The Research Of Mechanism Of Si-HMMR Inhibiting Glycolysis And Application To Radio-sensitivity In LUAD

Background:Lung cancer is one of the most severe cases of malignant tumors worldwide.Death-rate caused by lung cancer places at the top among all malignant tumors.Lung cancer’s high malignance is due to the fact that most patients seek medical care after occurrences of symptoms,and by the time of diagnosis,patients are already in the late stage and have missed the best time to get surgical treatments.Lung cancer treatments include radiotherapy,chemotherapy,and targeted therapy.The 5-year survival rate of all treatments mentioned above is 16.1%.Radiotherapy is an important method for local treatments,and thus plays a significant role in the overall treatment of lung cancer.60%-70% of patients of various stages are in need of receiving radiotherapy.However,due to radiation resistance factor,even though increasement in radiation enhances the control-rate of tumors,it also increases the acute and chronic toxic side effects of regular tissues and organs nearby.Hence,enhancing treatment of tumors and lowering harms to regular tissues have been questions central to radiologists’ research focus.In cellular energy metabolism,tumor cells require more glucose than regular cells.Glucose transforms into lactic acid through metabolic pathways.Even with abundant oxygen,tumor cells tend to supply energy through glycolysis.This cellular energy metabolism is aerobic glycolysis,also known as the Warburg effect.Aerobic glycolysis creates environment favorable for tumor cells’ development,slower apoptosis,increasing invasiveness,immune escape,and drug resistance.The flexibility of tumor cell metabolism guarantees tumor cells’ “fuel” in their development process in adapting to changing environment and external stimuli.Three rate-limiting enzymes in glycolysis regulate three significant irreversible reactions and affect the overall level of cellular glycolysis.In the glycolysis of pyruvate kinase(PK),the last significant rate-limiting enzyme catalyzes ATP to transfer energy-rich phosphate bond to pyruvic acid and forms phosphoenolpyruvate and ADP.Nevertheless,various isoforms of PK contain tissue specificities.PKM2 subtype becomes a glycolytic enzyme characteristic of tumor cells.PKM2 is therefore termed Tumor M2 pyruvate kinase,TUM2-PK.PKM2 significant function in tumor metabolism.The overexpression of PKM2 can increase tumor cells’ utilization of glucose and alter the balance of redox reaction.Such metabolic transformation enhances tumor cells’ syntheses of midbody and metabolite,which supply for tumor cells’ unlimited proliferation.Research shows that in NSCLC tumor cells,small-molecule inhibitors targeted at PKM2 can enhance cellular apoptosis.In nude mouse xenograft models for lung adenocarcinoma,restraining the enzymatic activity of PKM2 can decrease tumors in size.The above two instances demonstrate the significance of PKM2 in tumor cell glycolysis and tumor treatments.Research has shown that glycolysis and radiosensitivity are closely related.Due to the Warburg effect,tumor cells have natural resistance towards radiation compared to regular cells.We can target at the Warburg effect,create treatment methods based on tumor cell glycolysis and radioresistance,and invite further research in radiotherapy.Scholars have proven that restraining glycolysis can increase radiosensitivity and chemosensitivity in lung cancer,breast cancer,and cervical cancer.We can take advantage of bioinformatics,the High-Throughout Genomic Sequences data from the TCGA database,and GSEA enrichment to analyze the significance of glycolysis in lung cancer occurrence and development.In the meantime,we can enrich possible participating genes in the process of glycolysis and construct a set of patient prognosis molecular markers from AGRN,HMMR,AKR1A1,and DDIT4 by using R computation and risk factor regression model.Receptor of hyaluronic acid mediated motility(RHAMM,also known as HMMR or CD168)is the first known hyaluronic acid mediated receptor.It exists in various forms in cell surface,cytoplasm,and cell nucleus.Research shows that intracellular HMMR unites with both actin filament and microtubule cytoskeleton and interacts with ERK and SRC kinase.Intracellular HMMR also unites with protein and regulate microtubular dynamics and centrosome structure and function in order to facilitate microtubules-mediated cell polarity and cell migration.However,the relation between HMMR and glycolysis still remains unclear.According to our bioinformatics,HMMR may participate in the glycolysis process of lung cancer and could be a molecular marker for lung cancer patients’ prognosis.The purpose of this essay is to further explore the correlation between HMMR and glycolysis as well as its influence on lung cancer radiosensitivity,using proteomics to discuss possible relations between HMMR and PKM2,and using animal model to testify whether Si-HMMR has the effect of increasing radiosentivity for lung cancer.This research complements current research on HMMR gene,provides theoretical foundation for its possibility for lung cancer prognosis and molecular markers of radiosensitivity,and make it possible for clinicians to achieve individual and precise treatments of tumors.Methodology:1.Obtain information of 1038 cases of lung cancer tissue and 108 cases of normal tissue from the Cancer Genome Atlas(TCGA.)Use GSEA and R language for univariate and multivariate cox regression analyses.Create risk models according to the analyses and results,calculate the risk scores of 511 lung cancer patients,categorize these 511 cases into high-risk category and low-risk category,use multivariate cox regression and stratified analysis to estimate whether molecular markers of genes are independent prognostic factors.2.Use CCK8 to determine the influence of glycolysis inhibitor 2-DG with different concentration on lung adenocarcinoma cell lines H1299,A549,and H1975 proliferation and activity,and to determine the IC50 concentration in various cell lines.3.Apply 0,? IC50,and IC50 concentration gradients to dispose of lung adenocarcinoma,use q PCR to measure the changes in expression quantity of the four genes,AGRN,AKR1A1,DDIT4,and HMMR.Select one with the most stable and significant changes,HMMR,as the main gene under discussion in this essay.4.Use q PCR,Western Blot,and immunohistochemistry methodologies to test 3 lung adenocarcinomas,8 pairs of lung adenocarcinoma samples,and the expression quantity of 42 pairs of HMMR paraffin sections.Verify TCGA bioinformatics analysis results.5.Use RNAi technology,construct HMMR silent and overexpression vectors,q PCR,Western Blot,and immunofluorescence technique to verify changes in lung adenocarcinoma lactic acid,glucose,and related protein expression after silent and expression HMMR.Use hippocampal energy metabolizer to test how changes in HMMR6.H1299 and A549 cell lines were irradiated with different radiation doses(0Gy,2Gy,4Gy,6Gy,and 8Gy),and plate clone formation experiments were used to analyze the effect of changes expression of HMMR on radio-sensitivity.7.The effect of HMMR expression on radiosensitivity of lung adenocarcinoma was verified in vivo using nude mice tumor-bearing experiments.expression influence lung adenocarcinoma glycolysis.8.overexpression of HMMR in H1299,protein profiling and identification were performed to identify proteins that may interact with HMMR.According to the results of proteomic analysis,the interaction protein PKM2 related to glycolysis was selected for research.The method of q PCR,Western Blot,enzyme activity detection,immunofluorescence co-localization and CO-IP were used to analysis the interaction between HMMR and PKM2.After experiment of HMMR expression,we detect the changes on PKM2 m RNA level,protein level and enzyme activity;immunohistochemical method was used to analyze the synergistic expression of HMMR and PKM2.Conclusion: 1.HMMR can be used as an independent prognostic indicator for LUAD patients;2.Changes in HMMR expression affect glycolysis of LUAD cells;3.HMMR affects its enzymatic activity by directly interacting with PKM2 protein and participates in glycolysis process;4.Changes expression of HMMR can affect the radio-sensitivity of LUAD cells and may become a potential sensitization target for lung cancer radiotherapy.