ABSTRACT
Objective:To investigate the regulation effect of miR-125b in the gastric cancer cell growth mediated by apoptosis related protein (Fas)/apoptosis related protein ligand (FasL) signal.Methods:Gastric cancer SGC-7901 cells were cultured in vitro. MiR-125b inhibitor sequence, NC sequence and transfection reagent were transfected into SGC-7901 cells and divided into three groups: miR-125b inhibited group, NC group and control group. The expression of miR-125b in transfected cells was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and cell proliferation was detected by cell counting kit-8 (CCK-8) method. The colony formation was detected by plate cell clone formation assay. Cell apoptosis and cycle were detected by flow cytometry. The protein expression of Fas and FasL was detected by Western blot. The targeted regulation of Fas by miR-125b was detected by luciferase activity assay. Results:The expression level of miR-125 and the number of cell colony in miR-125b inhibited group was significantly lower than those in control group and NC group, and the inhibition rate of cell proliferation and apoptosis rate were significantly higher than that in control group and NC group (all P<0.05). The DNA content in G 1 phase in miR-125b inhibited group was significantly higher than that in control group and NC group, and the DNA content in S phase in miR-125b inhibited group was significantly lower than that in control group and NC group (all P<0.05). The expression of Fas and FasL protein in miR-125b inhibited group was significantly higher than that in control group and NC group (all P<0.05). The target site of miR-125b was found in 3′-UTR of Fas mRNA, and compared with the NC+ Fas 3′UTR-Wt group, the activity of luciferase in the miR-125b inhibited group+ Fas 3′-UTR-Wt group decreased significantly ( P<0.05). Conclusions:Inhibition of miR-125b expression can activate Fas/FasL signal and inhibit SGC-7901 cell proliferation, induce G 1 phase arrest of cell cycle and promote apoptosis.
ABSTRACT
Objective To explore the influence of down-regulating Daxx on cell cycle and chemotherapeutic drug resistance in human ovarian cancer cells.Methods SiRNA and NC negative control(NC) RNA of Daxx were constructed and divided into the NC group and silencing Daxx(siDaxx) group after transfecting to ovarian cancer cell line C13k.The doxorubicin concentration gradient of 0,0.15,0.30,0.60 μmol/L was set.The cellular cycle changes and apoptosis changes of these two groups were detected by using the flow cytometry.Western blot was used to detect the expression changes of apoptosis related protein cyclinB1 and cleavedparp.Results 0.30 μmol/L doxorubicin down-regulated Daxx to result in significant G2/M arrest(P＜0.05).Down-regulating Daxx resulted in doxorubicin resistance in C13k cells.Conclusion The effect of Daxx on ovarian cancer chemotherapy might be related to its regulation on cell cycle.
ABSTRACT
Objective To investigate the effects of gene silencing of Fas-associated death domain (FADD) with synthetic small interfering RNA (siRNA) on apomorphine-induced contralateral rotation,and the expression of Fas and caspase-8 in rat models of parkinsonism. Methods Sprague-Dawley rats were randomly divided into 5 groups:control group,Parkinson' s disease (PD) group,FADD siRNA group,FADD siRNA positive control group and FADD siRNA negative control group. Synthetic FADD siRNA sequences,siRNA positive sequences or siRNA negative sequences were infused into right substantianigra of midbrain using RNA interference and stereotactic techniques before parkinsonian rat model establishment.Apomorphine-induced contralateral rotations of the rats were observed after the injection.The protein and mRNA expression levels of FADD,Fas and caspase-8 were measured by Western blot and RT-PCR.Results In the control group,no rotation was observed after injecting apomorphine; however,in the rest groups,the number of rats respectively was 12 (12/14),3 (3/13),4 (4/15) and 11 ( 11/14 ) in apomorphine-induced contralateral rotation,which had significant statistical differences ( x2 ＝ 18.56,P =0.000).In parkinsonian substantia nigra,marked increases in the protein and mRNA levels of FADD,Fas and caspase-8 were observed,compared with control group.Furthermore,compared with PD group,FADD protein and mRNA levels were strongly suppressed by administration of FADD siRNA in FADD siRNA group. FADD siRNA administration also resulted in great attenuation of 6-hydroxydopamine-induced increases in expression and activation of caspase-8.However,no decrease in expression of Fas was observed in FADD siRNA group and FADD siRNA positive control group,compared with PD group.Conclusion Our results suggest that death receptor signaling pathway plays a critical role in the pathogenesis of PD.FADD siRNA is effective against this pathway and it may,at least in part,provide a potential neuroprotective effect.
ABSTRACT
Objective To examine the sensitivity of human colorectal carcinoma cells to 5-fluorouracil treatment by stable transfection of extrinsic Fas-associated death domain protein(FADD) gene，both in vitro and in vivo，so as to investigate the feasibility of combination therapy of FADD gene and 5-fluorouracil in human colorectal carcinoma.Methods①RT-PCR and Western blotting were used to detect the expressions of both mRNA and protein of FADD gene in SW480／FADD (stably transfected with FADD)，SW480／neo and SW480 cells.②After treatment with 5-fluorouracil as an apoptotic inducer，in vitro cell growth activities were investigated by MTT assay.Cell apoptosis and its rates were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)assay and flow cytometry of annexin V-FITC／PI staining.The expressions of caspase-8 and caspase-3 were examined by Western blotting.③To examine the inhibitory effect of FADD gene combined with 5-fluorouracil, tumor xenograft model was prepared for in vivo study.Results ① Compared with SW480 and SW480/neo cells, FADD mRNA and protein levels of SW480/FADD cells were higher (P<0.05). ② Inhibitory rate of SW480/FADD cells was remarkably higher than SW480 and SW480/neo cells (P＜0.05 ). ③ Forty-eight hours after treatment with 5-fluorouracil (10 mg/L), the apoptotic rate of SW480/FADD cells was (33.3 ± 4.5)%, which was higher than SW480 [(13. 9 ± 3. 2)%3 and SW480/neo [(14. 1 ± 3. 4)%], with significant difference (P< 0.05).④ Forty-eight hours after treatment with 5-fluorouracil (10 mg/L),procaspase-8 and procaspase-3 expressions of SW480 and SW480/neo cells were higher than SW480/FADD cells, whereas their cleaved caspase-8 and cleaved caspase-3 expressions were lower than SW480/FADD cells (P＜0. 05).⑤ In in vivo study, SW480/FADD cells increased the efficacy of fluorouracil-induced inhibition of tumor growth in nude mice. ConclusionsStable overexpression of FADD increases sensitivity of the cells to 5-fluorouracil and combination of FADD with 5-fluorouracil will he a promising alternative in colorectal cancer treatment.
ABSTRACT
Objective:To imestigate the effect of Shenxiong injection on neuronal aooptosis and Fasassociated death dormin protein(FADD)and its mRNA expression after ischernia-reperfusion in rats.Methods:Atotal of 100 SD rats wgre randomly allocated into normal(n=10),sham-operation(n=10),cerebral ischemia-reperfusion(n=40),and Shenxiong injection(n=40)groups.A model of middle cerebral wtery occlusion was induced by suture method.The neuronal apoptosis was detected by the TUNEL assay.The expressions of FADD protein and mRNA were detected by inmmnohistochemical staining and reverse tramcription-polymerase chain reaction(RT-PCR),respectively.Results:As compared with the normal and sham-operation groups.the numbers of apoptotic cell in the cerebral ischemia-reperfusion group were increased significantly(P＜0.01),and the expressions of FADD protein and mRNA were enhanced significantly(all P＜0.01).As compared with the ceretral ischemia-reperfusion group,the numbers of apoptotic cell were decreased significantly (P＜0.05,P＜0.001),and the expressiom of FADD protein and mRNA were reduced significantly in the Shenxiong group(P＜0.05,P＜o.001).Convlusions:Shertxiong injection may inhibit the neuronal apoptosis after ischemia-reprfusion in r-cas by down-regulaftng the expression of FADD.
ABSTRACT
Objective: To investigate the induction of Tca8113 cells to apoptosis by fadd gene.Methods: RT PCR and recombinant PCR were used to amplify human fadd gene and cloned into expression vector pcDNA3 and pIRES2 EGFP, then transfered into Tca8113 cells. The growth and apoptosis of the cells were tested by cell counting,fluorescent microscopy, electron microscopy and flow cytometery. Results: fadd gene was obtained and transfered into Tca8113 cells. After transfection of the gene the growth of the cells was inhibited by 25%～52%, cell number in G 1 phase increased and that in S phase decreased. Apoptosis of the cells was observed. Conclusion: fadd gene can effectively inhibite cell growth and induce Tca8113 cells to apoptosis.