Transactivation Of CENPA Via MYBL2 In Mitotic Catastrophe Of Hepatocellular Carcinoma Cells

Background and aims: Hepatocellular carcinoma Hepatocellular carcinoma(HCC)is a malignant tumor that seriously damages human health.Its incidence and mortality rank among the top five in the world.The reason for the high mortality of hepatocellular carcinoma is that,in addition to the lack of obvious symptoms in the early stage,the patients can not find it in time and thus delay the diagnosis and treatment,the hepatocellular carcinoma cells have a strong ability of self-division and proliferation and a reduced ability of regulatory cell death,which leads to rapid intrahepatic metastasis and diffusion of hepatocellular carcinoma cells.Mitotic catastrophe is an inherent tumor inhibition mechanism,which can lead to tumor cell death or aging as a precursor of regulatory cell death.Therefore,inducing tumor cell mitotic catastrophe is a very potential treatment strategy in cancer treatment.The key to the occurrence of mitotic catastrophe is that cell chromosomes cannot be properly allocated to daughter cells during mitosis,and the key element to regulate this process is the centromere kinetochore complex.Centromere protein A(CENPA),as the important members of the centromeric kinetochore complex,regulates the assembly and docking of the centromere and kinetochore,thus ensuring the correct distribution of chromosomes and facilitating the smooth progress of mitosis.Therefore,this study intends to explore whether the differential expression of CENPA causes mitotic catastrophe in hepatocellular carcinoma cells and its regulatory mechanism.Content and methods: Part 1:Correlation between expression level of CENPA and mitotic catastrophe of hepatocellular carcinoma cells 1.The expression of CENPA in hepatocellular carcinoma and its influence on survival rate and clinical stage of patients with hepatocellular carcinoma:(1)The expression of CENPA m RNA was detected by q PCR in cancer and paracancerous tissues of 20 patients with primary hepatocellular carcinoma collected from our hospital.(2)The expression of CENPA m RNA in hepatocellular carcinoma cells(Huh-7,SMC-7721,Hep G2)and normal hepatocytes(HL-7702)was detected by q PCR.(3)The correlation between the expression level of CENPA in 360 patients with hepatocellular carcinoma in TCGA database and the 10-year overall survival rate,disease-free survival rate and clinical stage of patients with hepatocellular carcinoma was analyzed using R studio software.2.The effect of differential expression of CENPA on mitotic catastrophe of hepatocellular carcinoma cells:(1)The cell model of CENPA knockdown was constructed using Sh RNA chronic toxin vector,and the expression level of CENPA in transfected and control groups was detected by Western Blot and q PCR to determine whether the cell model was successfully constructed.(2)To detect whether mitotic catastrophe occurs in hepatocellular carcinoma cells after inhibiting the expression of CENPA: to detect the distribution of chromosomes in the mitotic process of hepatocellular carcinoma cells after inhibiting the expression of CENPA through cell immunofluorescence assay,to detect the effect of inhibiting the expression of CENPA on the cycle of hepatocellular carcinoma cells through PI cell staining assay,and to detect the effect of knockdown of CENPA on the apoptosis of hepatocellular carcinoma cells using apoptosis assay;the division ability of hepatocellular carcinoma cells was detected by EDU test;the proliferation ability of hepatoma cells was evaluated by CCK8 experiment.Part 2:Correlation between expression level of MYBL2 and mitotic catastrophe of hepatocellular carcinoma cells 1.Screening the transcription factors of CENPA:(1)Query the 3000 bp upstream to 200 bp downstream base sequence of the transcriptional starting site of CENPA gene on the NCBI website as the promoter sequence,and predict the transcription factors that may combine with the sequence through the UCSC website.(2)TCGA data was used to analyze the correlation between the expression of predicted transcription factors and the expression of CENPA for further screening.2.The expression of the filtered transcription factor(MYBL2,MYB protooncogene like 2)in hepatocellular carcinoma and its influence on the clinical survival and stage of patients with hepatocellular carcinoma:(1)The expression of MYBL2 m RNA was detected by q PCR in the cancerous and paracancerous tissues of 20 patients with primary hepatocellular carcinoma collected from our hospital.(2)The expression of MYBL2 m RNA in hepatocellular carcinoma cells(Huh-7,SMC-7721,Hep G2)and normal hepatocytes(HL-7702)was detected by q PCR.(3)Using R studio software,the correlation between MYBL2 expression level of 364 hepatocellular carcinoma patients in TCGA database and the 10-year overall survival rate,disease-free survival rate and clinical stage of hepatocellular carcinoma patients was analyzed.3.The effect of MYBL2 differential expression on CENPA m RNA level and mitotic catastrophe of hepatocellular carcinoma cells:(1)Use Si RNA to construct the cell model of MYBL2 knockdown,and use Western Blot and q PCR to detect the expression level of MYBL2 protein and m RNA in the MYBL2 knockdown group and the control group to determine whether the cell model is successfully constructed,and detecting the expression level of CENPA m RNA using q PCR.(2)Detect whether mitotic catastrophe occurs in hepatocellular carcinoma cells after inhibition of MYBL2 expression(same methods as before).Part 3:Explore the mechanism of MYBL2 on mitotic catastrophe in hepatocellular carcinoma cells 1.Regulation mechanism of MYBL2 on CENPA:(1)Use JASPAR website to predict the binding site of MYBL2 on the promoter of CENPA gene.(2)Chromatin immunoprecipitation assay(CHIP)was used to detect whether MYBL2 binds to the predicted site of the CENPA gene promoter.(3)Through the double luciferase reporter gene experiment,construct the firefly luciferase reporter gene vector including the mutant and wild-type of the binding site of the CENPA gene promoter,and explore whether MYBL2 can activate the transcription of the CENPA gene by binding to the prediction site.2.Explore whether MYBL2 regulates the mitotic catastrophe of hepatocellular carcinoma cells by CENPA: compared with the control group,the expression level of MYBL2 in the si MYBL2 and the si MYBL2 + h-CENPA is reduced;compared with the control group,the CENPA level in the si MYBL2 reduced,and hepatocellular carcinoma cells occur mitotic catastrophe,however,CENPA level in the si MYBL2 + h-CENPA group recovered.The mitotic catastrophe of hepatocellular carcinoma cells was relieved.Results:Part 1:Correlation between expression level of CENPA and mitosis catastrophe in hepatocellular carcinoma cells 1.CENPA m RNA was significantly up-regulated in hepatocellular carcinoma tissues and cells.Patients with high CENPA expression,the total 10-year overall survival and disease-free survival of the were lower than those with low CENPA expression,in addition,CENPA level was higher in patients with advanced clinical stage.2.Western blot and q PCR results indicates that the knockdown of CENPA is successful.Inhibiting of CENPA causes the chromosomes could not be properly distributed into the daughter cells during the mitosis of hepatocellular carcinoma cells,the proportion of cells in G2/M phase increased,the apoptosis of hepatocellular carcinoma cells significantly increased,the division and proliferation ability significantly decreased.Part 2:MYBL2 positively regulates CENPA and participates in mitotic catastrophe of hepatocellular carcinoma cells 1.We predict that the transcription factor of CENPA is MYBL2.2.MYBL2 m RNA was significantly up-regulated in hepatocellular carcinoma tissues and cells.Patients with high MYBL2 expression,,the total 10-year overall survival and disease-free survival are lower than those with low expression,and the expression of MYBL2 was higher in patients with advanced clinical stage.Part 3: MYBL2 binds to the CENPA gene promoter-1055/-1041 site to transactivate CENPA and participate in the mitotic catastrophe of hepatocellular carcinoma cells 1.MYBL2 transactivates CENPA:(1)Using the JASPAR website to predict the binding site of MYBL2 on the CENPA gene promoter at-1055/-1041 sites.(2)CHIPPCR results shows that MYBL2 was positively bind to the-1055/-1041 site of the CENPA promoter.(3)The results of the double luciferase reporter gene experiment showed that the expression of firefly luciferase was activated when the firefly luciferase reporter gene plasmid containing the MYBL2 binding site promoter sequence was transfected into hepatocellular carcinoma cells.In the MYBL2 overexpression group,the expression of firefly luciferase was further enhanced.However,when the firefly luciferase reporter gene plasmid containing the promoter sequence of the MYBL2 binding site mutant was used to transfect hepatocellular carcinoma cells,the expression of firefly luciferase in the vector control group and the overexpression group was not activated.2.MYBL2 regulates the mitotic catastrophe of hepatocellular carcinoma cells by regulating CENPA: Comparing to the control group,the expression level of MYBL2 in the MYBL2 knockdown group and the MYBL2 knockdown + CENPA overexpression group downregulates;Comparing to the control group,the expression level of CENPA in the MYBL2 knockdown group downregulates,and hepatocellular carcinoma cells experienced mitotic catastrophe.However,the expression level of CENPA restores in the MYBL2 knockdown + CENPA overexpression group.And the mitotic catastrophe of hepatocellular carcinoma cells has been alleviated.Conclusions: 1.The abnormal expression of CENPA in hepatocellular carcinoma cells resists mitotic catastrophe.2.MYBL2 positively regulates the level of CENPA m RNA and participates in the mitotic catastrophe of hepatocellular carcinoma cells.3.MYBL2 regulates the mitotic catastrophe of hepatocellular carcinoma cells by binding to the-1055/-1041 site of the CENPA gene promoter and transactivating CENPA.Conclusion: MYBL2 can resist the mitotic catastrophe of hepatocellular carcinoma cells by binding to the-1055/-1041 site of the CENPA gene promoter to transactivate CENPA.