Hypoxia Response And The Clinicopathological Correlation Of The Expression Of PDHA1 And GBP1 In Non-small Cell Lung Cancer

Lung cancer is the leading cause of cancer mortality worldwide.In developing countries,lung cancer incidence trends increased in relation to rapidly increasing tobacco consumption.However,non-smokers account for 25%of all lung cancer cases.Although lung cancer affects men more than women traditionally,the increasing women smoking population leads to increased lung cancer incidence and more adenocarcinoma in women.Although there are many new treatments focused on the tumor,the overall survival rate is not significantly improved.Histologically,lung cancer is divided as non-small cell lung cancer and small cell lung cancer.Among them,non-small cell lung cancer is the most common histologic type of lung cancer,accounting for 80-85%of the total number of lung cancer.Although there are many new treatments focused on lung cancer,the overall survival rate is not significantly improved.It is important to identify the key genes and proteins that regulate the proliferation,invasion and metastasis of tumor cells,and to study the intrinsic mechanism of the tumor.Although previous studies by our team have suggested that GBP1 and PDHA1 play an important role in other tumors,there has been no report on the role of GBP1 and PDHA1 in non-small cell lung cancer.Pyruvate is the key metabolic intermediate of the tricarboxylic acid cycle,glycolysis and pentose phosphate pathway,and mitochondria-mediated pyruvate oxidation is the core of the metabolic pathway of sugar,fatty acids and amino acids in eukaryotic cells.In human cells,glucose glycolysis can produce pyruvate,and multiple carrier proteins and enzyme complexes are involved in pyruvate catabolism.PDHA1 is the pyruvate dehydrogenase?PDH?E1 alpha subunit,which belongs to the key hub.Pyruvate produced by glucose metabolism,first of all,is transported into the mitochondrial matrix by the mitochondrial pyruvate carrier?MPC?,which is located on the inner mitochondrial membrane.Oxidative decarboxylation was subsequently under the action of the pyruvate dehydrogenase complex?PDC?,generating acetyl-CoA into the tricarboxylic acid cycle and oxidative phosphorylation.The phosphorylation and dephosphorylation of pyruvate dehydrogenase E1 alpha subunit?PDHA1?determines the inactivation and activation of PDC.The normal expression of PDHA1 protein is a prerequisite for the regular running of the tricarboxylic acid cycle and oxidative phosphorylation in the mitochondria.The oxidative phosphorylation and cellular glycolysis are two major pathways that produce energy in the cells.To these two major pathways,pyruvate functions as a connection between glycolysis and the tricarboxylic acid cycle.Disturbance of pyruvate metabolism may change the relative ratio of oxidative phosphorylation and glycolysis of mitochondria.Otto Warburg made a striking discovery in the 1920s.He found that,even in the presence of ample oxygen,cancer cells prefer to metabolize glucose by glycolysis,a seeming paradox as glycolysis,when compared to oxidative phosphorylation,is a less efficient pathway for producing ATP.Although aerobic glycolysis has now been generally accepted as a metabolic hallmark of cancer,the internal mechanism of cancerprogression is still unclear.Although our emphasis on the Warburg effect reflects the focus of the field,we would also like to encourage a broader approach to the study of cancer metabolism that takes into account the contributions of all interconnected small molecule pathways of the cell,which is to choose a unique microenvironment that is beneficial to the survival and reproduction of tumor cells.When the activity of PDC get decreased,the activity of PDHA1 is decreased,and the effect of pyruvate conversion to acetyl-CoA is also reduced,which would result in a large amount of lactate accumulation extracellularly.If PDHA1 activity is inhibited,or protein expression is suppressed,the activity of PDC or PDH will be suppressed,thus the Warburg effect would be positively influenced.Our previous studies on PDHA1 gene in prostate cancer and ovarian cancer showed that hypoxia can lead to the decrease of PDHA1 expression and the increase of GBP1 expression,and the enhancement of tumor cell stemness.The human guanylate-binding protein 1?GBP1?is a member of of a major class of interferon induced genes.GBPs,P47 family and the MX family,belong to the super large family of guanosine triphosphatase?GTPase?.GBPs can strengthen the host immune protein function,including phagocytic oxidase,antibacterial peptide and autophagy effect protein to eliminate intracellular pathogens.GBP1 is among the proteins the most highly induced by inflammatory factors and interferon-??IFN-??.It also endorses cells will good resistance to pathogenic microorganisms.Through the study of relationship between GBP1 and tumorprogression,there are studies indicating that GBP1 can increase the expression of epidermal growth factor?EGF?to stimulate and promote the expression of EGFR.GBP1 expression can be stimulated by P38MAPK signaling and EGFR signaling.The expression of GBP1 can increase cell proliferation,angiogenesis and decrease apoptosis.Therefore,GBP1 may be a new tumor oncogene.Previous studies have shown that tumor cells can enhance glycolysis by metabolic reprogramming,including reducing the activity of PDHA1,and promote the proliferation and metastasis of tumor cells.Therefore,the expression of PDHA1 and GBP1 may be in a certain degree of reverse correlation.The expression pattern of these factors is worthy of studying in terms of the biological behavior,invasion and metastasis of non-small cell lung cancer.Tumor cells often face hypoxic microenvironment.Hypoxia can maintain the undifferentiated state of tumor,slow down the growth of tumor and keep it in a dormancy status.In particular,low grade tumor cells can be"dedifferentiated"and obtain higher stemness.There is growing evidence pointing to the possibility that cancer is a disease associated with stem cells.The growth,invasion,metastasis and recurrence of the tumor are enhanced by the enhancement of the tumor cells stemness.The aims of this study were to explore the relationship between the expression of PDHA1/GBP1 and hypoxia in vitro,investigate their clinicopathological correlation of NSCLC in clinical samples,and explore their gene activity interactions between PDHA1 and GBP1 with siRNA technology.Our study discovered that low expression of PDHA1 and high expression of GBP1 are associated with malignant clinical pathological factors and poor prognosis in non-small cell lung cancers;Non-small cell lung cancer cells surviving from hypoxia down-regulated PDHA1 expression,but GBP1 expression was significantly up-regulated in these cells;Further gene silencing experiments with non-small cell lung cancer cell lines revealed that the gene and protein expression of GBP1 was significantly up-regulated 48 hrs after PDHA1 was successfully silenced,while no PDHA1 expression alteration was observed 48 hrs after GBP1 was successfully suppressed.In summary,PDHA1 can directly or indirectly participate in the negative feedback regulation of GBP1 expression,while the low expression of PDHA1 and the high expression of GBP1 are poor prognostic factors for non-small cell lung cancers.Part ? PDHA1 and GBP1 protein expression and its correlation with Clinicopathological characteristics and prognosis in NSCLCObjectiveTo explore the clinical significance of PDHA1 and GBP1 protein expression and its relationship with the prognosis in non-small cell lung cancer.Methods1.An analysis of 102 cases non-small cell lung cancer patients from January2011 to December 2013 in The Department of Pathology,The First Affiliated Hospital of Zhengzhou University was carried out retrospectively.All cases were confirmed by pathological examination of surgical operation.2.Immunohistochemical method was used to detect the expression of PDHA1and GBP1 proteins in cancer tissues and adjacent tissues.3.The relationship between the expression of PDHA1 and GBP1 protein and its clinicopathological features was analyzed,and was also statistically analysed.4.The influence of expression of PDHA1 and GBP1 protein,the degree of differentiation,the lymph nodemetastasis,and the TNM staging on the total survival time was determined.Results1.The positive expression rate of PDHA1 protein in non-small cell lung cancer was 39.2%?40/102?,and the positive expression rate in the corresponding adjacent tissues was 82.4%?84/102?.The positive expression rate of PDHA1 protein in cancer tissues was significantly lower than that of adjacent tissue in non-small cell lung cancer??²=39.813,P<0.05?.The positive expression rate of GBP1 protein in non-small cell lung cancer was 65.7%?67/102?,and the positive expression rate in the corresponding adjacent tissues was 33.3%?34/102?.The positive expression rate of GBP1 protein in cancer tissues was significantly higher than that of adjacent tissue in non-small cell lung cance??²=21.355,P<0.05?.2.In non-small cell lung cancer,the expression of PDHA1 protein and GBP1protein is not related to gender,smoking history and pathological type,but is related to histological differentiation,lymph node metastasis and TNM stage.There was a negative correlation between the expression of PDHA1 and GBP1 protein.3.The expression of PDHA1 and GBP1 protein,the degree of tissue differentiation,lymph node metastasis and TNM staging were all related to the prognosis of non small cell lung cancer?P<0.05?.ConclusionAlong with the progress of non-small cell lung cancer,the expression of PDHA1protein is decreased,and the expression of GBP1 protein is increased.Both of them can affect prognosis and can be used as a potential index to predict prognosis.Part ? Detection and clinical significance of the PDHA1 and GBP1expression related to mRNA and protein level in non-small cell lung cancer:an prospective studyObjectiveStudy on the expression level and clinical significance of PDHA1 and GBP1mRNA and protein in fresh non small cell lung cancer tissues.Methods1.20 paired lung cancer and adjacent tissues were collected.The expression level of PDHA1 and GBP1 gene mRNA in non-small cell lung cancer and its adjacent tissues were detected by Realtime RT-PCR,and the correlation between the two mRNA levels was analyzed.2.The expression of PDHA1 and GBP1 protein in cancer tissues and adjacent tissue was detected by Western bloting in non-small cell lung cancer.Results1.In paired lung cancer specimens,the expression of PDHA1mRNA in the early stage was lower than that in the adjacent tissues,and the expression of advanced cancer was even further lower?P<0.05?.The expression of GBP1mRNA in early cancer was increased and the expression in advanced cancers was even further increased?P<0.05?.There was a significantly negative correlation between the PDHA1 and GBP1 mRNA?P<0.05?.2.There were similar significant differences in the expression of PDHA1 and GBP1 proteins in different TNM stages of the non-small cell lung cancer tissues and adjacent tissues?P<0.05?.ConclusionLow expression of PDHA1 and high expression of GBP1 are related to the occurrence and development of non-small cell lung cancer.It can be used as an important reference index for judging the biological behavior of non-small cell lung cancer.Part ? The effect of hypoxia on the expression of PDHA1 and GBP1 in non-small cell lung cancer cell linesObjectiveTo explore the correlation between hypoxia,PDHA1 and GBP1 expression,and to analyse their effect on malignant behaviors in vitro.Methods1.Lung squamous cell carcinoma cell line LC-42 and lung adenocarcinoma cell line SELS were cultured under normal oxygen?20%O₂?and hypoxia?1%O₂?respectively.2.The morphological observation,growth curve and DT?doubling time?of the two lung cancer cell lines were made when the cells were cultured in 1%O₂,and the cells cultured in 20%O2 as controls.3.Western blot was used to detect the expression of PDHA1 and GBP1 proteins in the cell lines cultured in different oxygen concentrations.4.Transwell invasiveness test in vitro was used to detect the invasiveness of the cell lines either cultivated in 20%O₂or pretreated in 1%O₂.5.Colony formation ability of the cells cultured in 20%O₂ or pretreated in 1%O2 was detected by the conventional colony formation test.Results1.Under 1%O₂ cultivation,the cell growth was slower and the DT?doubling time?was prolonged.The differences in DT of two lung cancer cell lines in group20%O₂ and 1%O₂ were statistically significant?P<0.05?.2.With the decrease of oxygen in the environment,the expression of PDHA1protein decreased and the expression of GBP1 protein increased.There were significant differences in the expression of PDHA1 and GBP1 proteins in two lung cancer cell lines under 20%O₂ compared to the expression in the cells under 1%O₂?P<0.05?.3.The results of Transwell cell invasion test showed that the number of lung squamous cell carcinoma cell line LC-42 cells penetrating membrane in the 1%O₂group was?3014.6±521.1?,and the number of the same penetrating membrane cells in the 20%O₂ group was?1837.2±796.4?,and the difference between the two groups was statistically significant?P<0.05?.The number of the lung adenocarcinoma cell line SELS cells penetrating membrane in 1%O2 group was?3812.2±809.9?,and the number of the same cell line penetrating membrane in 20%O₂ group was?1501.7±378.2?.The difference was statistically significant?P<0.05?.4.The colony formation rate of lung squamous cell carcinoma cell line LC-42was 15.6±4.2%in the 20%O₂group,and the colony formation rate was 32.9±6.9%in the 1%O₂ group for the same cell line,showing that the difference was statistically significant?P<0.05?.The colony formation rate of the lung adenocarcinoma cell line SELS was 18.1±5.6%in the 20%O₂group,and the colony formation rate was 36.4±5.6%in 1%O₂group for the same cell line.The difference was statistically significant?P<0.05?.Conclusion1.Hypoxia leads to the decrease of PDHA1 protein expression and increase of GBP1 protein expression in lung squamous cell carcinoma and adenocarcinoma cells in vitro.2.Hypoxia,downregulation of PDHA1 and upregulation of GBP1 protein expression together result in rather aggressive features of lung squamous cell carcinoma and adenocarcinoma cells in vitro.Part ? Interactions between PDHA1 and GBP1 gene expressions in non-small lung cancer cellsObjective:To explore their genes activity relationship with siRNA technology by firstly silencing the expression of PDHA1 gene and investigating the gene and protein expression of GBP1,and then silencing the expression of GBP1 gene and investigating the gene and protein expression of PDHA1.Methods1.Cell preparation.Non-small cell lines LC-42 and SELS were cultivated with6-wells plate at 37??5%CO₂?saturation wetness with RPMI 1640 containing10%FBS for 24hrs,so that the cells reached 70%confluence.2.Gene silencing was performed with corresponding siRNA reagents from Sant Cruz Biotechnology.3.Cells were collected 48 hrs after successfully tranfered with the reagents.4.RT-PCR analyses of GBP1 and PDHA1 RNA expression of the experimental and control cells.5.Immunocytochemical analyses of GBP1 and PDHA1 protein expression of the experimental and control cells.6.Western blot analyses of GBP1 and PDHA1 protein expression of the experimental and control cells.Results1.When PDHA1 gene was susccessfully silenced with the siRNA technology,GBP1 expression in both RNA and protein levels was significantly upregulated.2.However,when GBP1gene was successfully silenced with the siRNA technology,there was no apparent PDHA1 expression change,neither in RNA nor in protein levels.3.Both the squamous cell line LC-42 and the adenocarcinoma cell line SELS showed the same alterations.Conclusions1.GBP1is the down-stream gene of PDHA1.2.PDHA1 directly or indirectly participates in the negative regulation of GBP1expresssion.