demonstraram interrupção do ciclo celular na fase S. As células de carcinoma basocelular tratadas com mitomicina C mostraram maiores taxas de apoptose (p=0,002) e significativa regulação positiva do gene TP53 (p=0,0001). Além disso, o gene CDKN1A foi positivamente regulado e o gene CDK6 foi negativamente regulado em células de carcinoma basocelular tratadas com 5fluorouracil (respectivamente, p=0,0001 e p=0,01) ou com a combinação de medicamentos (respectivamente, p=0,007 e p=0,001). Injeções posteriores de mitomicina C na paciente em questão levaram à melhora significativa da lesão do carcinoma basocelular. Conclusão: Nossos resultados in vitro otimizaram as doses efetivas de mitomicina C e 5fluorouracil na cultura de células de carcinoma basocelular e mostraram que a mitomicina C tem mais eficácia na apoptose de células de carcinoma basocelular do que o 5fluorouracil e a combinação de ambos. Injeções intralesionais de doses otimizadas de mitomicina C podem ser propostas para o tratamento não cirúrgico do células de carcinoma basocelular avançado de pálpebra.
Subject(s)
Aged , Female , Humans , Skin Neoplasms , Carcinoma, Basal Cell , Carcinoma, Basal Cell/drug therapy , Antineoplastic Combined Chemotherapy Protocols , Survival Analysis , Mitomycin , FluorouracilABSTRACT
@#Objective: To investigate the effect of panax japlcus var polysaccharide (PJPS) on the proliferation and apoptosis of gastric cancer MKN45 cells and its regulatory mechanism. Methods: Human gastric cancer cell lines (HGC27, MGC803, MKN45) and gastric mucosal epithelial cell line GES-1 were selected for this study. Let-7a mimics and let-7a inhibitor were transfected into MKN45 cells; Gastric cancer cell lines were treated with 100 μg/ml PJPS and MKN45 was selected as the subsequent experimental cell line. MKN45 cells were cultured with 0, 10, 50, 100 and 120 μg/ml PJPS, respectively. The proliferation and apoptosis rate of MKN45 cells were detected by CCK-8 and flow cytometry, respectively. Expressions of cell cycle dependent kinase 6 (CDK6) and apoptosis-related proteins in MKN45 cells were detected by Western blotting, and the expression level of miRNAs regulating the proliferation of gastric cancer cells was detectedbyReal-timequantitativePCR(qPCR).TheDualluciferasereportergeneassaywasusedtovalidatethetargeting relationship between let-7a and CDK6. Results: Compared with other gastric cancer cells, 100 μg/ml PJPS significantly inhibited the proliferation of MKN45 cells (P<0.01). At the same time, 100 μg/ml PJPS significantly up-regulated the expression of let-7a in MKN45 cells (P<0.01). The Dual luciferase reporter gene assay confirmed that CDK6 was the target gene of let-7a. Furthermore, PJPS inhibited the expression of CDK6 by up-regulating let-7a, thereby inhibiting the proliferation and inducing apoptosis of MKN45 cells (all P<0.01). Conclusion: PJPS inhibits proliferation and induces apoptosis of gastric cancer MKN45 cells by regulating the let-7a/ CDK6 axis.
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Aim To explore whether deguelin can block cell cycle and cell migration, inhibit the proliferation of non-small cell lung cancer cells.Methods H1299 cells were treated with 1.562 5, 3.125, 6.25, 12.5, 25, 50 μmol·L-1 deguelin for different time(24, 48, 72 h); cell viability was detected by CCK-8 assay, and cell migration ability was tested by scratch assay.H1299 cells were treated with 1.5, 3, 6 μmol·L-1 deguelin for 24 h.Flow cytometry with PI single staining and Annexin V-FITC/PI double staining experiment were used to evaluate cell cycle and apoptosis.qPCR was used to detect the regulatory effects of deguelin and its carbamate derivative on the Cyclin D-CDK4/6 complex at the gene level.Results Deguelin inhibited cell growth and the IC50 value of deguelin was(5.47±0.97),(4.01±0.45),(2.86±0.19)μmol·L-1 when treated with 24, 48, 72 h respectively.Deguelin also inhibited the healing ability of H1299 cells and the migration of H1299 cells significantly(P<0.05).Deguelin could block H1299 cell cycle in G1 phase.Flow cytometry combined with Annexin V-FITC/PI double staining showed that deguelin could induce apoptosis of H1299 cells.qPCR experiments showed that deguelin could down-regulate the expression of CDK4, CDK6 and Cyclin D1 genes significantly(P<0.05).Conclusions Deguelin may regulate cell cycle by down-regulating CDK4, CDK6 and Cyclin D1 genes in the cell cycle regulation system, and reduce the migration ability of tumor cells to induce apoptosis.
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Objective To study the expression level and function of micro RNA (microRNA)-218 in hepatocellular carcinoma (HCC) .Methods 46 cases of HCC surgery in the hepatobiliary surgery department of this hospital were selected and divided into the transfection group and nontransfection group .The expression ,proliferation and apoptosis of microRNA-218 and the expression level of B cell specific Maloney leukemia virus insertion site 1(Bmi-1) and cycling-dependented kinase 6(CDK6) in HepG2 cells were compared between the two groups .Results The expression level of microRNA-218 in HCC tissue was significantly lower than that in paracancerous tissues (P＜0 .05);the microRNA218 expression level was closely correlated with the clinicopathological characteristics such as tumor size and TNM stage(P＜0 .05);the HepG2 cell proliferation rates at 24 ,48 ,72 h after transfection in the transfection group were significantly lower than those in the nontransfection group(P＜0 .05);the HepG2 cell apoptosis rate in the transfection group was significantly higher than that in the nontransfection group(P＜0 .05);the Bim-1 and CDK6 expression levels after HepG2 cell transfection in the transfection group were significantly lower than those in nontransfection group(P＜0 .05) . Conclusion microRNA-218 can suppress the proliferation of HCC cells and promotes HCC cells apoptosis by down-regulating the Bim-1 and CDK6 expression level in potential targets .
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Objective The aim of our study was to investigate the influence of hexavalent chromium exposure on mRNA expression of cell cycle related genes in electroplating workers, and to provide population data for investigating the toxic mechanisms of hexavalent chromium. Methods A total of 155 cases of workers occupationally exposed to hexavalent chromium were selected, including 89 males and 66 females, and the average age of workers was 39.65±8.856 years old. Questionnaire was used to collect essential information of workers. Peripheral blood was collected from electroplating workers. The inductively couple plasma mass spectrometry (ICP-MAS) was used to measure total blood chromium content. The workers were divided into four groups according to the blood chromium content. After extracting total RNA from whole blood and reverse transcription, the mRNA expression levels of p16 and CDK6 genes were detected by real-time quantitative PCR. Meanwhile, the levels of blood chromium (BCr) and the mRNA expression of p16 and CDK6 genes were compared among four groups. The impact of BCr, smoking habits, drinking habits, gender on the mRNA expression of CDK6 gene was analyzed. Results The levels of BCr in group 1 to 4 were 0.04ppb, 0.47±0.29 ppb, 2.76±1.16 ppb, 9.36 ±4.38 ppb, respectively, and the difference between every two groups was significant (P<0.05) . The median of p16 gene expression in four groups was 4.22, 7.19, 7.47, and 14.60, respectively, and the difference between every two groups was not significant (P>0.05) . The mRNA expression levels of CDK6 gene in groups 2 to 4 were 15.05, 8.03 and 24.81, respectively, which were significantly higher than that in group 1 (P<0.05) . The results of logistic regression showed that the level of Bcr was the main influence factor, while smoking habits, drinking habits and gender had no obvious impact on the mRNA expression of CDK6 gene. Conclusions Long-term exposure of hexavalent chromium led to higher mRNA expression of CDK6 gene, and it may serve as a biomarker for workers occupationally exposed to hexavalent chromium.
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Objective:To investigate the relationship between miR-211 and the occurrence of epithelial ovarian cancer,and its influence on the proliferation of ovarian cancer cells. Methods:To analyze the expression miR-211,CDK6 and Cyclin D1 of 30 cases of ovarian cancer and ovarian cancer cell lines,and 30 cases of non-ovarian cancer tissues and the normal ovarian epithelial cells were selected as the control group,and to analyze effects of miR-211 on the proliferation of ovarian cancer cells,as well as the Cyclin D1 and CDK6. Results:miR-211 relative expression level of ovarian cancer tissue was significantly lower than that in the normal group ( P<0. 05). Relative expression level of miR-211 of ovarian cancer cells was significantly lower than that in normal ovarian epithelial cells (P< 0. 05);in epithelial ovarian cancer cell line HO8910,cell number of miR-211 on the 3-day and the 4-day was significantly lower than that of miR-Ctrl group (P<0. 05);relative expression levels of Cyclin D1 and CDK6 in epithelial ovarian cancer were significantly higher than those in normal ovarian epithelial tissues (P<0. 05);miR-211 of epithelial ovarian cancer cell lines significantly inhibited Cyclin D1 and CDK6 expression;in ovarian cancer tissues,Spearman correlation analysis results showed that relative expression levels of miR-211 and Cyclin D1 and CDK6 was negatively correlated ( r=-0. 583, P= 0. 010 ) . Conclusion: miR-211 can inhibit the proliferation of ovarian cancer cells,and inhibit the expression of Cyclin D1 and CDK6;miR-211,Cyclin D1 and CDK6 in ovarian cancer may be involved in the regulation of ovarian cancer.
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Cell-cycle deregulation leading to excessive cellproliferation is an important mechanism of human tumorigenesis. CDK6 and CDK4 have been found to be significant regulators of cellcycle, particularly in promoting cell -cycle progress. Moreover, these proteins are usually overly active in most tumors and closely related to tumor development. Recently, research has confirmed CDK4/6 as prospective targets for cancer therapy. However, the mechanism of excessive CDK6 activation leading to tumorigenesis is not completely understood. Therefore, further understanding of the role of CDK4/6 in cell -cycle regulatory pathways and celldifferenti-ation is essential, as well as their overexpression in different types of tumors. This information will elucidate the mechanisms of tumor development and treatment. Therefore, this review intends to discuss the structure and biological function of CDK6, the role and mecha-nism of CDK6 in carcinogenesis, and the clinical application of CDK6 inhibitors.
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Objective:To study the role of FDCs-miR-548m-CDK6 axis on clonogenicity in mantle cell lymphoma. Methods:RT-qPCR and Western blot were used respectively to test the expression of miR-548m and CDK6. Bioinformatics assay was applied to predict the targets of miR-548m, and Western Blot was used to test the expression level of CDK6 after miR-548m overexpression or in-hibition. Luciferase report assay was performed to test whether CDK6 was a direct target of miR-548m. Colony forming assay was used to test the colony forming activity in MCL after overexpression of miR-548m or knockdown of CDK6. Results:Cell adhesion to FDCs induced downregulation of miR-548m and CDK6 expression in MCL. Bioinformatics assay revealed that miR-548m could target the 3'-UTR of CDK6 and that a negative correlation exists between the level of miR-548m and the CDK6 expression. Luciferase report as-say confirmed that miR-548m directly targeted 3'-UTR of CDK6. Colony forming assay showed that overexpression of miR-548m or knockdown of CDK6 significantly suppressed MCL colony formation. Conclusion:This study reveals that FDC-enhanced mantle cell lymphoma clonogenicity is mediated by the miR-548m/CDK6 axis.