Effects Of HMTERF3 Gene Silencing On Proliferation And Apoptosis Of Human Cervical Cancer HeLa Cells

Objective Cervical cancer is one of the common malignant gynecologic tumors,with the incidence rate only second to breast cancer,ranking the second among all female malignant tumors.In recent years,although the screening and diagnosis methods for cervical cancer have been continuously improved,and surgical treatment and chemoradiotherapy have made great progress,the long-term survival rate and quality of life of patients are still not optimistic.To study the mechanism of cervical cancer at the molecular level is beneficial to discover new molecular targets and provide the basis for targeted and precise treatment of cervical cancer.hMTERF3 plays a negative regulation role in the expression of mtDNA gene.This gene can bind to the promoter in the D-loop region of mitochondrial DNA and participate in the regulation of replication,transcription and translation of mtDNA.It has been reported that hMTERF3 is highly expressed in a variety of solid tumors,such as breast cancer,pancreatic cancer,gastric cancer,non-small cell lung cancer and other types of tumors,suggesting that hMTERF3 may be closely related to the occurrence,development and prognosis of malignant tumors.However,the role and mechanism of MTERF3 in the development of cervical cancer remain unclear.This study is to analyze the expression of hMTERF3 gene in cervical cancer and its clinical value using TCGA datasets,and Oncomine and GEPIA databases.Through the construction and identification of recombinant eukaryotic expression vector pSi-hMTERF3,the effect of transfected recombinant eukaryotic expression vector pSi-hMTERF3 on the mitochondrial DNA copy number,mitochondrial transcription level,mitochondrial protein expression level,cell proliferation,cell apoptosis,oxidative phosphorylation and other biological behaviors of cervical cancer HeLa cells is further studied.Thisstudy provides the theoretical and experimental basis for further studies on the interaction between hMTERF3 and cervical cancer.Methods: The information related to hMTERF3 gene was retrieved based on Oncomine and GEPIA and TCGA databases to analyze the expression of hMTERF3 in cervical cancer;the clinical data related to hMTERF3 was downloaded from TCGA datasets to analyze the expression level of hMTERF3 in cervical cancer and prognosis.four recombinant interfering plasmids psi-MTERF3-1~psi-MTERF3-4 were obtained.It was identified by PCR and DNA sequencing.The recombinant interfering plasmid was transfected into HeLa cells by liposome-mediated transient transfection.The expression level of hMTERF3 protein was detected by Western blot and the expression level of hMTERF3 mRNA was detected by qPCR 48 hours after transfection.Changes in the amount of mitochondrial DNA copies were detected by semi-quantitative PCR,the effect of mitochondrial DNA transcription was detected by fluorescence quantitative PCR,the effect of pSi-hMTERF3 on cell apoptosis and mitochondrial ROS was detected by flow cytometry,and the effect of pSi-hMTERF3 on cell growth was detected by MTT assay.Results (1)The analysis of Oncomine and GEPIA databases shows that hMTERF3 mRNA is highly expressed in cervical cancer.The analysis of TCGA datasets shows that the expression of MTERF3 mRNA was not significantly correlated with age,primary tumor,regional lymph node metastasis,distant metastasis and pathological type(P>0.05),but significantly correlated with clinical stage and pathological grade of AJCC(P<0.05).Univariate analysis showed that primary tumor,regional lymph node metastasis,distant metastasis and AJCC stage could affect the prognosis of the patients(P<0.05).Age,pathological grade,pathological type and expression of hMTERF3 mRNA had no correlation with the prognosis of cervical cancer(P>0.05).Cox multivariate regression analysis showed that N stage was an independent factor affecting the prognosis of cervical cancer patients(P<0.05).(2)The results of gene correlation analysis based on GEPIA database shows that the expression level of hMTERF3 gene in cervical cancer tissue is significantly positively correlated with the expression levels of TFAM,TFB1 M,TFB2M,MTERF1,MTERF2,TEFM and,PTEN genes(P<0.01,R>0),and is not correlated with theexpression level of MTERF4 and FHIT gene.(3)PCR and DNA sequencing confirmed that the target DNA sequence had been inserted into the recombinant plasmid.After screening,pSi-MTERF3-2 and pSi-MTERF3-4 have the best interference effect on hMTERF3 gene expression.(4)The results of semi-quantitative PCR indicate that the replication level of mitochondrial DNA in cervical cancer HeLa cells increases after the interference with hMTERF3 gene.(5)The results of both quantitative PCR and semi-quantitative PCR show that the down-regulation of hMTERF3 gene can increase the transcription level of mitochondrial DNA.(6)The effect of pSi-MTERF3 on mitochondrial ROS is detected by flow cytometry,and the results show that ROS level increases after the down-regulation of hMTERF3.(7)The results of cell proliferation by MTT assay show that the down-regulation of hMTERF3 inhibits cell proliferation.(8)The results of cell apoptosis detected by flow cytometry show that the down-regulation of hMTERF3 promotes apoptosis.Conclusion (1)The related information of hMTERF3 expression in human cervical cancer tissue was obtained based on TCGA,GEPIA and Oncomine database.The important clue and data support was provided for the clinical treatment of cervical cancer through a large number of data mining and analysis.(2)The shRNA sequence which effectively interfere the expression of hMTERF3 gene was screened in human cervical cancer HeLa cell line and the RNA interference eukaryotic expression vector targeting hMTERF3 gene was constructed,which provided an experimental basis for the further study of the effect of hMTERF3 gene on the malignant biological behavior of cervical cancer cell.(3)By interfering with hMTERF3 gene in HeLa cells,it can promote the replication of mitochondrial DNA and improve the transcription level,inhibit cell proliferation,promote apoptosis and mitochondrial ROS level.These studies provide theoretical guidance and experimental basis for in-depth study of the effects of hMTERF3 on malignant biological behavior of cervical cancer.