The Prognostic Evaluation Of KLF14 In Patients With Cervical Cancer And The Role And Mechanism Of KLF14 In Cell Cycle Regulation

BackgroundCervical Cancer（CC）is the second leading cause of cancer-related death in young women worldwide.In recent years,the incidence and mortality rate of cervical cancer increased significantly.KLF14（kriippel-like factor 14）is a newly discovered member of the SP/KLFs family of transcription factors and is localized to 7q32.3.Relevant researches show that KLF14 plays a vital role in the occurrence and progression of malignancies such as colorectal cancer,breast cancer and liver cancer.Our previous research founded that KLF14 may inhibit the proliferation and promote apoptosis of cervical cancer cells,and its role and mechanism in cervical cancer progression need to be thoroughly explored.In this study,cancer tissues of cervical cancer patients,cervical cancer cell lines and nude mice were the main experimental objects to explore the prognostic evaluation of KLF14 in patients with cervical cancer and the role and mechanism of KLF14 in cell cycle regulation,offering new insights into the diagnosis,treatment and prognosis of cervical cancer.Methods1.Immunohistochemical staining was used to detect the expression level of KLF14 protein in cancer tissues of cervical cancer patients.The relationship between KLF14 expression level with clinicopathological characteristics and overall survival was analyzed,and the pathologic factors affecting the patient prognosis were analyzed.2.Cervical cancer SiHa and HeLa cell lines were infected by lentivirus,and stable overexpression of KLF14 and KLF14 zinc finger mutant cervical cancer cell lines were constructed.3.The effect of KLF14 on cervical cancer cell cycle was detected by flow cytometry.4.The effect of KLF14 on early apoptosis of cervical cancer cell lines was detected by mitochondrial membrane potential test.5.The effect of KLF14 on CDK2 expression in cervical cancer cell lines was detected by qRT-PCR.6.The effect of KLF14 on the expression of CDK2,CyclinA2 and MAPK signaling pathway related molecules in cervical cancer cell lines were detected by Western blotting.7.Cervical cancer cells were treated with JNK MAPK pathway inhibitor or agonist to analyze the effect of pathway activation on cell cycle and expression of CDK2 and CyclinA2.8.The effect of KLF14 on cervical cancer cell proliferation in vivo was detected by subcutaneous tumor formation in nude mice.9.Western blotting was used to detect the expression of CDK2,CyclinA2 and MAPK signaling pathway related molecules in the tumor of nude mice.Results1.There was no statistical difference in age,pathological grade,lymph node metastasis,TNM grade and HPV between the high expression group and the low expression group.cervical cancer patients with high KLF14 expression had a good prognosis（P=0.038）.KLF14 expression level（P=0.020）,age（P<0.001）and TNM stage（P=0.001）were independent factors affecting the prognosis of cervical cancer patients.2.KLF14 decreased the proportion of G0/G1 phase(P_SiHa<0.001,P_HeLa=0.049)and increased the proportion of S phase(P_HeLa=0.005,P_HeLa<0.001)in cervical cancer cell lines.After zinc finger deletion mutation,the effect of KLF14 on the cycle was decreased（P<0.001）.3.KLF14 had no significant effect on early apoptosis of cervical cancer cell lines（P>0.05）.4.KLF14 promoted CDK2 mRNA expression in cervical cancer cell lines（P<0.001）.5.KLF14 promoted the activation of JNK MAPK pathway in cervical cancer cell lines(P_SiHa=0.014;P_HeLa=0.004),promoting CDK2(P_SiHa=0.043;P_HeLa<0.001)and CyclinA2(P_SiHa=0.012;P_HeLa=0.015)protein expression.6.When JNK MAPK pathway activation was inhibited in cervical cancer cell lines,the proportion of S phase decreased(P_SiHa=0.020,P_HeLa<0.001),the mRNA expression of CDK2 decreased（P<0.001）,the protein expressions of CDK2（P<0.001）and CyclinA2(P_SiHa=0.006,P_HeLa<0.001)decreased.7.When JNK MAPK pathway activation was promoted in cervical cancer cell lines,the proportion of S phase increased(P_SiHa<0.001,P_HeLa=0.014),the mRNA expression of CDK2 increased(P_SiHa<0.001,P_HeLa=0.030),the protein expressions of CDK2(P_SiHa=0.049,P_HeLa<0.001)and CyclinA2(P_SiHa=0.001,P_HeLa<0.001)increased.8.KLF14 inhibited cervical cancer cell proliferation in vivo（P=0.012）.9.KLF14 promoted the activation of JNK MAPK pathway and protein expression of CDK2 and CyclinA2 in vivo cervical cancer cells(P_p-JNK/JNK<0.001,P_CDK2=0.002,P_CyclinA2=0.009).Conclusions1.Cervical cancer patients with high KLF14 expression have a good prognosis.KLF14 expression level,age and TNM stage are independent factors affecting the prognosis of cervical cancer patients.2.KLF14 can decrease the proportion of G0/G1 phase,increase the proportion of S phase and induce S phase arrest in cervical cancer cell lines.KLF14 inhibited cervical cancer cell proliferation in vivo.3.The effect of KLF14 on cervical cancer cell cycle is related to the presence of its own zinc finger structure.KLF14 increases the proportion of S phase,induces S phase arrest and promotes CDK2 and CyclinA2 expression through activation of the JNK MAPK pathway.In conclusion,cervical cancer patients with high expression of KLF14 have a good prognosis.KLF14 may inhibit the proliferation of cervical cancer cells by inducing S phase arrest.The presence of zinc finger structure of KLF14 and activation of JNK MAPK signaling pathway play important roles in KLF14 induced S phase arrest of cervical cancer cells.KLF14 may be a new indicator for the diagnosis,treatment and assessment of prognosis of cervical cancer.