KAT6B Promotes The Malignant Biological Behavior Of Cervical Cancer And Its Molecular Mechanism

Background and ObjectivesCervical cancer ranks first in the incidence and death of gynecological tumors in China.More and more studies have found that abnormal gene epigenetic modification plays a vital role in cervical cancer development.Lysine acetyltransferases(KATs)histone acetylation is essential in the structure and function of chromosomes.In the gene encoding KATs,KAT6B is a very important tumor driver gene,whose amplification has been found in many kinds of tumors,such as ovarian cancer,breast cancer,lung cancer,and bowel cancer,etc.KAT6B is also identified as tumor suppressor genes.At present,there are few relevant studies on KAT6B.The malignant biological behavior of KAT6B and its molecular mechanism is not yet clear in cervical cancer.This project intends to study the biological role of KAT6B and its related signal mechanism in the development of cervical cancer by molecular and cell biology methods.The relationship between KAT6B expression and the clinical prognosis was studied further.Try to explore new potential targets for cervical cancer treatment(KAT6B-AMPK?).Methods and materialsFirst of all,6 cases of fresh primary cervical cancer tissues without radiotherapy or chemotherapy before surgery were collected from Kunshan Hospital affiliated to Jiangsu University.KAT6B mRNA in the tissues was detected by PCR.The western blot method was used to detect the expression of KAT6B protein and AMPK? protein in cervical cancer tissues.Subsequently,the Cell Bank of Shanghai Biological Institution provided the Hela and C33 a cervical cancer cell lines.Two different lentiviral si RNA constructs targeting KAT6B,KAT6B over-expression construct,two CRISPR/Cas9 KAT6B knockout constructs,and nonsense scramble vectors were transfected into cervical cancer cells.Cell proliferation was performed by the daily recording of cell number,clonogenicity,and EdU assay.Cell cycle was detected by flow cytometry.Cell scratch assay and transwell assay was performed to study the migration.Matrigel transwell assay detected cell invasion.Western blot was performed to detect the expression of different proteins,including KAT6B,AMPK?,phosphorylated AMPK?,phosphorylated AKT,phosphorylated m TOR,S6,and phosphorylated S6.EGFR,PDGFR?,and PDGFR? were also detected by Western blot assay.Finally,119 cervical cancer specimens(fresh and paraffin-embedded)after biopsy and surgery were collected from Kunshan Hospital affiliated to Jiangsu University.Surrounding normal tissues(35 cases)were taken as controls.The expression of KAT6B was detected by immunohistochemistry(IHC)staining.Analyzed the correlation between KAT6B,pathological type,stage,grade,prognosis,and other clinical characteristics.The protocols were approved by the Ethics Review Board in Kunshan Hospital affiliated to Jiangsu University.The cell number in the seed plate,the concentration of reagents,and specific experimental assays were based on literature and results from preliminary experiments.Statistical analysisThe in vitro experiments were replicated three times at least 3 auxiliary wells,One-way ANOVA,Scheffe and Tukey test,Chi-square test,Kaplan-Meier method,and COX regression analysis were performed by SPSS 22.0 and Graph Pad prism 7.0.p<0.05 was considered significant statistically.Results1.KAT6B expression in cervical cancer tissues increased compared with normal cervical tissues.KAT6B mRNA in cervical cancer tissues was significantly increased compared with surrounding normal tissues.Western blot method was performed to detect KAT6B protein and AMPK? protein in cervical cancer tissues and normal tissues.It showed that the KAT6B,EGFR,and PDGFR? protein increased significantly in cervical cancer tissues.AMPK? was reduced compared to normal tissues,but it was not significant statistically.(p>0.05).2.KAT6B promoted the growth,proliferation,migration,and invasion of cervical cancer.Cervical cancer cells expressing different levels of KAT6B were established.Counted cell number for 6 consecutive days.Cell proliferation rate was slowed down in KAT6B knock-down/knock-out cervical cancer cells.Cell proliferation was inhibited significantly on the 4th day.Clonogenicity and EdU assays also detected that cell proliferation was inhibited significantly after KAT6B down-regulated.The KAT6B overexpression promoted the growth and proliferation obviously in cervical cancer cells.After inhibiting KAT6B,cell migration and invasion were inhibited significantly compared with the control group by Scratch,Transwell,and Matrigel transwell assays.The KAT6B overexpression promoted cervical cancer migration and invasion.3.KAT6B promoted cell division and proliferationAfter knocking down or knocking out KAT6B,the number of cells in the G0/G1 phase increased and ones in the S phase decreased significantly.On the other hand,KAT6B overexpression cervical cancer cells showed the opposite results.Cells number decreased in G0/G1 phase but increased in the S phase significantly.4.KAT6B inhibited AMPK? signaling pathway.After knocking down or knocking out KAT6B,phosphorylated AMPK? increased,but phosphorylated m TOR,AKT,S6 decreased.Tyrosine kinase receptors EGFR,PDGFR?,PDGFR? have also detected expression decreased.KAT6B inhibited the activation of AMPK?,reduced the expression of EGFR,PDGFR?,and PDGFR?,and activated the AKTm TORC1 pathway.The opposite result appeared in KAT6B overexpressing cells.5.High expression of KAT6B is a poor prognostic factor of cervical cancer.KAT6B expression was detected in the cytoplasm and nucleus.The KAT6B high expression ratio in cervical cancer was 30.43%(cytoplasm)and 33.91%(nucleus).The difference was not significant statistically.The KAT6B high expression in the normal cervix accounted for 2.94%(cytoplasm)and 23.53%(nucleus).The difference is significant statistically.Kaplan-Meier analysis showed that the Overall Survival(OS)and ProgressionFree Survival(PFS)in high KAT6B expression(cytoplasm/nucleus)cervical cancer patients were shorter.COX regression method analyzed the correlation between KAT6B expression,tumor tissue type,grade,FIGO stage,and prognosis.It showed that FIGO stage,high KAT6B expression were independent prognostic factors in OS(FIGO stage: HR 3.531,95% CI 1.763-7.071,p<0.05;Cytoplasm: HR 2.949,95% CI 1.443-6.025,p<0.05;Nucleus: HR 2.529,95% CI 1.248-5.128,p<0.05)and PFS(FIGO stage: HR 3.531,95% CI 1.763-7.071,p<0.05;Cytoplasm: HR 2.968,95% CI 1.541-5.713,p<0.05;Nucleus: HR 2.529,95% CI 1.321-4.816,p<0.05).Conclusions1.The KAT6B expression was increased in cervical cancer tissues,and EGFR and PDGFR? protein was also increased.KAT6B may be one of the factors promoting the proliferation of cervical cancer.The expression of AMPK? was decreased compared with that one in surrounding normal tissues(p>0.05).2.After KAT6B knockdown/knockout,the proliferation,migration,and invasion abilities in cervical cancer cells were significantly decreased.It showed the opposite results for the cervical cancer cells with KAT6B overexpression.KAT6B played an important role in the proliferation,migration,and invasion of cervical cancer.3.KAT6B inhibited AMPK? activation,accompanied by increased expression of multiple tyrosine kinase receptors.KAT6B activated AKT-m TOR signaling pathway.This may be the signal mechanism that KAT6B promoted the proliferation in cervical cancer.4.The KAT6B expression was increased in cervical cancer tissues,and there was no significant difference between the expression in the cytoplasm and nucleus.The KAT6B expression in cervical cancer was correlated with age,FIGO stage,and lymph node status,but not with pathological type or tumor grade.Patients with KAT6B high expression had a poor prognosis with lower OS and PFS.FIGO stage and KAT6B were independent prognostic factors for OS and PFS.