A Study On The Mechanism Of PGAM1 Induced Paclitaxel Resistance Via Modulating Glycolysis Flux In Ovarian Cancer Cells

Ovarian cancer is one of the three most common malignant tumors in female reproductive system,but the mortality rate ranks the first,seriously threatened women's health.Due to the advent of new chemotherapeutic drugs such as paclitaxel and the widespread introduction to China since the beginning of the twenty-first Century,the short-term complete remission rate of ovarian cancer has improved significantly,but the overall mortality rate has not decreased.Chemo-resistance is one of the most dominant causes of treatment failure and death in patients with advanced ovarian cancer.It is a pivotal strategy to crack paclitaxel resistance to improve the survival rate of ovarian cancer patients.Paclitaxel as an anti-microtubule drug,is one of the most widely used first-line chemotherapy drugs.The mechanism of paclitaxel in killing cancer cells is,promoting microtubule polymerization and inhibiting depolymerization,maintaining microtubule stability,thus inhibiting tumor cell mitosis.The evidence showed that tumor cells exposed to paclitaxel could escape from being killed through a series of adaptive alteration,the mechanisms being studied mainly focused on microtubule associated gene mutation or abnormal protein expression,changes of microtubule stability,which makes paclitaxel-microtubule binding disorders.Other mechanisms involved are over expression of transmembrane efflux transporters which leads to the increase of paclitaxel pumpout,weakened function of apoptosis related molecules,and so on.But no strategy designed for these mechanisms has been used in clinical practice to date.Recent studies have also showed that the mechanism of paclitaxel resistance may be related to glycolysis.Cancer cells exhibit enhanced glycolysis.Enhanced glycolysis leads to chemo-resistance may have three mechanisms:First,enhanced glycolysis flux can help cancer cells obtain energy rapidly to gain survival advantage;Second,mitochondrial respiratory function is inhibited,oxidative phosphorylation pathway is weakened,and the oxygen free radicals production is reduced,cells escape from apoptosis and autophagy,resulting in decreased programmed cell death;third,glycolysis flux is enhanced concomitant with its bypass phosphoric acid pentose pathway,resulting in increased production of NADPH and phosphoric acid ribose to meet the need for biological macromolecules synthesis especially nucleic acid,to repair damaged DNA and rapid cell proliferation.In this study,we first demonstrated the high expression of PGAM1 in paclitaxel resistant ovarian cancer cells by Western blot,and observed the induction of paclitaxel on PGAM1 expression in ovarian cancer cells.Then down-regulated PGAM1 expression by siRNA and up-regulated PGAM1 expression by pCMV4-PGAM1 plasmid,and changing the concentration of final-product pyruvic acid,in order to observe the alteraration of paclitaxel sensitivity in ovarian cancer cells.AT last,we used RNA-seq transcriptomics,informatics analysis and gene function study to determine that DDIT4 could be involved in PGAM1 regulating paclitaxel sensitivity in ovarian cancer cells.The objective of this study is to elucidate the mechanism of ovarian cancer cells resistant to paclitaxel in the glucose metabolism pathway aspect,and to provide scientific evidences for exploring new approach of ovarian cancer targeted therapy.Part I PGAM1 expression and glycolysis flux regulates the paclitaxel sensitivity in ovarian cancer cellsObjective:To observe the impact on PGAM1 expression and glycolytic flux by paclitaxel inducing,and reversely observe the alteration of paclitaxel sensitivity by modulating PGAM1 expression or glycolytic flux in ovarian cancer cells,in order to investigate PGAM1 and glycolytic flux regulation on the paclitaxel sensitivity in ovarian cancer cells.Methods:In this part,we compared PGAM1 expression between paclitaxel resistant ovarian cancer cells SKOV3-TR30 and parental cells by Western blot.Observing alteration of PGAM1 expression after paclitaxel stimulated in SKOV3 cells.Then,we down-regulated PGAM1 expression by siRNA and up-regulated PGAM1 expression by pcMV4-PGAM1,observing the alteration of paclitaxel sensitivity of ovarian cancer cells.We test the final-product of glycolysis of pyruvic acid produced by paclitaxel resistant cell lines and parental cells,and compared the difference.Finally,we gradually increased the concentration of pyruvic acid in the culture medium and observed the alteration of paclitaxel sensitivity.Results:1.The expression of PGAM1 in paclitaxel resistant ovarian cancer cell line SKOV3-TR30 was higher than that of the parental cells,and the stimulation of paclitaxel could induce the expression of PGAM1 in ovarian cancer cell line.2.Down regulation of PGAM1 expression enhanced the sensitivity of paclitaxel in ovarian cancer cells,while up-regulation of PGAM1 expression increased the paclitaxel sensitivity.3.In paclitaxel resistant cell lines which the expression of PGAM1 is high,the final-product of glycolysis of pyruvic acid was significantly higher than the parental cells.We gradually increased the concentration of pyruvic acid within a certain arrangement in the culture medium could decrease the paclitaxel sensitivity of SKOV3.Conclusions:1.PGAM1 involved in the regulation of paclitaxel sensitivity in ovarian cancer cells.2.Changing the concentration of the final product of glycolysis can affect the paclitaxel sensitivity of ovarian cancer cells.Part ? Screening and validation for downstream signaling molecules in PGAM1 modulating paclitaxel sensitivity in ovarian cancer cellsObjective:To screen and verify the downstream signaling molecules involved in PGAM1 regulating paclitaxel sensitivity in ovarian cancer cells via RNA sequencing and bioinformatics analysis.Methods:In this part,we first down-regulated PGAM1 expression by siR-PGAM1 transfection in paclitaxel resistant ovarian cancer cells,interference efficiency was verificated by Western blot.Then the samples were prepared for RNA-seq detection(siRNA-PGAM1 and negative control group,each with 3 biological replicates).Then the total RNA was extracted and the cDNA library was constructed and sequenced by Illumina Hi-seq 2000 sequencing platform.The results of sequencing were compared and analyzed.The differentially expressed genes were analyzed by KEGG and GO cluster analysis.Then,the online software GeneMANIA was used to analyze the differentially expressed genes.Finally,we anchored one key molecule related to PGAM1 regulation of paclitaxel sensitivity in ovarian cancer cells.Results:1.RNA sequencing was carried out and 101 differentially expressed genes was discovered except PGAM1,including 43 up-regulated genes and 57 down-regulated genes.2.GO and KEGG cluster analysis showed that metabolic process is the main signal pathway.3.Bioinformatics analysis was carried out using an online software GeneMANIA to find out the network among these 100 differentially expressed genes.4 proteins(DDIT4,ADM,AP3S1 and FTL)were found co-expression with PGAM1.According to the function of these four proteins,DDIT4 has the most likelihood as the key downstream molecule involved in PGAM1 regulating paclitaxel sensitivity in ovarian cancer cells.4.In SKOV3-TR30 cells,the expression level of DDIT4 was concomitantly down-regulated after down-regulating PGAM1 expression by siR-PGAM1 transfection.Conclusions:1.There are many signal pathways involved in the regulation of PGAM1 on the paclitaxel sensitivity of ovarian cancer cells,and the metabolic pathway is the dominant pathway.2.DDIT4 may be a downstream signaling molecule involved in PGAM1 regulation of paclitaxel sensitivity in ovarian cancer cells.Part III DDIT4 is involved in PGAM1 regulation of paclitaxel sensitivity in ovarian cancer cellsObjective:To confirm DDIT4 involvement in PGAM1 regulating paclitaxel sensitivity in ovarian cancer cells by gene function study.To predict the structural mechanism of PGAM1 and DDIT4 interaction by computer simulation of molecular docking models.Methods:We observed the alteration of paclitaxel sensitivity of ovarian cancer cells after down-regulating DDIT4 expression by siRNA and up-regulating DDIIT4 expression by pcDNA3.1-DDIT4.Furthermore,we observed whether the effect of PGAM1 in modulating paclitaxel sensitivity of ovarian cancer cells could be reversed by DDIT4.DDIT4 is a newly discovered protein and its tertiary structure has not been resolved.We used homology modeling of DDIT4 protein,and docking DDIT4 and PGAM1 protein by computer simulation,predicted the stability of DD1T4-PGAM1 combination and protein-protein interaction sites.Results:1.Down-regulation of DDIT4 expression can enhance the paclitaxel sensitivity of ovarian cancer cells.Up-regulation of DDIT4 expression can reduce the paclitaxel sensitivity of ovarian cancer cells.2.DDIT4 could reverse the regulation of PGAM1 on paclitaxel sensitivity in ovarian cancer cells.3.DDIT4 binds to the extra domain of the PGAM1 receptor protein in a similar manner to the telechelic and interacts strongly with its amino acid residues by hydrogen bond.DDIT4 and PGAM1 may be stably bound by three major binding sites to form a protein-Protein complex.Conclusions:1.PGAM1 regulates the paclitaxel sensitivity of ovarian cancer cells by directly binding to DDIT4.2.The spatial structure of PGAM1 and DDIT4 protein could complement each other,thus has the possibility to form a stable combination at three major binding sites by hydrogen bonds between amino acid residues.