The Association Of Cervical Carcinogenesis And Human Papillomavirus(HPV) Infection With The Metabolic Regulation In Tumor Tissues

Objectives:Cervical cancer is high prevalent among Uyghur women in Xinjiang Autonomous Region,China.The infection by human papillomaviruses(HPVs)is the main etiological factor of cervical carcinogenesis,among which the HPV16 infection is the predominant one.In previous studies,our research team is engaged in the study of cervical carcinogenesis by serum-based metabolomics,and found a causal relationship between the regulation of small molecular metabolites and the development of cervical carcinoma,but its association with the metabolic regulation in tumor tissues and cells is not investigated so far.In this study,we will investigate the association of cervical carcinogenesis and HPV infection with changes in small molecular metabolite profile and the expression of key enzymes modulating key metabolic pathways in tumor tissues from patients and in cervical cancer cell lines,for revealing the mechanisms of cervical carcinogenesis related to metabolic regulation and the identification of molecular profile for prognosis of cervical carcinoma.Methods:(1)Fifty-two cases of fresh specimens of uterine cervix were collected form patients with cervical squamous cell carcinoma(CSCC),cervical intraepithelial neoplasia(CIN)??? and normal controls(NC),including 21 cases of CSCC,20 cases of CIN??? and 11 cases of NC.One-hundred and twenty three cases of paraffin-embedded tissue slices were collected from patients with cervical carcinoma and precancerous lesions,and normal controls(or cervicitis)by review of pathological specimen bank of the pathology department.(2)After tissue DNA extraction and the HPV detection,the tissue specimens of HPV negative normal controls and HPV16 positive cervical carcinoma and precursor lesions were subjected to the determination of the metabolomic spectra by high-resolution magic angle spinning nuclear magnetic resonance(HRMAS-~1H NMR),and the generated data was further analyzed by professional software and its online database to identify the differentially expression profile of metabolites common to,and altered in tumor tissues and HPV infection.(3)To improve the sensitivity and accuracy of metabolomics,we analyzed the tissue specimens of cervical carcinoma,precancerous lesions and normal controls by ultra high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry(UHPLC-MS/MS)to identify metabolites that are differentially expressed in,and common to,tumor specimens and HPV infection.The metabolomics data was additionally analyzed by professional softwares recognizing metabolic pathways(KEGG;MetaboAnalyst 2.0)and online databases,to identify certain metabolic pathways and key enzymes associated with the metabolic regulation.(4)After extraction of tissue RNA and designing of primair-pairs specific to mRNA-sequences coding for the identified key enzymes,we analyzed the transcription of these coding genes in tissue RNAs of cervical carcinoma and normal controls by quantitative RT-PCR,to reveal the association of cervical carcinoma with the mRNA expression level of candidate key enzymes.Meanwhile,we analyzed the association of cervical carcinoma and the protein expression level of candidate key enzymes in paraffin-embedded tissue specimens of cervical carcinoma and normal controls that were provided by the pathological tissue specimen bank.(5)We designed 6 different RNAi oligo nucleotide fragments identical with the sequences of HPV16 coding E6 and E7 genes,and cloned into expression vector pRNAi that can syncroneously express the green fluorescent protein(GFP)and the target RNAi fragment.After transient transfection of HPV16-positive SiHa cervical carcinoma cells with these expression vectors,the transfection efficiency was evaluated by checking the GFP fluorescence by confocal microscopy.The metabolomic spectra and the expression of candidate key enzymes were detected by nuclear magnetic resonance technique for metabolomics and quantitative RT-PCR,to reveal the dependence of the cellular metabolic regulation and the expression level of key enzymes on the HPV infection.Results:(1)Based on HPV test and the typing of high-risk HPVs,such HPV16,18,33,52,58 and 67 in 52 cases of tissue specimens,we detected HPV positivity in 20 cases of CSCC,18 cases of CIN ??? and 1 case of normal control,respectively.Among them,HPV16 positivity were found in 19 cases of CSCC,17 cases of CIN ??? and 1 case of normal controls,respectively.The high ratio of HPV16 to HPV(37/39)indicated that HPV16 infection was predominant among tissue specimens tested.(2)Based on HPV typing,tissue specimens of HPV16 positive cervical carcinoma and HPV negative normal controls were subjected to the determination of metabolomics spectra by nuclear magnetic resonance technique for metabolomics.This was resulted in the identification of 17 small molecular metabolites that were differentially expressed in specimens of cervical carcinoma with HPV infection.By further analysis of the metabolite data,we suggest that the pathological condition of patients with cervical carcinoma may be characterized by aberrantly regulated metabolism due to enhanced energy metabolism,which was reflected in increased catabolism of sugar,lipid and amino acids in the body.(3)By analysis of tissue speciemens of cervical carcinoma,precancerous lesions and normal controls using ultra high-performance liquid chromatrography coupled with triple quandropole mass spectrometry(UHPLC-MS/MS),we idientified 34 small molecular metabolites that were differentially expressed in cervical carcinoma.This profile was comprehensively analyzed by professional softwares for recognizing metabolic pathways(MetaboAnalyst and KEGG)and predicted 18 key metabolic pathways,among which cervical carcinogenesis was most probably associated with 4 key metabolic patways,including taurine and hypotaurine metabolism,alanine and aspartate metabolism,D-glutamine and D-glutamate metabolism,and thiamine metabolism.(4)Based on the metabolomics data and analysis of key metabolic pathways,we selected 4 key enzymes related to the key metabolic pathways of sugar,lipid or amino acids,including glutamate decarboxylase 1,(GAD1),glycogen synthesis kinase 3?(GSK-3?),pyruvate kinase muscle isozyme 2(PKM2)and carnitine palmitoyl transferase IA(CPTI A1).By analysis of transcriptions for candidate genes coding these key enzymes by quantitative RT-PCR,we showed that the expression of PKM2 and CPT1 was significantly upregulated,and GAD1 and GSK-3?downregulated,respectively,in tissue specimens of cervical carcinoma compared with normal controls(P<0.05).In immunohistochemical staining analysis further confirmed the result of quantitative RT-PCR,the protein expression of GAD1 and GSK-3?was gradually decreased,and PKM2 and CPT1 increased,respectively,from normal controls to precancerous lesions(CIN???)and cervical carcinoma with significant differences(P<0.05).(5)We constructed RNAi expression vector for HPV16 coding E6 and E7 genes,and transfected HPV16 positive SiHa cervical carcinoma cells to synchroneously express RNAi fragments and green fluorescent protein(GFP),and confirmed the transfection efficiency by checking the fluorescence using confocal microscopy,to establish an RNAi expressing cell models.By quantitative RT-PCR analysis and metabolomics spectra analysis,we identified 13 small molecular metabolites that were differentially expressed in RNAi model compared to normal controls,and in addition,found that the expression of GAD1 and GSK-3?was upregulated and the expression of PKM2 and CPT1downregulated,with such a tendence that was inversely correlated with the expression of these genes in tissue specimens of cervical carcinoma.These results suggest that the expression HPV 16 coding genes may have an effect on the regulation of metabolic pathways mediated by these key enzymes,and provide evidence for understanding the biological nature of HPV16 in the pathogenesis and carcinogenesis associated with metabolic regulation.Conclusions:(1)Cervical carcinogenenesis and HPV infection were accompanied with enhanced catabolism of sugar,lipid and amino acids,which reflects the deregulated metabolism associated with pathological condition of patients.(2)Cervical carcinogenesis and HPV infection is closely associated with the regulation of key enymes involved in key metabolic pathways in cervical carcinoma tissues.(3)The expression of HPV16 coding E6 and E7 genes may have an effect on the regulation of cellular metabolism and key enzymes related to key metabolic pathways in cervical cancer cells.