Preliminary Study On The Clinical Significance And Biological Role Of LDHA/B In NSCLC

Background: Worldwide,the incidence and mortality of lung cancer rank first among all types of tumors.Most patients with lung cancer are in the advanced stages at the time of diagnosis and lose the chance of surgery,which is an important cause of high mortality.With the development of lung cancer research,the treatment of lung cancer has made great progress.Treatments mainly include surgical treatment,radiotherapy,chemotherapy,molecular targeted therapy and immunotherapy,all of which bring huge benefits to patients.However,many problems,such as the adverse reactions of chemical drugs,the lack of known gene mutations and the emergence of drug resistance in sensitive cases,need to be resolved.Therefore,it is necessary to further study the pathogenesis of lung cancer.Reprogramming of energy metabolism is one of the characteristics of tumors,which can meet the needs of energy supply and biosynthesis of tumor cells.As a tumor metabolite,the role of lactate in the development of tumor has attracted a lot of scholars’ attention.Lactate can not only promote tumor migration and metastasis,enhance angiogenesis,contribute to the occurrence of immune escape,but also serve as the energy source of tumor cells.Lactate Dehydrogenase A(LDHA)and Lactate Dehydrogenase B(LDHB)are key enzymes involved in lactate production and play important roles in tumor.LDHA is highly expressed in a variety of tumors and plays a significant role in tumor proliferation,invasion,metastasis,immune escape and angiogenesis,which is associated with poor prognosis of lung cancer.However,the role of LDHB in tumors is not completely consistent.For example,the inhibition of LDHB expression by hypoxia can enhance the proliferation,migration and invasion of pancreatic cancer cells,while the high expression of LDHB in triple-negative breast cancer indicates a poor prognosis [3,4].In addition,the role of these two enzymes in NSCLC is not clear.Therefore,the role of LDHA and LDHB in NSCLC needs to be further elaboratedPurposes: To explore the expression profiles of LDHA/LDHB and their correlation with clinical characteristics of tumor in non-small-cell lung cancer.To investigate the correlation between LDHB and clinical features of lung adenocarcinoma.And the possible mechanism of LDHB promoting the malignant progression of lung adenocarcinoma also been investigated.Methods:(1)Transcriptional data sets of NSCLC,lung adenocarcinoma and lung squamous cell carcinoma were obtained from TCGA and GEO databases,and the expression profiles of LDHA/LDHB in cancer and normal tissues were analyzed by selecting a reasonable statistical method.(2)According to the clinical information of the lung adenocarcinoma and lung squamous cell carcinoma data sets in the TCGA database,Kaplan-Meier method and Logrank test were used for survival analysis to evaluate the correlation between LDHA and LDHB m RNA levels and overall survival and progression-free survival of patients.(3)The chi-square test was used to analyze the correlation between LDHA/LDHB transcription level and clinical characteristics of NSCLC.(4)Spearman rank correlation was used to explore the m RNA levels of LDHA and LDHB and proliferating cell nuclear antigen(PCNA),Ki-67,Cadherin 1,CDH1 and CDH2 in lung adenocarcinoma.(5)To investigate the expression of LDHB protein in clinical specimens of lung adenocarcinoma by immunohistochemistry.Appropriate cut-off values were used for the dichotomization of LDHB expression levels,and the correlation between LDHB protein levels and survival of patients with lung adenocarcinoma was analyzed based on this criterion.By using valid clinical information,the correlation between LDHB protein levels and clinical characteristics of lung adenocarcinoma was analyzed.(6)LDHB stable knockdown cell lines and E2F1 low expression cell lines were constructed by using RNA interference assay.The effects of LDHB on the proliferation,migration and invasion of lung adenocarcinoma cells were investigated by CCK8 and Transwell experiments.Western blotting was used to verify the effect of LDHB on tumor autophagy.(7)Using q RT-PCR experiments to explain the m RNA levels of E2F1 and LDHB in a nutrient deficient condition.Regulation of E2F1 on LDHB was revealed by Western blotting and q RT-PCR.Results:(1)In multiple data sets of NSCLC,lung adenocarcinoma,and lung squamous cell carcinoma,the LDHA and LDHB m RNA levels of tumor tissue were significantly higher than normal tissues.(2)The m RNA levels of LDHA and LDHB were related to the survival of lung adenocarcinoma patients.The overall survival and progression-free survival of patients with lung adenocarcinoma were the shortest when LDHA and LDHB m RNA levels were high.(3)In lung adenocarcinoma,the m RNA levels of LDHA and LDHB were correlated with the clinical features and tumor malignant biomarkers.(4)The overall survival time of lung adenocarcinoma patients with higher expression of LDHB protein was shorter.(5)LDHB had no impact on the proliferation of lung adenocarcinoma cells under normal condition.However,LDHB promoted the proliferation,migration and invasion of lung adenocarcinoma cells under low-glucose condition.Furthermore,LDHB promoted autophagy of lung adenocarcinoma cells.(6)LDHB and E2F1 m RNA levels of lung adenocarcinoma cells were up-regulated in a nutrient deficient condition.LDHB was regulated by E2F1 in lung adenocarcinoma cells.Conclusions:(1)Multiple transcription data sets suggested that the m RNA levels of LDHA and LDHB were significantly higher in NSCLC,lung adenocarcinoma,and lung squamous cell carcinoma than normal tissues.The m RNA levels of LDHA and LDHB in lung adenocarcinoma were related to the survival status of patients,especially when the m RNA levels of LDHA and LDHB are simultaneously high,the overall survival and progression-free survival of the patients are poorer.These clues suggested that there may be a synergistic effect between LDHA and LDHB during the progression of lung adenocarcinoma.In addition,the m RNA levels of LDHA and LDHB were associated with the clinical features of lung adenocarcinoma and tumor malignant biomarkers.LDHA and LDHB may be involved in the malignant progression of lung adenocarcinoma,and interventions targeting them may curb tumor progression and improve patient prognosis.(2)Our clinical data indicated that patients with higher LDHB protein levels have a worse prognosis.The LDHB protein levels had no obvious correlation with the clinical features of lung adenocarcinoma,which may be related to our small sample size.Cell experiments showed that LDHB did not affect the proliferation of lung adenocarcinoma cells under normal condition,but promoted the proliferation,migration and invasion of lung adenocarcinoma cells under low-glucose condition.In addition,LDHB can promote tumor autophagy.These clues suggested that LDHB may be a key gene that mediates nutritional tolerance of lung adenocarcinoma.(3)LDHB and E2F1 m RNA levels of lung adenocarcinoma cells were increased in a nutrient deficient condition.E2F1 positively regulated LDHB.In a word,the E2F1-LDHB axis may promote tolerance to nutrient deficient condition in lung adenocarcinoma by promoting autophagy.