| 1 Silencing CDC25AAffects Liver Cancer Cell Proliferation by Down-Regulating IL-6Objective:The purpose of this study was to explore the effect of cell division cycle 25A(CDC25A)on the proliferation of liver cancer cells and liver cancer xenografts,and further explore the interaction between CDC25A and IL-6 in liver cancer and the possible mechanism.Methods:HepG2 cells were transfected with the lentiviral LV-sh RNA-CDC25A to specifically silence CDC25A expression(CDC25A-sh RNA group),and the HepG2 cells were transfected with the lentiviral LV-sh RNA-NC as negative control(CDC25A-NC group).The untransfected HepG2 cells were used as blank control group(Control group).The transfection efficiency and the expression levels of IL-6 and IL-1β/NIK/NF-κB signal axis were detected by real-time fluorescent quantitative PCR(q RT-PCR)and western blot assays.Thirty female BALB/c nude mice of 4-6 weeks were randomly divided into 3groups(n=10):KD group,NC group and Control group,and three groups of cells were inoculated subcutaneously at 0.5-1.0 cm in the armpit of nude mice.The tumor sizes were observed and recorded every week,and the xenograft tumor tissues were obtained after 5 weeks.Immunohistochemistry,q RT-PCR and western blot assays were used to detect the expression levels of CDC25A and IL6 and the expression levels of IL-1β,NIK and NF-κB in xenograft tumors.Results:The HepG2 cells stably silencing the CDC25A gene were constructed.The q RT-PCR and western blot results showed that the relative expression levels of CDC25A m RNA and protein in the CDC25A-sh RNA group were significantly lower than those in the CDC25A-NC and Control groups,the difference was statistically significant(p<0.05).The relative expression levels of IL-6 m RNA and protein in CDC25A-sh RNA group(0.36±0.05,0.40±0.05)were significantly lower than those in CDC25A-NC group(1.16±0.12,1.31±0.03)and Control group(1.00±0.09,1.25±0.04)(p<0.05).Respectively,in the CDC25A-sh RNA group,the relative expression levels of IL-1β,NIK,NF-κB m RNA were 0.56±0.07,0.49±0.07,0.58±0.04,and the relative expression levels of proteins were 0.45±0.06,0.24±0.05,0.63±0.06.The relative expression levels of IL-1β,NIK,NF-κB m RNA in the CDC25A-NC group were 1.16±0.07,1.16±0.05,1.22±0.08,and the relative protein expression levels were 0.93±0.06,0.70±0.09,and 1.09±0.08.The relative expression levels of IL-1β,NIK,and NF-κB m RNA in the control group were 1.01±0.12,1.04±0.11,1.08±0.12,and the relative expression levels of proteins were 0.88±0.12,0.73±0.07,and1.08±0.11.It can be seen that the expression levels of IL-6 and IL-6 signal axis-related factors(IL-1β,NIK,NF-κB)m RNA and protein in CDC25A-sh RNA group were significantly lower than those in the negative control group(p<0.05)and the blank control group(p<0.05),the difference is statistically significant.The results of xenograft tumor growth curve showed that the tumor growth rate in the KD group was significantly lower than that in the NC and Control groups,but there was no significant difference in the growth rates between the NC group and the Control group.The average tumor volumes of the three groups were 25.96±9.62mm~3,79.00±10.56mm~3,and 81.96±14.75mm~3.The average tumor weights of the three groups were 0.21±0.09g,0.47±0.10g,and 0.52±0.11g.It can be seen that the average tumor volume and weight in the KD group were significantly smaller than those in the NC group and the Control group,and the differences were statistically significant(p<0.05).Immunohistochemical results showed that the expression levels of CDC25A and IL6 in KD group were significantly lower than those in NC and Control groups,and there was a significant positive correlation between CDC25A and IL6expression(r=0.669,p<0.05).The q RT-PCR and western blot results showed that the relative expression levels of CDC25A m RNA and protein in the KD group(0.32±0.07,0.41±0.06)were significantly lower than that in the NC group(0.99±0.05,1.10±0.15)and the Control group(1.05±0.10,1.06±0.11),the difference was statistically significant(p<0.05).At the same time,the relative expression levels of IL6 m RNA and protein in the KD group(0.42±0.06,0.54±0.06)were significantly lower than those in the NC group(1.28±0.09,1.26±0.10)and the Control group(1.00±0.04,1.21±0.07)(p<0.05),the difference was statistically significant.The q RT-PCR and western blot of xenograft tumor tissues showed that the relative expression levels of IL-1β,NIK,NF-κB m RNA in the KD group were 0.60±0.08,0.53±0.07,0.62±0.08,and the relative protein expression levels were 0.51±0.06,0.30±0.07,0.75±0.05.The relative expression levels of IL-1β,NIK,and NF-κB m RNA in the NC group were 1.23±0.14,1.13±0.09,1.46±0.10,and the relative protein expression levels were 0.94±0.02,0.69±0.05,and 1.32±0.02.The relative expression levels of IL-1β,NIK and NF-κB m RNA in the control group were 1.00±0.13,1.01±1.14,1.00±0.09,and the relative protein expression levels were 0.91±0.02,0.68±0.05,and 1.28±0.08.It can be seen that the m RNA and protein expression levels of the IL-6 signal axis related factors(IL-1β,NIK,NF-κB)in the KD group were significantly lower than those in the negative control group(p<0.05)and the blank control group(p<0.05),the difference was has statistically significant.Conclusion:1.Silencing the expression of CDC25A gene in HepG2 cells can down-regulate the expression of IL-6 and the expression of IL-1β/NIK/NF-κB signal axis.2.By constructing xenograft tumor models of liver cancer,the CDC25A gene in HepG2 cell line was clearly silenced,which significantly reduced the cell proliferation ability.There is a positive regulatory relationship between the CDC25A and IL-6.The effect of CDC25A on IL-6 may be related to the IL-1β/NIK/NF-κB signal axis.2 The Identification of CDC25 A Interacting Proteins by Tandem Affinity Purification Combined with Mass SpectrometryObjective: The purpose of this study was to explore the interaction proteins of CDC25 A in liver cancer cells,in order to provide new ideas for the follow-up study of its molecular mechanism and the diagnosis and treatment of liver cancer.Methods: The CDC25 A vector with Flag and TST dual tags(tandem affinity purification tags)was constructed,and the agar gel electrophoresis assay and sequencing technology were used to detect the Flag and TST.And the CDC25 A overexpression lentivirus with flag and TST dual tags were constructed and stably transferred to liver cancer HepG2 cells,the HepG2 cells were transfected with the overexpression lentiviral as experimental group,and transfected with the no-load lentivirus as control group.The western blot assay was used to verify CDC25 A protein expression.The tandem affinity purification technology was used to wash the two groups of cells twice.The western blot assay was used to verify the expression of CDC25A-Flag-TST fusion protein after purification.The tandem affinity purified proteins were identified by mass spectrometry,and the interaction proteins of CDC25 A were analyzed by bioinformatics.Co-Immunoprecipitation(Co-IP)was used to verify the interactions between CDC25 A and SPIN1 or hn RNPQ in HepG2 cells.Results: The results of agarose gel showed that the CDC25 A vector with Flag and TST dual tags(tandem affinity purification tags)was successfully constructed.Western blot results showed that the expression of CDC25 A protein in the experimental group was significantly higher than that in the control group,that is,the double-labeled CDC25 A overexpressing cell group was successfully constructed.And CDC25A-Flag-TST fusion protein was significantly expressed in the experimental group,but not in the control group.A total of 44CDC25A-related proteins were identified by tandem affinity purification mass spectrometry(TAP-MS).The GO analysis results showed that CDC25 A interacting proteins are involved in various physiological activities of cells,and the KEGG analysis results showed that CDC25 A interacting proteins are involved in multiple biochemical metabolic pathways and signal transduction pathways.The analysis of String protein interaction network revealed the connection between CDC25 A interacting proteins.Co-IP results showed that CDC25 A interacted with SPIN1 and hn RNPQ in HepG2 cells.Conclusion: CDC25 A interacting proteins in HepG2 cells were successfully screened by tandem affinity purification combined with mass spectrometry.Among them,CDC25 A interacts with SPIN1 and hn RNPQ. |
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