Roles And Mechanisms Of Glucose Metabolism Regulation In The Pathogenesis Of Melanoma And Condylomata Acuminata

Part? Overexpression of FAH in melanoma and its role in metabolic reprogramming regulationObjective: The purpose of this study was to explore the expression of FAH in melanoma and its role in the regulation of metabolic reprogramming.We first investigated whether FAH could affect the survival of melanoma patients or tumor-bearing mice,and then further studied the role of FAH in melanoma and the potential mechanism included at the cellular level.The research of cellular level mainly focused on the effects and mechanism included of FAH on the biological functions of melanoma cells,which including proliferation,cell cycle and apoptosis.The research of FAH function focused on the anaplerotic reactions in the TCA cycle,as well as other metabolic pathways including glucose metabolism,nucleotide and fatty acid synthesis.Further research was performed to explore the driving factors of FAH expression in melanoma cells,and mainly focused on the CDC5 L protein.Through these study above,we hoped to further understand the characteristics of metabolic reprogramming in melanoma,and the role of FAH in these process.Meanwhile,we also speculated the possibility of FAH becoming a new therapeutic target and prognostic indicator of melanoma.Methods:(1)Immunohistochemistry was performed in tissue microarrays containing 100 human melanomas and benign pigmented nevus samples to detect intracellular FAH content of these samples.Two datasets of human specimens,including melanoma,benign pigmented nevus and normal skins,obtained from the Oncomine database,were used to analyzed the m RNA values of FAH;(2)Detailed information of 278 melanoma cases was extracted from the TCGA database.Then Kaplan-Meier survival analysis was used to survival rate(OS)or disease free survival(DFS).Human melanoma cells stably deficient in FAH were constructed by lentivirus and then they were used to establish the tumor-bearing mouse model.Then the survival time of tumor-bearing mice constructed by FAH-deficient melanoma cells and normal melanoma cells respectively was compared;(3)The change of proliferation,cell cycle and apoptosis of human melanoma cells were detected after silencing the expression of FAH using si RNAs or overexpressing using FAH-pc DNA3.1 plasmids.(4)m RNA expression profiling microarrays in control and FAH-knockdown A375 cells were performed after being transfected with si RNAs.Gene Ontology(GO)analysis,Pathway analysis and Pathway-net construction were used to analyze changes of the key cellular functions and pathways.(5)Subcellular localization of FAH and fumarase(FH)in human melanoma cells was analyzed by confocal microscopy and western blot,respectively.(6)Filter out the m RNA data of related enzymes or transporters from the tyrosine metabolism to TCA cycle from m RNA expression profiling of FAH-silent A375 cells.After being silenced by si FAHs or overexpressed by FAH-pc DNA3.1 plasmids,the fumarate content in human melanoma cells was detected,and the related enzymes and transporters from tyrosine metabolism to TCA cycle in A375 cells was verified by real-time RT-PCR and western blot;(7)Detailed data of 278 melanoma cases were extracted from the TCGA database,and the correlation between FAH expression in tumor tissues and the expression of the enzymes in TCA cycle was analyzed.Then we filtered out the m RNA data of enzymes in TCA cycle from m RNA expression profiling of FAH-silent A375 cells.After regulating the expression of FAH in human melanoma cells by si FAHs and FAH-pc DNA3.1 plasmids respectively,the rate-limiting enzymes in TCA cycle were detected by real-time RT-PCR and western blot.Meanwhile,the related metabolic flow was analyzed using 13C-D-glucose tracing;(8)The m RNA data of related enzymes in citrate-derived fatty acid synthesis from m RNA expression profiling of FAH-silent A375 cells was filtered out.Then the changes of related enzymes in citrate-derived fatty acid synthesis was detected by real-time RT-PCR and western blot in analyze the relationship between the expression of FAH in tumor tissue and the overall the human melanoma cells,of which the FAH expression levels was either downregulated by si FAHs or upregulated by FAH-pc DNA3.1 plasmids;(9)The m RNA data of glucose transporters and related enzymes of glycolysis,pentose phosphate pathway and nucleotide synthesis from m RNA expression profiling of FAH-silent A375 cells were also filtered out.After being silenced by si FAHs or overexpressed by FAH-pc DNA3.1 plasmids,we use real-time RT-PCR and western blot to detected the glucose transporters and rate-limiting enzymes of glycolysis,pentose phosphate pathway and nucleotide synthesis;Meanwhile,the metabolic flow of glycolysis was also analyzed using 13C-D-glucose tracing;(10)After being silenced by si FAHs or overexpressed by FAH-pc DNA3.1 plasmids,glucose consumption rate of the human melanoma cells was evaluated using enzymatic methods;(11)We searched the SABiosciences database and found the binding site and sequence of the CDC5 L protein in the upstream of the transcription start point of the FAH gene.After A375 cells were transfected with the HA-tagged CDC5 L high expression plasmids(p CMV-C-HA-CDC5L-CDS),we conducted chromatin immunoprecipitation(Ch IP)and real-time PCR to verify the effective bind between the two.Then we further assessed FAH expression in melanoma cells after CDC5 L was silenced using si RNAs.Results:(1)The expression of FAH in human melanoma cells was significantly higher than that in benign pigmented nevus at both protein and m RNA levels;(2)The overall survival rate(OS)and disease-free survival(DFS)of melanoma patients were negatively correlated with the expression of FAH,that is,patients with high FAH expression levels exhibited obviously shortened OS and DFS than those with low levels.Meanwhile,the survival time of FAH-deficient melanoma tumor-bearing mice was significantly longer than the normal groups;(3)After inhibiting FAH expression using si RNAs,human melanoma cells showed a decrease in both proliferation ability and the number of cells in G2+S,and an increase in apoptosis and the number of cells in G1.In addition,after overexpressing FAH using FAH-pc DNA3.1 plasmids,human melanoma cells showed no changes in proliferation and apoptosis,while the number of cells in G2+S slightly increased and the number of cells in G1 slightly reduced;(4)After FAH expression in A375 cells was inhibited by si RNAs,m RNA expression profiling microarrays were carried out and the subsequent bioinformatic analysis was performed.Among them,GO analysis showed that some functionally annotated clusters were significantly enriched,which included small molecule metabolism,cell cycle arrest,cell proliferation,apoptosis,TCA cycles and biosynthetic process.Consistent with GO analysis,pathway analysis revealed significant enrichment of annotated clusters including metabolic pathways,cell cycle,apoptosis,HIF-1 signaling,TCA cycle,tyrosine and phenylalanine metabolism;(5)Confocal microscopy and western blot analysis showed that FAH in human melanoma cells mainly localized in the cytosol,rather than the mitochondria,while fumarase(FH)was distributed in both mitochondria and cytoplasm;(6)After FAH expression was silenced by si RNAs,the content of fumarate in human melanoma cells was significantly decreased.Consistently,the content of fumarate was increased after FAH was overexpressed by FAH-pc DNA3.1 plasmids.The m RNA expression profiling microarrays of A375 cells after si RNA-mediated FAH silencing showed that the m RNA expression level of the related enzymes and transporters from tyrosine metabolism to TCA cycle was decreased.After inhibiting FAH expression using si RNAs or upregulating FAH expression using FAH-pc DNA3.1 plasmids,the m RNA expression of the related enzymes and transporters from tyrosine metabolism to TCA cycle in A375 cells was significantly decreased or slightly increased,which was confirmed by real-time RT-PCR and western blot.(7)Analysis of 278 melanoma cases from TCGA database showed that there was a significantly positive correlation between the expression of FAH and the expression of the enzymes in TCA cycle in tumor tissues.The m RNA expression profiling microarrays of A375 cells after si RNA-mediated FAH silencing showed that the m RNA expression of each enzyme in TCA cycle was decreased.Further analysis using real-time RT-PCR and western blot demonstrated that the expression of the rate-limiting enzymes in TCA cycle was significantly decreased after FAH depletion using si RNAs,and slightly increased after FAH overexpression using FAH-pc DNA3.1 plasmids.Simultaneously,analysis of 13C-D-glucose tracing showed that the metabolic flow of the TCA cycle was significant deceleration after FAH silencing,but no significant change was observed after FAH overexpression.(8)The m RNA expression profiling microarrays of A375 cells after si RNA-mediated FAH silencing showed that the m RNA expression of each related enzyme in citrate-derived fatty acid synthesis was decreased.Further analysis using real-time RT-PCR and western blot demonstrated that the expression of the related enzymes in citrate-derived fatty acid synthesis were significantly decreased after FAH depletion using si RNAs,and slightly increased after FAH overexpression using FAH-pc DNA3.1 plasmids.(9)The m RNA expression profiling microarrays of A375 cells after si RNA-mediated FAH silencing showed that the m RNA expression of glucose transporter and the related enzymes of glycolysis,pentose phosphate pathway and nucleotide synthesis were decreased.Further analysis using real-time RT-PCR and western blot demonstrated that the expression of glucose transporter and the rate-limiting enzymes of glycolysis,pentose phosphate pathway and nucleotide synthesis was significantly decreased after FAH silencing using si RNAs,and slightly increased after FAH overexpression using FAH-pc DNA3.1 plasmids.Meanwhile,analysis of 13C-D-glucose tracing showed that the metabolic flow of the glycolysis was significant deceleration after FAH silencing,but no change was observed after FAH overexpression;(10)After FAH depletion using si RNAs,the glucose consumption rate of A375 cells was decreased.However,there was no obvious change in glucose consumption rate after FAH overexpression using FAH-pc DNA3.1 plasmids;(11)Ch IP-q PCR demonstrated that CDC5 L protein can effectively bind to the predicted binding site,which localized in the upstream of the transcription start point of FAH gene,and FAH expression in human melanoma cells was also significantly decreased after CDC5 L silencing using si RNAs.Conclusions: In the development of melanoma,overexpression of FAH in tumor cells,which may be driven by CDC5 L,can effectively promote the anaplerotic reactions and thus accelerate the metabolic flux in TCA cycle.Meanwhile,FAH can also promote the uptake of glucose and accelerate the metabolic flux of glycolysis,pentose phosphate pathway,nucleotide synthesis and citrate-derived fatty acid synthesis in melanoma.These metabolic pathways are essential components of biosynthesis and energy metabolism and are extremely important for maintaining the homeostasis,survival and development of melanoma cells.Consistently,we have demonstrated that high expression of FAH can significantly affect multiple biological functions in melanoma cells,such as promote proliferation,glucose consumption rate and cell cycle progression and inhibit apoptosis.We also found that high expression of FAH was correlated with poor survival of melanoma patients and tumor-bearing mice.Thus,FAH may be an important tumor-promoting factor in melanoma,and may emerge as both an attractive therapeutic target and an useful independent prognostic indicator.However,we also observed that there was no obvious effect on the regulation of melanoma cellular functions after the expression of FAH was further increased using overexpressing plasmids,we attributed the mainly cause to the existed overexpression of FAH and limited substrate.The study of FAH can also provide new ideas for elucidating the underlying metabolic changes and effects in the development of melanoma and provide new directions for the treatment of such tumors.Part? Changes and roles of hypoxia-induced glycogen metabolism in the development of condyloma acuminatumObjective: This study was to explore the changes in the glucose metabolism of keratinocytes in warts during the development of condylomata acuminata(CA),including which we mainly focused on the role and regulation of glycogen metabolism.Meanwhile,we also explored the changes of oxygen concentration in CA warts and their effects on glycogen metabolism.We first collected the CA patients' warts and normal human foreskins,and then we analyzed the keratinocytes of the two groups above using m RNA expression profiling microarrays,with the purpose of exploring the key genes,functions,and pathway changes that occured in warts' keratinocytes during the development of CA.Then the keratinocytes of the two groups above were analyzed by PCR,western blot and immunohistochemical analysis to further verify the characteristics of glucose metabolism,including which we emphased on the glycogen metabolism and glycolysis.Simultaneously,we also analyzed the changes in TCA cycle,pentose phosphate pathway and nucleotide synthesis.Then glycogen levels in CA tissue and the role of GYS1 on human keratinocyte cell line were further studied to explore glycogen metabolism and its impact on the development of CA.Meanwhile,we also explored whether there was hypoxia changes existed in CA warts,and whether hypoxia could affect glycogen metabolism using multiple molecular biology methods.Through these researches above,we hoped to help further understand the characteristics and its impact of glucose metabolism in the development of CA,and also to provide a new idea for developing new strategies for CA prevention and treatment.Methods:(1)After getting informed consent of patients and volunteers and with the approval of the Clinical Research Ethics Board of the Tongji Hospital,the forty-six warts in foreskin from diagnosed CA patients in clinic of Tongji Hospital were obtained.Meanwhile,normal foreskins from thirty-two healthy young male volunteers were collected by circumcision surgery as control group.Then,the keratinocytes of warts and foreskins were surgically removed and analyzed using the m RNA expression profiling microarrays.Bioinformatics analysis including GO analysis,Pathway analysis,Pathway-net and Signal-net construction was performed to explore the changes of the key genes,functions and pathways.(2)We filtered out the m RNA data of enzymes in TCA cycle from m RNA expression profiling of keratinocytes in CA and normal foreskin,then we further verified the microarray results in keratinocytes from dozens of CA patients' warts and normal foreskins using real-time RT-PCR;(3)After filtering out the m RNA data of glucose transporters and related enzymes of glycolysis,pentose phosphate pathway and nucleotide synthesis from m RNA expression profiling of keratinocytes in CA and normal foreskin,we further confirmed the microarray results in keratinocytes from dozens of CA patients' warts and normal foreskins using real-time RT-PCR;(4)When the m RNA data of key enzymes in glycogen metabolism from m RNA expression profiling of keratinocytes in CA and normal foreskin were filtered out,the expression of these enzymes in keratinocytes from dozens of CA patients' warts and normal foreskins were subsequently confirmed by real-time RT-PCR;(5)After GYS1 expression in Ha Ca T cells was silenced by si RNA,the cells were cultured under normoxia(20% O2)and hypoxia(1% O2)conditions respectively.Then the proliferation and apoptosis of Ha Ca T cells were analyzed by flow cytometry using Ki-67 and Annexin/PI,respectively.(6)After filtering out the m RNA data of hypoxia inducible factor-la(HIF-1?)and carbonic anhydrase-9(CA-9)from m RNA expression profiling of keratinocytes in CA and normal foreskin,we subsequently performed immunohistochemical staining using HIF-1? in both CA and normal foreskin tissues to further determine the hypoxic condition in their keratinocytes.Further PAS staining,CA-9 immunohistochemical staining and their co-staining were performed to determine the glycogen content,hypoxic condition and whether there is a direct relationship between the two in the keratinocytes of CA tissue and normal foreskin;(7)Ha Ca T cells were cultured under normoxia(20% O2)and hypoxia(1% O2)conditions at different timepoints,respectively,and the glycogen content was determined by PAS staining.Results:(1)The mRNA expression profiling microarrays of keratinocytes from CA warts and foreskins showed that cell cycle,small molecule metabolism,apoptosis,cellular response to hypoxia,glucose metabolism and transport,lipid and nucleotide metabolism,proliferation of keratinocytes and other biological functionally annotated clusters were significantly enriched in GO analysis.Consistent with the GO analysis,pathway analysis revealed obvious enrichment of metabolic pathways,cell cycle,HIF-1 signaling,purine and pyrimidine metabolism,glycolysis / gluconeogenesis and lipid metabolism pathways.Consistent with this,the Singal-net also showed that the expression of metabolic genes such as HK2,HIF-1?,LDHA and SLC2A1 in CA warts' keratinocytes were significantly up-regulated compared with the normal group,suggesting that these genes might be the main metabolic-regulation genes in the CA development;(2)The m RNA expression profiling microarrays of keratinocytes from CA warts and foreskins showed that there was no significant change in the expression of each enzyme in TCA cycle.Further analysis using real-time RT-PCR in keratinocytes from CA warts and normal foreskins was consistent with the microarrays results,and the expression of rate-limiting enzymes in TCA cycle did not change significantly at m RNA level;(3)The m RNA expression profiling microarrays of keratinocytes from CA warts and foreskins showed that the expression of glucose transporters and related enzymes of glycolysis,pentose phosphate pathway and nucleotide synthesis was significantly higher in warts than in normal foreskins.The results of real-time RT-PCR of keratinocytes in CA warts and normal foreskins were consistent with those of the microarrays;(4)The m RNA expression profiling microarrays of keratinocytes from CA warts and foreskins showed that the expression of key enzymes in glycogen metabolism was obviously higher in warts than in normal foreskins.The results of real-time RT-PCR of keratinocytes in CA warts and normal foreskins were consistent with those of the microarrays;(5)The GYS1-silent Ha Ca T cells showed a decrease of Ki-67 expression and a increase of apoptotic cells in both normoxia(20% O2)and hypoxia(1% O2)culture;(6)The m RNA expression profiling microarrays of keratinocytes from CA warts and foreskins showed that the expression of HIF-1? and CA-9 in warts was significantly higher than that in normal foreskins.Immunohistochemical staining also showed that the expression of HIF-1? in CA warts was significantly higher than that in normal foreskins.Meanwhile,PAS staining showed that the glycogen content in keratinocytes from warts was obviously higher than that in normal foreskins,and the expression of CA-9 in warts was also significantly higher than that in normal foreskins using immunohistochemical staining.And PAS positive areas showed co-localization expression with CA-9 positive area;(7)The positive rate of PAS staining in Ha Ca T cells cultured in hypoxia(1% O2)condition was higher than that in normoxia(20% O2),especially after 48 hours culturing.Conclusions: In the occurrence and development of CA warts,the infected keratinocytes will undergo local hypoxia and glycogen accumulation,and hypoxia is directly related to the glycogen accumulation.Meanwhile,the metabolic fluxes of glucose uptake,glycolysis,pentose phosphate pathway,glycogen and nucleotide synthesis of infected keratinocytes were also significantly enhanced,while the metabolic flux of TCA cycle was not significantly enhanced.This phenomenon suggests that uptake of glucose does not more flow into the TCA cycle through glycolysis,but more flow into the branch of glycolysis-pentose phosphate pathway to enhance nucleotide synthesis,and also more flow into the glycogen metabolism to increase glycogen synthesis.These changes in glucose metabolism may be associated with localized hypoxia in infected keratinocytes.Several studies have reported that hypoxia can induce the expression of GYS1,thus promoting glycogen accumulation,whereas increased glycogen is crucial for maintaining cell survival under hypoxic conditions.Therefore,these metabolic changes can provide a theoretical basis for the discovery of local hypoxia and related metabolic changes,as well as the role of glycogen accumulation in the development of CA,and also help to find the key metabolic therapeutic targets.