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OBJECTIVES@#This study aimed to explore the effects of silencing isoprenylcysteine carboxyl methyltransfe-rase (Icmt) through small interfering RNA (siRNA) interference on the proliferation and apoptosis of tongue squamous cell carcinoma (TSCC).@*METHODS@#Three siRNA were designed and constructed for the Icmt gene sequence and were then transfected into TSCC cells CAL-27 and SCC-4 to silence Icmt expression. The tested cells were divided as follows: RNA interference groups Icmt-siRNA-1, Icmt-siRNA-2, and Icmt-siRNA-3, negative control group, and blank control group. The transfection efficiency of siRNA was detected by the fluorescent group Cy3-labeled siRNA, and the expression of Icmt mRNA was screened by quantitive real-time polymerase chain reaction (qRT-PCR) selected the experimental group for subsequent experiments. The expression of Icmt, RhoA, Cyclin D1, p21, extracellular regulated protein kinases (ERK), and phospho-extracellular regulated protein kinases (p-ERK) were analyzed by Western blot. The proliferation abilities of TSCC cells were determined by cell counting kit-8 assay. The change in apoptosis was detected by AnnexinV-APC/propidium staining (PI) assay. Cell-cycle analysis was conducted by flow cytometry.@*RESULTS@#The expression of Icmt mRNA and protein in TSCC cells significantly decreased after Icmt-siRNA transfection (@*CONCLUSIONS@#Silencing Icmt can effectively downregulate its expression in TSCC cells, reduce the RhoA membrane targeting localization and cell proliferation, and induce apoptosis. Thus, Icmt may be a potential gene therapy target for TSCC.
Subject(s)
Humans , Apoptosis , Carcinoma, Squamous Cell , Cell Line, Tumor , Cell Proliferation , Protein Methyltransferases , RNA, Small Interfering , Tongue , Tongue NeoplasmsABSTRACT
Objective To investigate the effects of Rapamycin and mammalian target of rapamycin - small in-terfering RNA (mTOR siRNA)on the proliferation,apoptosis and collagen Ⅰ(COLⅠ),collagen Ⅲ(COLⅢ)and fi-bronectin(FN)in premature rats lung fibroblasts exposed to hyperoxia. Methods 900 mL/ L volume fraction of oxygen was used to establish hyperoxia - damaged cell models,and the premature rats lung fibroblasts were divided into air control group,hyperoxia group,hyperoxia + rapamycin group and mammalian target of rapamycin - small interfering RNA transfection group. Cell proliferation was assessed by using 3 -(4,5 - Dimethylthiazol - 2 - yl)- 2,5 - dipheny-ltetrazolium bromide assay. Apoptosis were detected by Annexin V - FITC and propidium lodide (PI)double staining. The expressions of COLⅠ,COLⅢ and fibronectin was assessed by using enzyme linked immunosorbent assay and Bcl - 2,P53 and pro - fibrotic factors of connective tissue growth factor(CTGF)and transforming growth factor β(TGF - β)by using Western blot. Results Compared with the air control group,the proliferation of lung fibroblasts decreased and the apoptosis increased in the hyperoxia group,while the contents of COLⅠ(28. 30 ± 0. 53 vs. 17. 43 ±0. 37),COLⅢ(27. 86 ± 1. 02 vs. 17. 43 ± 0. 37)and fibronectin(32. 87 ± 0. 42 vs. 21. 57 ± 0. 47),P53(0. 810 ± 0. 119 vs. 0. 160 ± 0. 018),TGF - β(0. 580 ± 0. 108 vs. 0. 210 ± 0. 008)and CTGF(0. 590 ± 0. 017 vs. 0. 220 ± 0. 007)were also increased but the expression of Bcl - 2(0. 150 ± 0. 004 vs. 0. 600 ± 0. 130)protein was decreased, and the differences were all statistically significant (all P < 0. 01). Compared with the hyperoxia group,the proliferation of lung fibroblasts was increased in the hyperoxia + rapamycin group,but the apoptosis was decreased,the contents of COLⅠ(23. 17 ± 0. 60 vs. 28. 30 ± 0. 53),COLⅢ(17. 09 ± 0. 58 vs. 27. 86 ± 1. 02)and fibronectin(28. 11 ± 0. 68 vs. 32. 87 ± 0. 42),P53(0. 430 ± 0. 008 vs. 0. 810 ± 0. 119),TGF - β(0. 380 ± 0. 008 vs. 0. 580 ± 0. 108)and CTGF (0. 040 ± 0. 006 vs. 0. 590 ± 0. 017)were decreased while the expression of Bcl - 2(0. 290 ± 0. 009 vs. 0. 150 ± 0. 004) protein was increased,and the differences were all statistically significant (all P < 0. 01). In the mTOR siRNA transfec-tion group,compared with the hyperoxia + rapamycin group,the proliferation of lung fibroblasts was increased,but the apoptosis was decreased;the contents of COLⅠ(15. 71 ± 0. 34 vs. 23. 17 ± 0. 60),COLⅢ (13. 85 ± 1. 36 vs. 17. 09 ± 0. 58)and fibronectin(20. 18 ± 0. 28 vs. 28. 11 ± 0. 68),P53(0. 300 ± 0. 006 vs. 0. 430 ± 0. 008),TGF - β(0. 150 ± 0. 002 vs. 0. 380 ± 0. 008)and CTGF(0. 140 ± 0. 004 vs. 0. 040 ± 0. 006)were decreased while the expression of Bcl - 2 (0. 460 ± 0. 012 vs. 10. 290 ± 0. 009)protein was increased,and the differences were all statistically significant (all P < 0. 01). Conclusion Rapamycin and mTOR siRNA can protect lung injury caused by hyperoxia and have a certain inhibitory effect on pulmonary fibrosis,and mTOR siRNA effect is more obvious,so the mechanism may be through the inhibition of mTOR signaling pathway.
ABSTRACT
AIM:To investigate the effect of nuclear factor E2-related factor 2 (Nrf2) activation by 18α-gly-cyrrhetinic acid (18α-GA) on the proliferation and self-renewal of adult neural stem cells (aNSCs), and to explore an ef-fective way of maintaining the viability of aNSCs .METHODS:NSCs were dissociated from subventricular zone of the mice at postnatal days 0, 60, and 300.The expression levels of Nrf2 in the NSCs at various ages were compared .After treatment with 18α-GA, the expression of Nrf2 was examined by real-time PCR and Western blot.shRNA lentiviral vector (LV) car-rying green fluorescent protein (GFP) gene was constructed to knock down Nrf2 expression.The knockdown efficiency in the aNSCs was detected by real-time PCR and Western blot .Subsequently , the aNSCs were divided into DMSO group , 18α-GA group, LV-GFP group and LV-Nrf2-shRNA group.BrdU incorporation assay , Tuj1 staining, CCK-8 assay, Hoechst 33342/PI staining and detection of reactive oxygen species ( ROS) were performed to analyze the proliferation , dif-ferentiation, viability, apoptosis and oxidative stress levels of the NSCs .RESULTS:The mRNA expression level of Nrf2 in adult and aged NSCs was significantly lower than that in newborn NSCs (P<0.01), while the ROS level of aNSCs was significantly higher (P<0.05).After treatment with 18α-GA, the expression level of Nrf2 in the aNSCs was significantly up-regulated as compared with DMSO group ( P<0.01).Increased number of BrdU +and Tuj1 +cells was observed in 18α-GA group, indicating that 18α-GA-treated cells had higher viability (P<0.05).Meanwhile, there were fewer apop-totic cells and lower ROS level in 18α-GA group than those in DMSO group (P<0.05).After knockdown of Nrf2 in aNSCs and then treated with 18α-GA, there were less BrdU +and Tuj1 +cells, as well as the aNSCs with lower viability in LV-Nrf2-shRNA group (P<0.05).Moreover, the ROS level was increased in LV-Nrf2-shRNA group as compared with LV-GFP group (P<0.05).CONCLUSION:Activation of Nrf2 by 18α-GA elevates the antioxidant capacity of aNSCs , thus ameliorating the cell proliferation and differentiation potentials .
ABSTRACT
AIM:To investigate the effect of nuclear factor E2-related factor 2 (Nrf2) activation by 18α-gly-cyrrhetinic acid (18α-GA) on the proliferation and self-renewal of adult neural stem cells (aNSCs), and to explore an ef-fective way of maintaining the viability of aNSCs .METHODS:NSCs were dissociated from subventricular zone of the mice at postnatal days 0, 60, and 300.The expression levels of Nrf2 in the NSCs at various ages were compared .After treatment with 18α-GA, the expression of Nrf2 was examined by real-time PCR and Western blot.shRNA lentiviral vector (LV) car-rying green fluorescent protein (GFP) gene was constructed to knock down Nrf2 expression.The knockdown efficiency in the aNSCs was detected by real-time PCR and Western blot .Subsequently , the aNSCs were divided into DMSO group , 18α-GA group, LV-GFP group and LV-Nrf2-shRNA group.BrdU incorporation assay , Tuj1 staining, CCK-8 assay, Hoechst 33342/PI staining and detection of reactive oxygen species ( ROS) were performed to analyze the proliferation , dif-ferentiation, viability, apoptosis and oxidative stress levels of the NSCs .RESULTS:The mRNA expression level of Nrf2 in adult and aged NSCs was significantly lower than that in newborn NSCs (P<0.01), while the ROS level of aNSCs was significantly higher (P<0.05).After treatment with 18α-GA, the expression level of Nrf2 in the aNSCs was significantly up-regulated as compared with DMSO group ( P<0.01).Increased number of BrdU +and Tuj1 +cells was observed in 18α-GA group, indicating that 18α-GA-treated cells had higher viability (P<0.05).Meanwhile, there were fewer apop-totic cells and lower ROS level in 18α-GA group than those in DMSO group (P<0.05).After knockdown of Nrf2 in aNSCs and then treated with 18α-GA, there were less BrdU +and Tuj1 +cells, as well as the aNSCs with lower viability in LV-Nrf2-shRNA group (P<0.05).Moreover, the ROS level was increased in LV-Nrf2-shRNA group as compared with LV-GFP group (P<0.05).CONCLUSION:Activation of Nrf2 by 18α-GA elevates the antioxidant capacity of aNSCs , thus ameliorating the cell proliferation and differentiation potentials .