Study On The Mechanism Of UMtCK In The Development Of Gastric Cancer

Gastric cancer(GC)is the fifth most common and third most deadly malignancy worldwide.Due to lacking of effective early diagnosis technology,most GC patients are already in advanced stage at the time of diagnosis,and their prognosis is often poor.Tumor metastasis is also considered to be the main cause of death in GC patients.Therefore,it is urgent to study the prognosis and risk predictors of recurrence and metastasis for GC patients.Our previous studies have confirmed that Ras association domain family member 6(RASSF6)acted as a tumor suppressor in GC and after upregulated the expression of RASSF6 in GC cells,we identified that ubiquitous mitochondrial creatine kinase(uMtCK)was significantly decreased in GC cells compared with control groups by using a loss of heterozygosity analysis and cDNA microarrays.In recent years,many studies have found that uMtCK has abnormally high expression in breast cancer,liver cancer,prostate cancer and other malignant tumors,which is significantly associated with poor prognosis.However,up to now,there is no relevant research report on the clinical significance of uMtCK in GC as well as its effect on the clinical prognosis of GC patients.In order to explore the expression level of uMtCK in GC and its impact on postoperative survival of GC patients,quantitative polymerase chain reaction and western blot were used to detect the differential expression of uMtCK in GC tissues and their paired adjacent non-neoplastic tissues at the mRNA level and protein level,respectively.We found that uMtCK expression levels were significantly higher in GC tissues than in paired adjacent non-neoplastic tissues,which is also consistent with results from two independent GC datasets in the Oncomine public database.To further investigate the relationship between uMtCK expression and GC clinical factors,immunohistochemical staining was used to detect uMtCK protein expression in a set of tissue microarray.We demonstrated that the abnormally high expression of uMtCK in GC tissues was significantly associated with tumor invasion and lymph node metastasis,distant metastasis,UICC stage,histological differentiation and tumor recurrence.To explore the role of uMtCK in GC patient survival,a Kaplan-Meier analysis with a log-rank test for disease-free survival and overall survival was first conducted to evaluate the predictive role of uMtCK in GC patients.The results suggested that GC patients with high uMtCK expression had worse survival prognosis than those with low uMtCK expression.Cox proportional hazard ratio regression model further verified that uMtCK expression remained an independent prognostic factor for both disease-free survival and overall survival,indicating that high uMtCK expression was an independent prognostic biomarker for GC.We further evaluated uMtCK expression in one normal gastric mucosa cell line and seven different GC cell lines,respectively.We found that the expression level of uMtCK in GC cell lines was significantly higher than that in normal gastric mucosa cells,and the expression level of uMtCK in AGS/MKN28 cells was relatively lower,while the expression level of uMtCK in SGC-7901/MGC-803 cells was relatively higher.Then,stable uMtCK overexpressing GC cell lines(AGS/LV-UMTCK)and knockdown GC cell lines(SGC-7901/SI-UMTCK-3#)were constructed by cell transfection for further study.In vitro cell function experiments,wound-healing assays and transwell assays demonstrated that uMtCK could significantly promote the invasion and metastasis ability of GC cells.In vivo,uMtCK significantly increased the number of liver metastatic lesions in nude mice,and significantly shortened the overall survival of the experimental group compared with the control group.Notably,Gene set Enrichment analysis(GSEA)based on TCGA database showed that the increased expression of uMtCK in GC cells was closely related to glycolysis/gluconeogenesis.To explore the role of uMtCK in aerobic glycolysis of GC cells,the changes of glucose consumption,adenosine triphosphate(ATP)production,lactate production and extracellular acidification rate of GC cells were detected with uMtCK overexpression or knockdown.We found that overexpression or knockdown of uMtCK in GC cells significantly increased or decreased glucose consumption and ATP and lactate production,respectively.By measuring the extracellular acidification rate(ECAR),we found that GC cells with uMtCK overexpressing or knockdown displayed an enhanced or weakened glycolysis phenotype,respectively.Subsequently,we found that the expression levels of hexokinase 2(HK2)were significantly increased or decreased after uMtCK overexpression or knockdown in GC cells.In view of this,we transfected HK2 knockdown plasmids or overexpression vectors into uMtCK overexpressing or knockdown cells,respectively,to further verify whether uMtCK promotes glycolysis in GC cells in an HK2-dependent manner.As expected,knockdown or overexpression of HK2 in uMtCK overexpressing or knockdown cells partially reversed the promoting role of uMtCK on the glycolytic in GC cells.To reveal the mechanism of uMtCK promoting the progression of GC,we implemented RNA-sequencing through stable uMtCK knockdown or scramble control cells.According to cell RNA sequencing data combined with KEGG analysis,we preliminarily identified the MAPK signaling pathway as the key pathway upon uMtCK knockdown in GC cells.The results of western blot confirmed that overexpression or knockdown of uMtCK in GC cells could significantly promote or inhibit the expression of HK2 and the phosphorylation of JNK and JUN,while the inhibitors or activators of JNK-MAPK/JUN signaling pathway could partially offset the above effects,respectively.Finally,we used the JASPAR CORE database tool to predict the potential transcriptional binding regions and specific binding sites of JUN to HK2 and found that only mutant binding site 1 could downregulate luciferase reporter gene activity of HK2 and inhibit HK2 transcriptional expression.To further investigate the impact of the JNK-MAPK/JUN pathway on the glycolysis regulated by uMtCK in GC cells,we added JNK-MAPK/JUN signaling pathway inhibitors and activators to GC cells with stable uMtCK overexpression or knockdown,respectively,and the results suggest that JNK-MAPK/JUN pathway inhibitor or activator can partially counteract the promoting effects of abnormally expression of uMtCK on glucose uptake,ATP production,lactate production and extracellular acidification rate in GC cells.To explore whether uMtCK promotes the invasion and metastasis of GC cells in a HK2-mediated manner,we transfected HK2 knockdown plasmids or overexpression vectors into uMtCK overexpressing or knockdown cells,respectively.We found that the promotion or inhibition of migration,metastasis and invasion of GC cells after overexpression or knockdown of uMtCK could be partially reversed by HK2 knockdown or overexpression,respectively.In conclusion,this study identified the abnormally high expression of uMtCK in GC patients and its correlation with poor prognosis in GC patients,suggesting that uMtCK can be used as an independent prognostic factor for disease-free survival and overall survival in GC patients after radical resection.In addition,uMtCK enhances the glycolysis of GC cells in an HK2-dependent manner and further promoted their migration,invasion,and liver metastasis by activating the JNK-MAPK/JUN axis.We conclude that uMtCK overexpression in tumor tissues of GC patients can be used as a novel molecular biomarker for poor prognosis in GC patients,especially for GC patients with advanced UICC and tumor recurrence.The regulatory effect of uMtCK on malignant progression of GC and its molecular mechanism may provide new gene targets and theoretical basis for GC besides traditional VEGF,and guide the development of new antineoplastic drugs,so as to improve the therapeutic effect and prognosis of GC patients.