Role And Related Mechanisms Of Mi R-181a-5p-targeted Regulation Of High Mobility Group B2 Expression In The Malignant Behavior Of Cervical Cancer Cell

BackgroundCervical cancer(CC)is an essential public health problem which seriously threatens women’s health worldwide,and its pathogenesis is the focus of research and difficulty in the field of cancer.As the global incidence and death rate of CC remains high among female malignancies and the trend of young age is obvious,the malignant behaviors of CC cells(proliferation,migration,invasion,aerobic glycolysis and iron death resistance)are still the main reasons for the poor prognosis of advanced and metastatic CC patients,so it is important to investigate its developmental mechanisms.In this study,the mechanisms associated with malignant behaviors of CC are studied in depth,which can help to identify new targets for CC and have important scientific implications for intervening in the malignant transformation of CC.Human papillomavirus(HPV)infection is the main cause of CC,and the degradation of p53 protein is thought to be the pathogenesis of CC carcinogenesis associated with HPV infection.As a member of the high mobility group box(HMGB)protein family,HMGB2 takes part in transcription and DNA repair of various oncogenes.In particular,in CC,HMGB2 interferes with p53 degradation in CC cells to arrest the CC cell cycle in the G1 phase,and regulates p53 protein-dependent changes by up-or down-regulating HMGB2 expression in CC,suggesting that it is vitally essential to investigate the role of HMGB2 in regulating the progression of CC pathogenesis.Increasing evidence suggests that HMGB2,an important oncogene,is upregulated in a variety of malignancies and is closely associated with tumor malignant behavior.The high expression of HMGB2 in cervical squamous carcinoma tissues promotes CC cell cycle progression and accelerates CC cell proliferation by activating the AKT signaling pathway,suggesting that HMGB2 may be a key target gene for CC progression and is expected to be a new target for CC.However,the mechanism of HMGB2 action in CC is currently unreported.Hence,this thesis attempts to elucidate the regulatory mechanism by identifying key upstream target genes regulating HMGB2,which may provide a theoretical basis for developing new clinical treatment strategies for CC with research value and significance.MicroRNA(MiRNA)is a small non-coding RNA that regulates the expression level of protein-coding genes by pairing with complementary nucleotide sequences of target m RNAs,and specifically prevents m RNA translation or degrades them.According to a recent report in cell,there is extensive accumulation of heterodimers of aberrant miRNAs in tumors,and extensive antitumor effects can be achieved by editing and repairing defective miRNAs.Therefore,exploring miRNA regulatory mechanisms holds promise for potential new strategies for CC therapy.As a member of miRNA family,miR-181a-5p has been reported to be involved in proliferation,migration,cell cycle,EMT,chemoresistance and glycolytic pathways in a variety of cancers,however,the study of miR-181a-5p in CC is still in its early stage and the potential application of miR-181a-5p in CC needs further development.The miRNA with binding site and possible negative regulatory relationship with HMGB2 3’-UTR was found to be miR-181a-5p by our group through preliminary high-throughput sequencing combined with database prediction analysis.Based on this,we proposed the following hypothesis: "miR-181a-5p target binding and negative regulation of HMGB2 expression inhibits the progression of malignant behavior in cervical cancer cells".Thus,in this study,we propose to investigate the effects of miR-181a-5p on the malignant behavior of CC cells(proliferation,migration,invasion,aerobic glycolysis and iron death resistance)using cervical cancer He La and Si Ha cells and nude mice transplantation tumor models,and to provide novel ideas and theoretical basis for the prospective application of miR-181a-5p and HMGB2 in the clinical diagnosis and treatment of CC.This study is divided into three parts as follows:Part Ⅰ Mechanisms related to the promotion of malignant behavior of cervical cancer by high mobility group B2ObjectiveTo investigate the association of HMGB2 expression in cervical cancer with clinicopathological parameters and survival as well as the effects of HMGB2 on proliferation,migration,invasion and glycolysis of cervical cancer.Methods1.Bioinformatic analysis: TCGA and Human Protein Atlas HPA database to analyze HMGB2 m RNA and protein levels in pan-cancerous tissues;GEPIA database to analyze HMGB2 m RNA expression in cervical cancer tissues and normal cervical tissues;TCGA database to analyze the relationship between HMGB2 expression and clinicopathological parameters of cervical cancer patients;CCLE database to analyze HMGB2 m RNA expression in cervical cancer cell lines.2.Cervical cancer tissue: q RT-PCR and Western Blot assay to detect HMGB2 expression in cervical cancer tissue,χ2 test to analyze the relationship between HMGB2 expression and clinicopathological parameters of cervical cancer patients;Kaplan-Meier method to plot survival curves,Log rank test to analyze the relationship between HMGB2 protein expression and patient survival.The relationship between HMGB2 protein expression and patient survival was analyzed by Kaplan-Meier method.3.In vitro experiments: q RT-PCR and Western Blot assays to detect HMGB2 expression in CC cell lines;lentiviral transfection techniques to construct HMGB2 overexpression and knockdown in cervical cancer He La and Si Ha stable transgenic strains;CCK8,plate clone formation,cell scratch repair,Transwell assays to detect the effect of HMGB2 and glycolysis inhibitor 2-DG on proliferation,migration and invasion of cervical cancer cells;glucose uptake and lactate production assays to analyze the effects of HMGB2 and glycolysis inhibitor 2-DG on the glycolytic capacity of cervical cancer cells;Western Blot assay to detect the correlation between HMGB2 and the expression levels of GLUT-1,lack of oxygen-inducible factor HIF-1α and glycolytic key enzymes HK2,PKM2,and LDHA expression levels in CCcells.Results1.Results of bioinformatic analysis: HMGB2 expression was higher in cervical cancer tissues than in normal tissues of the uterine cervix;HMGB2 expression was closely correlated with clinical stage of CC patients,and there was no significant difference with patient age,tumor grade and lymph node metastasis.2.Cervical cancer tissue results: HMGB2 expression was up-regulated in cervical cancer tissues;HMGB2 was closely related to clinical stage and lymph node metastasis of CC patients,and overall survival of patients with high HMGB2 expression was reduced.3.in vitro results: HMGB2 expression was upregulated in cervical cancer HeLa and Si Ha cell lines.The expression of GLUT-1,hypoxia-inducible factor(HIF-1α)and glycolytic key enzymes HK2,PKM2 and LDHA was higher in CC cells with overexpressed HMGB2,and the opposite result was obtained by knocking down HMGB2 expression.The addition of the glycolysis inhibitor 2-DG along with overexpressed HMGB2 was able to reverse the effects of HMGB2 on proliferation,migration,invasion and glycolysis of CC cells.Conclusion1.High expression of HMGB2 in cervical cancer is closely associated with poor patient prognosis.2.HMGB2 promotes proliferation,migration and invasion of cervical cancer by enhancing glycolytic capacity.Part Ⅱ Mechanisms related to Mi R-181a-5p suppresses malignant behavior incervical cancer by targeting and regulating the expression of high mobility group B2ObjectiveTo investigate the effects of targeted binding and regulatory relationship between miR-181a-5p and HMGB2 on cervical cancer cell proliferation,migration and invasion.Methods1.Bioinformatic analysis: This study applied Target Scan and ENCORI database search and analysis,and combined with the group’s pre-sequencing results for Venn analysis,to screen miRNAs that bind to HMGB2 target and have a regulatory relationship with.2.In vitro experiments: q RT-PCR assay to detect the expression of miR-181a-5p in cervical cancer cell lines;lentiviral transfection technique to construct miR-181a-5p overexpression and knockdown of cervical cancer He La and Si Ha stable transgenic strains;Western Blot and IF assay to detect the regulatory relationship between miR-181a-5p and the regulatory relationship of HMGB2;Dual luciferase assay to verify the target binding between miR-181a-5p and HMGB2;CCK8,plate clone formation,cell scratch repair,and Transwell assays to verify the effects of miR-181a-5p on proliferation,migration,and invasion of cervical cancer cells;Western Blot assay to detect the correlation between miR-181a-5p and the expression levels of GLUT-1,HIF-1α and HK2,PKM2 and LDHA,the key enzymes of glycolysis in CC cells.3.In vivo experiments: a transplantation tumor model was constructed in nude mice with cervical cancer,and the effect of miR-181a-5p on the growth of transplantation tumors in nude mice was examined by measuring the length and diameter of the tumors and plotting the growth curve.The effect of miR-181a-5p on HMGB2 and Ki67 protein expression was examined by immunohistochemical staining.Results1.Bioinformatic analysis: miR-181a-5p was screened as the miRNA targeting and negatively regulating HMGB2 3’-UTR.2.In vitro results: the expression of miR-181a-5p was down-regulated in HeLa and Si Ha cell lines.HMGB2 expression was decreased in CC cells overexpressing miR-181a-5p,and the reporter gene activity of HMGB2 3’-UTR wild type was reduced.The proliferation,migration and invasion ability of cervical cancer cells were reduced by overexpression of miR-181a-5p,and knockdown of miR-181a-5p showed the opposite results.Overexpression of miR-181a-5p along with HMGB2 reversed the effects of HMGB2 on CC proliferation,migration and invasion.3.In vivo results: a cervical cancer transplantation tumor model was successfully constructed in nude mice;overexpression of miR-181a-5p inhibited the formation of transplantation tumors in nude mice with significantly less growth of transplantation tumor volume;the expression of HMGB2 and Ki67 in transplantation tumors of nude mice with cervical cancer overexpression of miR-181a-5p was reduced.Conclusion1.HMGB2 is a direct target of miR-181a-5p.2.MiR-181a-5p promotes the proliferation,migration,invasion and tumorigenic ability of cervical cancer cells in nude mice in vivo by negatively regulating the expression of HMGB2.Part Ⅲ Mi R-181a-5p affects Erastin-induced iron death in cervical cancer by regulating glycolysisObjectiveTo investigate whether Erastin can induce iron death in cervical cancer,and the influence of miR-181a-5p on glycolysis and iron death in cervical cancer.MethodsGlucose uptake and lactate production assays were performed to detect the effects of miR-181a-5p on the glycolytic capacity of cervical cancer cells.Western Blot assays were performed to detect the effects of overexpression and knockdown of miR-181a-5p on the expression levels of GLUT-1,hypoxia-inducible factor HIF-1α and key glycolytic enzymes HK2,PKM2 and LDHA.Optical microscopy observed the effects of miR-181a-5p and glycolysis inhibitor 2-DG on the morphological changes of iron death induced by Erastin in cervical cancer cells.CCK8 assay to verify the effect of miR-181a-5p and glycolysis inhibitor 2-DG on the survival rate of Erastin-induced cervical cancer cells.Fe2+ concentration assay to validate the effect of miR-181a-5p and glycolysis inhibitor 2-DG on Erastin-induced changes in Fe2+ concentration.JC-1 assay to detect the effects of miR-181a-5p and glycolysis inhibitor 2-DG on Erastin-induced changes in mitochondrial membrane potential in cervical cancer cells.Flow cytometry was performed to detect the effects of miR-181a-5p and glycolysis inhibitor 2-DG on Erastin-induced lipid ROS production.Western Blot assay was performed to detect the effects of miR-181a-5p and glycolysis inhibitor 2-DG on Erastin-induced expression of iron death protective protein GPX4.ResultsOverexpression of miR-181a-5p decreased glucose consumption and lactate production in cervical cancer cells,and knockdown of miR-181a-5p obtained the opposite result;the expression levels of GLUT-1,hypoxia-inducible factor HIF-1α and key enzymes of glycolysis HK2,PKM2 and LDHA were decreased by overexpression of miR-181a-5p in CC cells,and the opposite result was obtained by knocking down miR-181a-5p.Morphological changes in CC cell death,decreased cell survival,Fe2+ accumulation,decreased mitochondrial membrane potential,increased lipid ROS production and decreased expression of iron death protective protein GPX4 were induced by Erastin.Knockdown of miR-181a-5p while adding Erastin reversed Erastin-induced morphological changes in iron death and reduced cell survival in CC cells,and protected CC cells from Erastin-induced Fe2+ accumulation,mitochondrial damage,increased lipid ROS production and reduced expression of iron death protective protein GPX4.Addition of Erastin and knockdown of miR-181a-5p together with the glycolysis inhibitor 2-DG counteracted the effect of miR-181a-5p on iron death in CC cells.Conclusion1.Erastin induced cervical cancer cell death accompanied by Fe2+ accumulation,mitochondrial damage,increased lipid peroxidation and decreased expression of the iron death protective protein GPX4.2.Mi R-181a-5p promotes Erastin-induced iron death in cervical cancer cells by regulating glycolytic capacity. Innovative points and research significance1.HMGB2 promotes cervical cancer proliferation,migration and invasion by enhancing glycolytic capacity.2.Erastin induces cervical cancer cell death,accompanied by mitochondrial damage,enhanced lipid peroxidation and decreased expression of iron death protective protein GPX4.Mi R-181a-5p promotes Erastin-induced iron death in cervical cancer cells by regulating glycolytic capacity.3.The discovery and confirmation of the important role of Mi R-181a-5p in the malignant behavior of cervical cancer cells through targeting and regulating the expression of high mobility family protein B2 may be a novel mechanism mediating its malignant progression with original significance.