Study On The Mechanism Of PKM2 Involved In The Malignant Phenotype Of Esophageal Squamous Cell Carcinoma

Objective: To explore the expression of Pyruvate Kinase M2(PKM2)in esophageal squamous cell carcinoma(ESCC)and the relationship between PKM2 and clinicopathological parameters of ESCC patients.Meanwhile,to verify the effect of PKM2 on the malignant phenotype of ESCC cells.Furthermore,to discuss the regulation pathway which PKM2 involved in ESCC.Methods:(1)PKM2 expression was examined in ESCC and paired normal adjacent tissues(NAT)by Immunohistochemistry(IHC).The relationship between PKM2 and clinicopathological parameters(age,sex,infiltration depth,lymph node metastasis and prognosis)were analyzed concomitantly.PKM2 expression was comfirmed in serum samples of ESCC and normal controls based on ELISA.(2)The basic expression of PKM2 was detected in ESCC cell lines by Quantitative Real Time PCR(qRT-PCR).Then Eca109 cells were transfected with three kinds of siRNA.After transfection,the mRNA and protein expression levels of PKM2 were analysed with qRT-PCR and Westem blot.Cell proliferation,cell cycle,apoptosis,migration and invasion ability were dected by MTT,Flow cytometry(FCM),Wound healing and Transwell assays.(3)Specific lentiviral with PKM2 knockdown or overexpression in Eca109 cells were constructed.To verify the effect of PKM2 on proliferation,migration and invasion of ESCC cells,MTT,Wound healing and Transwell assays were performed after lentivirus transfection.(4)4 to 5-week-old BALB/c female nude mice were randomized into five groups.PKM2 knockdown and overexpression Eca109 cell lines were used to constructed xenograft model.2×107 cells/0.2m L/mouse was injected to the subcutaneous part of right hind limb of nude mice.Tumor volumes and body weights were measured every week.The longest diameter(A)and shortest diameter(B)were measured by vernier caliper.Tumor volumes were calculated with the using of the equation: volume=(longest diameter×shortest diameter2)/2.Paraffin embedded tissuesamples were fixed and sectioned for HE staining.The expression of PKM2 in mice tumor were detected by IHC.(5)To investigate the mechanism of PKM2 regulating the malignant phenotype of ESCC,PKM2 siRNA was transfected into Eca109 and KYSE150 cell lines.Eca109 cells and KYSE150 cells were cultured as control groups.RNA was extracted 48 h after transfection.The regulation pathway changes were examined by mRNA microarray after PKM2 siRNA transfection.Results:(1)Expression of PKM2 was significantly higher in ESCC samples(69/75,92.0%)compared with NAT(9/75,12.0%,P<0.05).Kaplan-Meier analysis showed a negative correlation trend between PKM2 expression level and poor prognosis of ESCC.Meanwhile,expression of PKM2 was significantly higher in Kazakh's ESCC samples(37/54,68.5%)compared with NAT(8/54,14.8%,P<0.05).Kaplan-Meier analysis showed that patients of Kazakh's ESCC with high level of PKM2 expression has a poor prognosis(P<0.05).(2)PKM2 expressed in all ESCC cell lines(Eca109,EC9706,KYSE30,KYSE150,KYSE450,and T4),KYSE450 showed the highest PKM2 expression level,KYSE30 showed the lowest PKM2 expression level.Both mRNA and protein levels of PKM2 were down regulated after PKM2 siRNA transfected into Eca109(P<0.05).Knockdown of PKM2 could suppress the proliferation,migration and invasion,increase apoptosis,and influence cell cycle at G0/G1 phase of ESCC cell.(3)In order to verify the effect of PKM2 expression on the function of ESCC cells,specific lentiviral with PKM2 knockdown or overexpression in Eca109 cells were constructed.Cells were divided into five groups: PKM2-siRNA group(transfected with PKM2-siRNA lentivirus),siRNA-scramble group(transfected with scramble sequence lentivirus),PKM2 over expression group(transfected with PKM2 overexpression lentivirus),PKM2-scramble group(transfected with scramble lentivirus),negative control group(normal Eca109 cells).Cell proliferation was significantly inhibited after transfected with PKM2-siRNA.Overexpression of PKM2 promoted the proliferation of Eca109 cells.Wound healing assay showed that the relative migration distance was decreased in PKM2 knockdown Eca109,and increased in cells with PKM2 overexpression.Transwell assay showed that the number of invasive cells were reduced after PKM2 knockdown and increased after PKM2 overexpression.(4)The subcutaneous xenografts of human ESCC in nude mice were formed 1 week later after injection.Expression of PKM2 was lower in the group of injected with PKM2-siRNA interference cell,while expression of PKM2 was higher in the group of injected with PKM2 overexpression cell.Moreover,tumor volume was larger in PKM2 overexpression group than that of the control group(P<0.05).These results suggested that the expression of PKM2 in vivo may promote the proliferation of ESCC cells.(5)The screening standards of mRNA microarray was FC(abs)more than 2 folds.According to the standard,4592 genes up-regulated and 2497 genes down regulated were found in Eca109 cells after knockdown of PKM2;1796 genes up-regulated and 1936 genes down regulated were found in KYSE150 cells after knockdown of PKM2.There were 298 genes up regulated,and 277 genes down regulated both in Eca109 cells and KYSE150 cells after transfected with siRNA-PKM2.Furthermore,there were 16 genes up regulated with a difference of more than 5 folds,and 8 genes down regulated with a difference of more than 4 folds both in these two cell lines.Meanwhile,affected cell function and signaling pathways include lysosome,p53 signaling pathway,Protein digestion and Absorption,Steroid hormone biosynthesis,Complement and Coagulation cascades and Chemical carcinogenesis were found in Eca109 cells,and Ribosome biogenesis in eukaryotes,N-Glycan biosynthesis,Apoptosis,TNF signaling pathway,Notch signaling pathway,DNA replication,Transcriptional misregulation,and Cell cycle were found in KYSE150 cells.Conclutions: The expression of PKM2 was high in ESCC,high expression of PKM2 in Kazakh's esophageal cancer had poor prognosis.PKM2 can promote the proliferation,migration and invasion of ESCC cells,inhibit cell apoptosis,and affect the cell cycle distribution.The effect of PKM2 on the malignant phenotype of esophageal cancer cells may be influenced by multiple factors,such as p53 signaling pathway,TNF signaling pathway and Notch signaling pathway.