The Functions Of LncRNAs In Regulating Glucose Metabolism In Lung Cancer Cells

Objective: Lung cancer is one of the leading tumor sites among all malignant tumors and has both the highest incidence as well as mortality rate.It is a serious threat to human health and lives.Although considerable progress has been made in the diagnosis and treatment of lung cancer,the five year survival rate of patients with lung cancer is still very low.Therefore,we should further explore the mechanism of lung cancer from the molecular level,and find novel biomarkers related to biological activity of lung cancer,which will help to improve the survival rate of lung cancer patients.Abnormal glucose metabolism is one of the important characteristics of cancer cells.In 1930 s,Warburg found that even under conditions of abundant oxygen,tumor cells underwent mostly glycolysis and consumed large amounts of glucose,with lactate dehydrogenase played an important role in the process.Long non-coding RNAs(lnc RNAs)are a class of RNAs that are larger than 200 nt and have no protein coding function.In recent years,studies have found that lnc RNAs are abnormally expressed in many cancers,and involved in many important life processes.They also play an important role in the pathogenesis of lung cancer.Ionizing radiation can cause a series of toxicological effects,such as DNA damage,cell cycle pertubation,and cell apoptosis.However,the role of lnc RNAs in modulating the response of cells to ionizing radiation is still unknown.Therefore,the present study to elucidate the functions of lnc RNAs and their related mechanisms will provide potential targets for the treatment of tumor.We mainly focused on the change of lnc RNAs expression,the effects of lnc RNAs on the level of glucose metabolism,and the effects of ionizing radiation(X-rays)on the expression of lnc RNAs and glucose metabolism in lung cancer cells.METHODS:This project is divided into four sections.(1)The biological effects of LNC CRYBG3 in lung cancer cells.Early studies from our laboratory showed that the expression of LNC CRYBG3 in He La cells could be induced by heavy ion irradiation.RT-q PCR was used to determine the expression of LNC CRYBG3 in a variety of cells,and suitable cell lines with LNC CRYBG3 expression were selected as the experimental model.The expression of LNC CRYBG3 in lung cancer tissue and normal human lung tissue was analyzed by absolute quantitative analysis.The effects of LNC CRYBG3 on the migration and proliferation of lung cancer cells were measured by scratch test and cell proliferation assay.All these studies were to determine the expression of LNC CRYBG3 in lung cancer cells and its effects on the biological effects of lung cancer cells.(2)The relationship between LNC CRYBG3 and LDHA.Lactate dehydrogenase(LDHA)plays an important role in glycolysis.The relationship between LDHA and the survival rate of patients with lung cancer was analyzed using the ATCG database.By overexpressing or knocking down the expression of LNC CRYBG3,the activity and expression of LDHA in lung cancer cells were detected by Western blotting and LDHA enzyme activity kit.The expression of LNC CRYBG3 was intervened in the tumor and the effect of LNC CRYBG3 on the expression of LDHA was analyzed by immunohistochemical technique.RNA pull down technology and RNA Binding Protein Immunoprecipitation(RIP)technology were used to analyze the interaction between LNC CRYBG3 and LDHA.(3)The effects of LNC CRYBG3 on glucose metabolism in lung cancer.By knocking down or overexpressing the level of LNC CRYBG3,its role on metabolites involved in glycolysis and Krebs cycle in lung cancer cells was determined by gas chromatography-mass spectrometry(GC-MS).The effects of the changes of LNC CRYBG3 on glucose metabolism in lung cancer cells were also analyzed.(4)Irradiation affects the glucose metabolism of lung cancer cells by changing the expression of LNC CRYBG3.After X-ray treatment,the changes of LNC CRYBG3 in A549 and H1299 lung cancer cells were analyzed by RT-q PCR.At the same time,the changes of metabolites associated with glycolysis and Krebs cycle were detected by GC-MS.RESULTS:(1)The biological effect of LNC CRYBG3 in lung cancer cells.The expression levels of LNC CRYBG3 in A549,Calu-1,92-1,H460,H1299,Kyse150,U251,Hep G2 and Eca109 were analyzed by RT-q PCR.The expression of LNC CRYBG3 in lung cancer cells was significantly higher than that in other cancer cells.As such,we chose the lung cancer cells A549 and H1299 as cell models for all subsequent studies.By absolute quantification,we found that the expression of LNC CRYBG3 in normal lung tissues was relatively low,while the expression in lung cancer patients was significantly higher(p<0.01).After knocking down the expression of LNC CRYBG3,the proliferation rate of A549 cells or H1299 cells decreased significantly(p<0.05).The scratch test of A549 and H1299 cells with knocking down of LNC CRYBG3 showed a reduced healing rate or cell migration.These results suggest that LNC CRYBG3 can promote the growth of lung cancer cells.(2)The interaction between LNC CRYBG3 and LDHA.Base on the ATCG database,we found that when the expression level of LDHA was high,the survival rate of lung cancer patients was significantly lower than those with low LDHA level..Furthermore,the expression of LDHA in lung cancer cells was significantly higher than that in normal lung epithelial cells.At the same time,we found that there was a significant positive correlation between the expression of LNC CRYBG3 and the expression of LDHA(R2=0.8419)in that overexpression of LNC CRYBG3 resulted in high LDHA activities(p<0.01)and protein levels.Similarly,in LNC CRYBG3 knock down tumor cells,the LDHA activity as well as protein levels were significantly reduced(p<0.01).In mice transplanted tumor cells,we found that the expression of LDHA increased after overexpression of LNC CRYBG3,whereas the expression of LDHA decreased significantly after knocking down the expression of LNC CRYBG3.These results suggest that there is a positive correlation between the expression of LNC CRYBG3 and LDHA,and LNC CRYBG3 can promote the expression of LDHA.According to the CO-IP and RIP experiments,we found that LNC CRYBG3 can interact with LDHA.(3)The effects of LNC CRYBG3 on glucose metabolism in lung cancer.Overexpression of LNC CRYBG3 in A549 and H1299 cells resulted in a decrease in pyruvate and a corresponding increase in lactic acid production.In contrast,knocking down of LNC CRYBG3 resulted in a down-regulation of lactic acid production.The related metabolites of three carboxylic acid,fumaric acid,malic acid,α-ketoglutaric acid,succinic acid and citric acid levels were not statistically changed,indicating that LNC CRYBG3 had little effect on the aerobic respiration and Krebs cycle in lung cancer cells.(4)Irradiation affects the glucose metabolism of lung cancer cells by changing the expression of LNC CRYBG3.After X-irradiation treatment,there was no significant change in the expression of LNC CRYBG3 in A549,whereas the expression of LNC CRYBG3 in H1299 cells increased with time.In addition,the expression of LDHA was significantly higher in lung cancer H1299 cells than in A549 cells.After X-rays irradiation,the levels of metabolites including pyruvate and lactic acid associated with glycolysis and intermediates including fumaric acid,malic acid,α-ketoglutaric acid,succinic acid and citric acid related to Krebs cycle did not change significantly in H1299 and A549,indicating that irradiation had little effects on this process.CONCLUSIONS:(1)The expression of LNC CRYBG3 is increased in lung cancer and lung cancer tissues;LNC CRYBG3 can promote the growth of lung cancer cells;(2)LDHA is highly expressed in lung cancer cells.Moreover the survival rate of lung cancer patients with relatively high expression of LDHA is significantly lower than that of lung cancer patients with relatively low expression of LDHA;LNC CRYBG3 can promote the expression of LDHA,and there is a positive correlation between LNC CRYBG3 and LDHA;(3)LNC CRYBG3 can promote glycolysis process while it has little effect on the Krebs cycle in lung cancer cells.(4)After X-ray irradiation,the expression of LNC CRYBG3 in A549 cells remained the same,while the expression level in H1299 cells gradually increased with time.Furthermore,radiation has little effects on glycolysis and Krebs cycle metabolites of lung cancer cells.