| Cervical cancer is a malignant tumor originating from the cervix,mainly caused by repeated infection of high-risk Human Papilloma Virus(HPV).Cervical cancer is insidious and asymptomatic in early stage.By the time most patients are diagnosed,the disease has advanced.Surgical excision and adjuvant chemoradiotherapy are the most effective treatment methods at present.But the cone of the cervix,the removal of the uterus and fallopian tubes,caused a great degree of harm to the female psychology.At the same time,the toxic side effects of radiotherapy and chemotherapy also seriously affect the quality of life of patients.Pristimerin(Pri)is a natural quinone methylated triterpenoid compound.Because of its anti-inflammatory and antitumor effects,it has been widely studied in recent years.microRNAs(miRNAs)are non-coding Rnas that are 19 to 22 nucleotides long.It was found that miR-144/451(microRNA-144 and microRNA-451)is involved in regulating the growth of a variety of tumors.Human Tumor necrosis factor α-inducible protein 8(TNFAIP8)is involved in tumor growth,invasion and metastasis,and plays an important role in regulating necrosis factor.In recent years,studies have found that TNFAIP8 is highly expressed in esophageal cancer,gastric cancer and breast cancer,and has a close relationship with the prognosis of the disease.In this study,we mainly investigated the expression and interaction between miR144/451 and TNFAIP8 in human cervical cancer cells.Secondly,the in vivo and in vitro studies were conducted to investigate whether pristimerin can synergistically target TNFAIP8 with miR-144/451 and thus inhibit the migration and tumor growth of human cervical squamous carcinoma SiHa cells.The study is divided into three parts as follows.Part I:Expression of miR-144/451 and TNFAIP8 in human cervical cancerObjective:To investigate the expression of miR-144/451 and TNFAIP8 in human cervical cancer clinical samples,and analyze the relationship between cervical cancer and the expression level of miR-144/451,and the relationship between cervical cancer and TNFAIP8 expression,respectively.Methods:HE staining was used to observe the differentiation degree of cervical cancer cells.Immunohistochemical assay was performed to detect the expression of TNFAIP8 protein in surgical specimens of cervical cancer with different degrees of differentiation.Quantitative real-time PCR(qRT-PCR)was used to compare the expression levels of miR-144/451 and TNFAIP8 mRNA in 14 cervical cancer tissues and adjacent normal tissues.The expression level of TNFAIP8 protein in cervical cancer and adjacent normal tissues was detected by western blot.Results:Immunohistochemical experiments confirmed that the expression of TNFAIP8 protein in cervical cancer with different degrees of differentiation.The results of qRT-PCR and western blot showed that the expression of TNFAIP8 mRNA and TNFAIP8 protein were significantly increased compared with those in adj acent normal tissues.The mRNA expression level of miR-144/451 in cervical cancer cells was significantly lower than that in adjacent tissues(P<0.05).Conclusion:Compared with paracancer tissues,the mRNA expression level of miR-144/451 in cervical cancer decreased,while the mRNA and protein expression level of TNFAIP8 increased.Part Ⅱ:Explore the relationship between miR-144/451 and TNFAIP8Objective:To investigate the relationship between miR-144/451 and TNFAIP8 in cervical tissues.The effects of miR-144/451 on the migration ability of SiHa cells and the expression of TNFAIP8 were explored by using knock out(KO)mouse model of miR-144/451 and SiHa cell model of human cervical cancer with high expression of miR-144/451.Methods:3D models of TNFAIP8 and miR-144/451 were constructed by AlphaFold and RNAfold software,respectively.AutoDock Vina software was used to simulate molecular docking,and the binding force between miR-144/451 and TNFAIP8 was calculated.The cervical tissues of three miR-144/451 KO mice and the control C57BL/6 wild mice of the same strain were taken separately and the expression of TNFAIP8 protein was analyzed by immunohistochemistry.Western Blot and qRT-PCR were used to detect the expression of TNFAIP8 protein and mRNA in the cervical tissues of miR-144/451 KO mice,respectively.Meanwhile,human cervical cancer SiHa cells with high expression of miR-144/451 were constructed to detect the changes of mRNA and protein expression levels of TNFAIP8 in the cells.The changes of proliferation and migration ability were detected by transwell and wound healing assays in the human cervical cancer SiHa cells with high expression of miR-144/451.Results:MiR-144/451 had strong binding force with TNFAIP8.The cervical tissues of miR144/451 KO mice and C57BL/6 mice showed no significant changes in HE staining.Immunohistochemical results showed that the expression of TNFAIP8 protein in the cervical tissues of miR-144/451 KO mice was positive.Compared with the cervical tissues of C57BL/6 mice,the expressions of TNFAIP8 protein and mRNA in the cervical tissues of miR-144/451 KO mice were significantly increased(P<0.05).Compared with wild-type human cervical cancer SiHa cells,after high expression of miR-144/451(named as SiHa/miR-144+and SiHa/miR-451+,respectively),cell proliferation and migration ability decreased,and TNFAIP8 mRNA and protein expression levels also decreased significantly.Conclusion:MiR-144/451 has a strong binding force with TNFAIP8 and TNFAIP8 may be one of the target proteins directly affected by miR-144/451.In the cervical tissues of mice,the expression level of TNFAIP8 increased significantly after the knockout of miR-144/451.Compared with wild-type human cervical cancer SiHa,the migration ability of SiHa/miR144+and SiHa/miR-451+cells was decreased,and the expression level of TNFAIP8 was significantly decreased.Part Ⅲ:The effect and molecular mechanism of pristimerin synergistic miR-144/451 on the proliferation and migration in human cervical cancer cellsObjective:Combined with in vitro and in vivo experiments,the effect and molecular mechanism of pristimerin synergistically with miR-144/451 in inhibiting the proliferation and migration ability of human cervical cancer SiHa cells we investigated.Methods:In vitro assay,MTT assay was performed to detect the effects of different concentrations of pristimerin(0.25,0.5,1,2 and 4 μM)on the proliferation of SiHa/miR-144+and SiHa/miR-451+ cells.Cell scratch assay,Transwell assay and EDU assay were used to observe the changes of cell migration and proliferation of SiHa/miR-144+ and SiHa/miR-451+after treatment with different concentrations of pristimerin.Western Blot assay was used to detect the expression levels of PI3K/Akt/mTOR signal transduction pathway related proteins in cells treated with pristimerin.In vivo study,42 female nude mice were divided into 7 groups(6 mice per group):human cervical cancer SiHa cells were control group,SiHa/miR-144+cells(or SiHa/miR-451+ cells)were negative control group,DMSO lysate control group(1 mg/kg),pristimerin low(0.25 mg/kg),medium(0.5 mg/kg),and high(1 mg/kg)groups.And DDP positive drug group(1 mg/kg).The cell suspension(1.5 × 106 cells/mL)was injected into the right armpit of nude mice for tumor bearing,and the injection volume was 0.2 mL.Each group was injected intraperitoneally once every three days.On the 28th day,the animals were sacrificed,and the therapeutic effect was recorded and analyzed.Results:In vitro studies,pristimerin significantly inhibited the proliferation of SiHa/miR-144+and SiHa/miR-451+ cells in a concentration-dependent manner.Meanwhile,pristimerin significantly inhibited the migration of SiHa/miR-144+ and SiHa/miR-451+cells,and decreased the expression levels of PI3K,Akt,mTOR and TNFAIP8 proteins.In vivo studies showed that compared with wild-type SiHa cells,tumor volume and weight were significantly reduced after SiHa/miR-144+and SiHa/miR-451+cells were injected.Meanwhile,compared with SiHa/miR-144+and SiHa/miR-451+cell groups,the tumor volume and weight were further reduced in the high-concentration pliotimerin treatment group and the DP-positive drug group.Conclusion:Pristimerin can synergistic with miR-144/451 targeting TNFAIP8 to inhibit the proliferation and migration of human cervical cancer SiHa cells,and its molecular mechanism is closely related to the PI3K/Akt/mTOR signal transduction pathway. |
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