Objective To investigate the role and mechanism of microRNA-21-5p(miR-21)in cisplatin(CDDP)resistance in non-small cell lung cancer.Methods The expression of miR-21 in cancer tissues and paracancerous tissues of non-small cell lung cancer patients was detected by real-time fluorescence quantitative PCR.Non-small cell lung cancer CDDP-resistant cells H1299/CR and H1975/CR were constructed using non-small cell lung cancer cell lines H1299 and H1975.CCK-8 was used to detect the cell activity of each group of cells under CDDP treatment,and apoptosis was analyzed by flow cytometry.Dual luciferase was used to detect the role of miR-21 in relation to PTEN in H1299 cells,RT-qPCR was used to detect the miR-21 level,and Western blotting was used to detect the protein expression level of PTEN and PTEN downstream Wnt/β-catenin signaling pathway.H1299 and H1299/CR were used to construct a hormonal nude mouse model to verify the effect of miR-21 on the sensitivity of CDDP treatment in non-small cell lung cancer.Results The expression of miR-21 was significantly higher in cancer tissues than in adjacent normal tissues(P＜0.001).The expression of miR-21 in drug-resistant cells H1299/CR and H1975/CR was significantly elevated compared to that in H1299 and H1975 cells(P＜0.05).After transfection with miR-21 inhibitor,cell viability in the inhibitor group was significantly lower than in the inhibitor NC group when treated with CDDP ≥12 pmol/L(P＜0.001).Flow cytometry analysis showed that while the apoptosis rate in the CDDP and miR-21 inhibitor groups did not significantly differ from that in the untreated group,the apoptosis rate in the CDDP+miR-21 inhibitor group was significantly higher(P＜0.001).In contrast,after transfection with miR-21 mimic,the cell viability of the mimic-NC group was significantly reduced compared to the miR-21 mimic group when treated with CDDP ≥12 pmol/L(P＜0.05).TargetScan predicted that miR-21 could bind to the 3-UTR region of PTEN.Dual-luciferase reporter assays confirmed that miR-21 directly targeted PTEN.Overexpression of PTEN(PTEN-OE)together with miR-21 mimic co-transfection in H1299 cells resulted in decreased PTEN expression and increased levels of p-β-catenin(Ser552,Ser675)and β-catenin,while the PTEN expression in the miR-21 mimic+PTEN-OE group was elevated,with a corresponding decrease in p-β-catenin andβ-catenin levels.Flow cytometry showed that apoptosis was significantly increased in the mimic-NC group after CDDP treatment(P＜0.05).In H1299 xenograft models,after treatment with miR-21 mimic and CDDP,tumor growth was slower in the control+CDDP group than in the control group;tumors were larger in the miR-21+CDDP group than in the control+CDDP group at all time points.TUNEL staining revealed that apoptosis in the tumor tissues of the control+CDDP group was higher than in the control group,while apoptosis was reduced in the miR-21+CDDP group compared to the control+CDDP group.In the H1299/CR xenograft model,after 5 days of treatment,the tumors were smaller in the H1299/CR+CDDP group than in the H1299/CR group,and those in the H1299/CR+CDDP+inhibitor group were smaller than in the H1299/CR+CDDP group.TUNEL staining showed that apoptosis was increased in the H1299/CR+CDDP group compared to the H1299/CR group,and further increased in the H1299/CR+CDDP+inhibitor group.Conclusion In non-small cell lung cancer,miR-21 overexpression inhibits PTEN level and activates the Wnt/β-catenin pathway involved in the development of CDDP resistance in non-small cell lung cancer.Inhibition of miR-21 expression in tumor cells will enhance the sensitivity of tumor cells to CDDP.

Objective To investigate the role and mechanism of microRNA-21-5p(miR-21)in cisplatin(CDDP)resistance in non-small cell lung cancer.Methods The expression of miR-21 in cancer tissues and paracancerous tissues of non-small cell lung cancer patients was detected by real-time fluorescence quantitative PCR.Non-small cell lung cancer CDDP-resistant cells H1299/CR and H1975/CR were constructed using non-small cell lung cancer cell lines H1299 and H1975.CCK-8 was used to detect the cell activity of each group of cells under CDDP treatment,and apoptosis was analyzed by flow cytometry.Dual luciferase was used to detect the role of miR-21 in relation to PTEN in H1299 cells,RT-qPCR was used to detect the miR-21 level,and Western blotting was used to detect the protein expression level of PTEN and PTEN downstream Wnt/β-catenin signaling pathway.H1299 and H1299/CR were used to construct a hormonal nude mouse model to verify the effect of miR-21 on the sensitivity of CDDP treatment in non-small cell lung cancer.Results The expression of miR-21 was significantly higher in cancer tissues than in adjacent normal tissues(P＜0.001).The expression of miR-21 in drug-resistant cells H1299/CR and H1975/CR was significantly elevated compared to that in H1299 and H1975 cells(P＜0.05).After transfection with miR-21 inhibitor,cell viability in the inhibitor group was significantly lower than in the inhibitor NC group when treated with CDDP ≥12 pmol/L(P＜0.001).Flow cytometry analysis showed that while the apoptosis rate in the CDDP and miR-21 inhibitor groups did not significantly differ from that in the untreated group,the apoptosis rate in the CDDP+miR-21 inhibitor group was significantly higher(P＜0.001).In contrast,after transfection with miR-21 mimic,the cell viability of the mimic-NC group was significantly reduced compared to the miR-21 mimic group when treated with CDDP ≥12 pmol/L(P＜0.05).TargetScan predicted that miR-21 could bind to the 3-UTR region of PTEN.Dual-luciferase reporter assays confirmed that miR-21 directly targeted PTEN.Overexpression of PTEN(PTEN-OE)together with miR-21 mimic co-transfection in H1299 cells resulted in decreased PTEN expression and increased levels of p-β-catenin(Ser552,Ser675)and β-catenin,while the PTEN expression in the miR-21 mimic+PTEN-OE group was elevated,with a corresponding decrease in p-β-catenin andβ-catenin levels.Flow cytometry showed that apoptosis was significantly increased in the mimic-NC group after CDDP treatment(P＜0.05).In H1299 xenograft models,after treatment with miR-21 mimic and CDDP,tumor growth was slower in the control+CDDP group than in the control group;tumors were larger in the miR-21+CDDP group than in the control+CDDP group at all time points.TUNEL staining revealed that apoptosis in the tumor tissues of the control+CDDP group was higher than in the control group,while apoptosis was reduced in the miR-21+CDDP group compared to the control+CDDP group.In the H1299/CR xenograft model,after 5 days of treatment,the tumors were smaller in the H1299/CR+CDDP group than in the H1299/CR group,and those in the H1299/CR+CDDP+inhibitor group were smaller than in the H1299/CR+CDDP group.TUNEL staining showed that apoptosis was increased in the H1299/CR+CDDP group compared to the H1299/CR group,and further increased in the H1299/CR+CDDP+inhibitor group.Conclusion In non-small cell lung cancer,miR-21 overexpression inhibits PTEN level and activates the Wnt/β-catenin pathway involved in the development of CDDP resistance in non-small cell lung cancer.Inhibition of miR-21 expression in tumor cells will enhance the sensitivity of tumor cells to CDDP.

Objective To explore the effects of vitamin C and niacinamide on the growth and differentiation of human primary cultured keratinocytes.Methods Normal human foreskin was used in this study.The epidermis was separated enzymatically from the dermis by thermolysin,and keratinocytes were isolated from the epidermis by digestion with trypsin plus EDTA.The single keratinocytes were cultured with undedying NIH-3T3 cells as feeder cells in a complete medium supplied with 50 mg/L (vitamin C group),niacinamide of 400 μmol/L(niacinamide group)or vehicle(control group).Immunocytochemistry and immunodot blot were performed using monoclonal antibodies directed against C-myc,cyclin D1,filaggrin and involucrin.Results The colony number was highest in vitamin C group,followed by the control group and niacinamide group,and the colony morphology in vitamin C group was similar to that in the control group,but distinct from that in the niacinamide group.A significant increase was noticed in the expression of C-myc,cyclin D1,filaggrin and involucrin in vitamin C-treated keratinocytes compared with the control keratinocytes(all P＜0.05);however,in niacinamide-treated keratinocytes,the expression of filaggrin was significantly enhanced(P＜0.01),that of involucrin remained unchanged(P＞0.05),while that of C-myc was depressed(P＜0.05).Conclusions These results demonstrate that vitamin C has a favorable effect on both the growth and differentiation of human keratinocytes,while niacinamide seems to only promote the differentiation but attenuate the growth of human keratinocytes.

Objective To compare the difference in quercetin against oxidative stress response in mouse and in NIH-3T3 cells before and after H2O2 treatment,to explore the underlying mechanism for the quercetin antioxidant.Methods The cultured NIH-3T3 cells were randomly divided into 4 groups: quercetin(Q) pre-protective group(Qb) firstly treated with quercetin for 24 h followed by incubation with H2O2 for 30 min;post-protective group(Qa) treated with H2O2 for 30 min followed by incubation with quercetin for 24 h;H2O2 group(H2O2) after exposure to H2O2 for 30 min,incubated with DMEM medium and the control group(C) only cultured with DMEM medium.The survival rate and apoptotic rate were detected respectively with MTT and TUNEL in NIH-3T3 cell sus-pension samples.The expression of cyclin D1,PTEN,NF-?B,HSP-70,BCl-2,BAX and caspase-3 were examined with immunocytochemistry and immunoblotting.Besides,20 Wistar rats were divided into control group and experimental group,the latter was given with quercetin in the doze of 0.13 mmol/kg.The levels of T-AOC,SOD,GSH-Px,GSH,MDA,NOS and NO2-/NO3-were detected both in the cleaved NIH-3T3 cells and in the plasma from both experimental and control animals prior to and post-1 h,2 h and after 24 h.Results When the Qb group was compared with H2O2 or Qa group,the survival rate was higher and the apoptotic rate was lower.When the H2O2 group was compared with C group,the expression of cyclin D1、PTEN or BCl-2 was down-regulated;while that of BAX、HSP-70、NF-?B or caspase-3 was up-regulated;the level of T-AOC,SOD,GSH-Px or GSH was decreased;that of NOS、NO2-/NO3-or MDA enhanced in the cleft NIH-3T3 cells.When the plasma level of the anti-oxidative enzyme system prior to-compared with post-1h and 2h-treatment with Q,the level of T-AOC,SOD,GSH-Px and GSH,especially the former two,were higher;MDA,lower;NOS or NO2-/NO3-promoted.However,the above parameters basically became normal 24 h after treatment with Q.Conclusion Quercetin down-regulates the promoted expression of HSP70,NOS,NO2-/NO3-and NF-?B etc.in H2O2-treatment NIH-3T3 cells.Qb could reverse the H2O2 damage effects more markedly.Moreover,the quercetin exerts anti-oxidant protective effect through modulating the anti-oxidative enzyme system both in vivo and in vitro.However,based on the cell heterogeneity in none-or pre/post-H2O2-treatment state,a difference in quercetin antioxidant response is noted.