Expression Of TKTL1 In Esophageal Squamous Cell Carcinoma And Its Implication In The Regulation Of Proliferation,Cell Cycle,Apoptosis And Invasion Of Esophageal Squamous Cell Carcinoma

Esophageal cancer(EC)accounted for sixth of global cancer related death,has a wide geographical distribution,the highest incidence of Chinese distribution in the northern and central regions,especially Henan and Shanxi.At present,esophageal cancer including two main types of tissue,squamous cell carcinoma(esophageal squamous cell instant tube carcinoma,ESCC)and esophageal adenocarcinoma(esophageal,adenocarcinoma,EA).More than 90% of esophageal squamous cell carcinoma distributed in East Asian countries.Although tremendous advancesin diagnosis and treatment of EC,a 5-year survival rateof the patients with EC ranges from 15 to 25 %.Most notably,50–60 % of patients with EC display advanced ormetastatic phase at the time of diagnosis.Therefore,it isdramatically imperative to seek novel molecular targets anddevelop effective targeted therapeutic strategies for improvementof survival outcomes of the patients with EC.Re-programming of metabolic pathways is an essential hallmark of physiological changes in tumor cells,in which pentose phosphate pathway(PPP)exerts important roles in the lipogenesis and nucleotide synthesis,which has been demonstrated to contribute to tumor development and progression of several tumors.TKTL1,a kind of crucial metabolism enzyme,participates in the nonoxidative pathway of PPP,implying its tight correlation with the occurrence and developmentof a variety of tumors.Currently,three human transketolase genes including TKT,TKTL1,and TKTL2 have been recognized in the human genome,but TKTL1 was the only one upregulated in tumors.Stepwise investigation revealed that elevated TKTL1 expression was tightly correlated with the occurrence,development,and progression of a large number of tumors,including bladder cancer,lung cancer,endometrial cancer,and uterine cervix cancer.These findings shed light on the pivotal role of TKTL1 in these tumors thereby may be a novel molecular target for the therapy of many tumors.However,to date,correlations of TKTL1 with the development and progression of ESCC remain unknown.Therefore,the purpose of the current study was to investigate the expression of TKTL1 and preliminarily elucidate its underlying biological functions in ESCC,which will provide novel molecular target for therapy of the patients with ESCC.Part I Expression of TKTL1 in ESCC and its association with prognosis of the patients with ESCCMethods(1)Immunohistochemistry was employed to detect the TKTL1 protein expression in 139 cases of ESCC tissues and paired normal esophageal epithelial tissues.(2)Real-time qPCR and Western blot methods were utilized to investigate the expressions of TKTL1 mRNA and protein in randomly selected 8 cases of ESCC tissues and corresponding normal esophageal epithelial tissues.(3)Semi-quantitative RT-PCR was used to detect the relative level of TKTL1 mRNA in ESCC patients with different clinic staging and various metastatic status.(4)SPSS 17.0 statistical software was used to investigate the associations of TKTL1 protein expression with clinicopathological features of ESCC patients.(5)Kaplan-Meier survival curve was utilized to verify the correlations of TKTL1 protein expression with the prognosis of the patients with ESCC.(6)Statistical assay: All data expressing as (x|-) ±s were investigated by SPSS17.0 statistical software.The result of immunohistochemistry was determined by chi-square,the results of Western blot,real-time qPCR and semi-quantitative RT-PCR were performed using One-way ANOVA,and the associations of TKTL1 protein expression with the prognosis of the patients with ESCC were investigated by Kaplan-Meier survival curve.P < 0.05 indicates significant difference.Results(1)Positive expression ratio of TKTL1 protein in ESCC tissues was 64.0%(89/139),which was significantly higher than that in paired normal esophageal epithelial tissues(11.5%,16/139),and there was statistical difference(X2=81.556,P=0.000).(2)Expressions of TKTL1 mRNA and protein in randomly selected 8 cases of ESCC tissues were markedly higher than those in corresponding normal esophageal epithelial tissues,and there were statistical differences(P<0.05).(3)TKTL1 mRNA in ESCC patients with I and II staging(0.496±0.034)was obviously lower than that in ESCC patients with III and IV staging(0.940±0.048),and there was statistical difference(P<0.01).(4)TKTL1 mRNA in ESCC patients with lymph node metastasis(1.015±0.056)was evidently higher than that in ESCC patients without lymph node metastasis(0.557±0.033),and there was statistical difference(P<0.01).(5)TKTL1 expression wasn’t associated with the patients’ gender and age(both P>0.05),but tightly correlated with histological differentiation,clinical staging and lymph node metastasis(all P=0.000).(6)The result of Log-rank(Mantel-Cox)demonstrated that the survival time of the patients with negative TKTL1 expression was significantly higher than that with positive TKTL1 expression(P<0.01).Part II Effects of TKTL1 downregulation on cell proliferation,cell cycle,apoptosis and invasion ability of ESCC cells and its related molecular mechanismsMethods(1)Western blot was used to detect the TKTL1 expression in several ESCC cell lines(Eca109,EC1 and EC9706)and normal esophageal epithelial cell Het-1A.(2)TKTL1 siRNA was utilized to transfect EC1 cells,and cells were harvested at 48 h.Subsequently,total proteins were extracted and Western blot was used to check TKTL1 protein expression at 48 h after transfection with TKTL1 siRNA.(3)Expressions of TKTL1,TKT and TKTL2 mRNA were detected in different treatment groups using Real-time qPCR method.(4)The enzyme-linking method was used to detect the transketolase activities in different treatment groups.(5)CCK-8 regeant was utilized to investigate the cell proliferation in different treatment groups.(6)Flow cytometry was employed to investigate cell cycle distribution and cell apoptosis in different treatment groups.(7)Boydem chamber was used to detect the changes of cell invasion ability in different treatment groups.(8)Caspase-3 activity detection kit was used to investigate the activity of caspase-3 in different treatment groups.(9)Western blot was used to analyze the expressions of cyclin D1,cdk4,Bcl-2,Bax,MMP-2 and MMP-9 proteins in different treatment groups.(10)Statistical assay: All data expressing as (x|-) ±s were investigated by SPSS17.0 statistical software.Experimental data were investigated by One-way ANOVA,and P < 0.05 indicates significant difference.Results(1)The expression of TKTL1 protein in ESCC cells were significantly higher than that in normal esophageal epithelial cell Het-1A,and there was significant difference(P<0.05).Besides,the expression of TKTL1 protein in EC1 cells was markedly higher than that in Eca109 and EC9706,and there was significant difference(P<0.05).(2)Compared with untreated group and control siRNA transfection group,expression of TKTL1 protein was significantly downregulated in TKTL1 siRNA group,and there was statistical difference(P<0.05).(3)Compared with untreated group and control siRNA transfection group,expression of TKTL1 mRNA was significantly decreased in TKTL1 siRNA group(P<0.05),whereas the expressions of TKT and TKTL2 mRNA didn’t change among three groups(P>0.05).(4)Compared with blank control group and control siRNA transfection group,thetransketolase activities was markedly reduced in TKTL1 siRNA group,and there was statistical difference(P<0.05).(5)Compared with blank control group and control siRNA transfection group,theproliferation of EC1 cells were significantly suppressed in TKTL1 siRNA group at24 h,48h,72 h and 96 h after transfection with TKTL1 siRNA,and there was statistical difference(P<0.05).(6)Compared with untreated group(52.57 ± 1.22%)and control siRNA transfection group(52.97±2.49%),the percentage of cell number in G0/G1 phase was obviously increased in TKTL1 siRNA group(63.83 ± 0.96%),and there was statistical difference(F=42.566,P=0.000).Conversely,the percentage of cell number in S phase in TKTL1 siRNA group(24.23±1.29%)was significantly lower that in untreated group(32.30±0.75%)and control siRNA transfection group(31.70 ±0.92%),and there was statistical difference(F=59.145,P=0.000).(7)Compared with untreated group(7.39±0.68%)and control siRNA transfection group(7.67±0.67%),the early apoptotic ratio was obviously increased in TKTL1 siRNA group(17.65±1.41%)(F=106.897,P=0.000).The ratio of viable cell number in TKTL1 siRNA group(67.25±1.92%)was significantly lower that in untreated group(88.22±1.39%)and control siRNA transfection group(87.33±1.31%)(F=172.627,P=0.000).(8)Compared with blank control group and control siRNA transfection group,the activity of caspase-3 were significantly elevated in TKTL1 siRNA group,and there was statistical difference(P<0.05).(9)Compared with untreated group(115.67±11.68)and control siRNA transfection group(102.33±8.08),the invading cell numbers were obviously decreased in TKTL1 siRNA group(43.67±10.02),and there was statistical difference(F=43.726,P=0.000).(10)Compared with blank control group and control siRNA transfection group,the expressions of cyclin D1,CDK4,Bcl-2,MMP-2 and MMP-9 proteins were significantly reduced in TKTL1 siRNA group,whereas the expression of Bax protein was markedly increased,and there was statistical difference(all P<0.01).Part III The effects of TKTL1 downregulation on the growth of ESCC xenografted nude mice and its preliminary mechanismsMethods(1)ESCC EC1 cells at a density of 5×106was subcutaneously injected into nude mice,when tumor volume reached around 100mm3,nude mice were divided into three group,including PBS treatment group,control siRNA treatment group and TKTL1 siRNA treatment group.Tumor volume was measured twice every week,and tumor growth curve was made.Besides,tumor weight was determined after measurement termination.(2)Western blot was utilized to detect the levels of TKTL1,cyclin D1,CDK4,Bcl-2,Bax,MMP-2 and MMP-9 proteins in different treatment groups.(3)Immunohistochemistry was used to investigate the Ki-67 protein level in different treatment groups,and the changes of proliferation index was determined by counting the positive cell numbers of Ki-67 staining in randomly selected 40 fields.(4)Statistical assay: All data expressing as (x|-) ±s were investigated by SPSS17.0 statistical software.Experimental data were investigated by One-way ANOVA,and P < 0.05 indicates significant difference.Results(1)Compared with PBS treatment group and control siRNA treatment group,tumor growth was significantly suppressed in TKTL1 siRNA treatment group,and there was statistical difference(P<0.05).(2)Tumor weight in TKTL1 siRNA treatment group(0.801 ± 0.231)was markedly lower than that in PBS treatment group(2.316±0.264)and control siRNA treatment(2.201±0.209),and there was statistical difference(F=63.915,P=0.000).(3)The proliferation index in TKTL1 siRNA treatment group was 33.35±2.14%,which was significantly lower than that in PBS treatment group(76.44±5.62%)and control siRNA treatment group(74.46±3.76%),and there was statistical difference(F=105.729,P=0.000).(4)Compared with PBS treatment group and control siRNA treatment group,the expressions of TKTL1,cyclin D1,CDK4,Bcl-2,MMP-2 and MMP-9 proteins were evidently upregulated in TKTL1 siRNA treatment group,whereas Bax protein expression was dramatically upregulated,and there was statistical difference(P<0.05).Conclusions(1)High TKTL1 level may be tightly associated with the occurrence,development,progression and prognosis of ESCC.(2)TKTL1 downregulation mediated the decrease of transketolase activity may trigger the alterations of PPP function in ESCC.(3)The proliferation inhibition,the alteration of cell cycle distribution,cell apoptosis and the decrease of cell invasion evoked by TKTL1 downregulation may be closely correlated with the alterations of cyclin D1,CDK4,Bcl-2,Bax,MMP-2 and MMP-9 protein expressions.(4)The growth suppression of xenografted tumors of ESCC mediated by TKTL1 siRNA may be tightly associated with the changes of TKTL1,cyclin D1,CDK4,Bcl-2,Bax,MMP-2 and MMP-9 protein expressions.