The Different Concentration Of Zinc Regulates The Expression Of Solute-linked Carrier SLC39A5(ZIP5)in Occurrence And Progression Of Esophageal Cancer

Part one The different concentration of zinc regulates the expression of solute-linked carrier SLC39A5(ZIP5)in occurrence and progression of esophageal cancer in cellularObjective:Esophageal cancer is a malignant common cancer with high incidence and mortality that affects human beings’ health.It’s reported that the lack of trace element zinc may be associated with the occurrence of esophageal cancer,the gene of SLC39A5(ZIP5)acts as a transporter of the solute-linked carrier family,it is closely related to the level of zinc in the body.In the early stage of our research,we discovered the potential relationship of ZIP5 and occurrence and development of esophageal cancer.In this research,we explore the potential role of trace element zinc and the gene of ZIP5 in esophageal cancer at cellular which may reveal the pathogenesis of esophageal cancer and provide new methods for clinical treatment and prevention.Method:1 We used the RT-PCR,Western Blot to verify the expression of ZIP5 in the cells of KYSE170S and KYSE170K.2 We used the cells of KYSE170S and KYSE170K cultured in deficient zinc,normal concentration of zinc,25umol/l of zinc to continue the subsequent experiment.2.1 We used the RT-PCR.Western Blot to explore the expression of ZIP5 in the cells of KYSE170S and KYSE170K.2.2 We used the MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium),colony-formi ng unit assays,Flow Cytometry,Transwell and scratch test to test the capacity of proliferation,migration and invasion in the cells of KYSE170S and KYSE170K cultured in different concentration of zinc.Results:1 The expression of ZIP5 reduced 37.5%and 22.0%in KYSE170K,which means the ZIP5 expression in KYSE170K is lower than in KYSE170S.2 The results of KYSE170S and KYSE170K cultured in different concentration of zinc in the subsequent experiment.2.1 The expression of ZIP5 in KYSE170S and KYSE170K cultured in different concentration of zinc.2.1.1 When cultured in deficient zinc,the results of RT-PCR showed that the expression of ZIP5 in KYSE170S and KYSE170K were 0.25±0.01,0.14±0.02,respectively,and there was a statistical difference between them.The results of Western Blot showed that the expression of ZIP5 in KYSE170S and KYSE170K were 0.68±0.01,0.50±0.02,respectively,and there was a statistical difference between them.When cultured in 25umol/l of zinc,the results of RT-PCR showed that the expression of ZIP5 in KYSE170S and KYSE170K were 0.16±0.04,0.11±0.01,respectively,and there was a statistical difference between them.The results of Western Blot showed that the expression of ZIP5 in KYSE170S and KYSE170K were 0.62±0.02,0.42±0.04,respectively,and there was a statistical difference between them.These results revealed that ZIP5 expression in KYSE170K is lower than in KYSE170S when cultured in deficient zinc and 25umol/l of zinc.2.1.2 When cultured in different concentration of zinc,the results of RT-PCR and Western Blot showed the expression of ZIP5 in KYSE170S were 0.25±0.01,0.17±0.02,0.16±0.04,respectively,there was a statistical difference between deficient zinc and normal concentration of zinc;deficient zinc and 25umol/l of zinc.When cultured in different concentration of zinc,the results of RT-PCR and Western Blot showed the expression of ZIP5 in KYSE170K were 0.14±0.02,0.10±0.01,0.11±0.01,respectively,there was a statistical difference between deficient zinc and normal concentration of zinc;deficient zinc and 25umol/l of zinc.These results revealed that ZIP5 expression became higher with the lower concentration of zinc in KYSE170S and KYSE170K.2.2 The capacity of proliferation in the cells of KYSE170S and KYSE170K cultured in different concentration of zinc.2.2.1 When cultured in normal concentration of zinc by using MTS,the value of OD in KYSE170S in Oh,24h,48h,72h,96h were 0.35±0.09,0.64±0.02,0.92±0.02,1.27±0.02,1.68+0.04,respectively;and the value of OD in KYSE170K in Oh,24h,48h,72h,96h were 0.34±0.02,0.40±0.01,0.65±0.02,0.85±0.01,1.19±0.38,respectively.When compared in same time point,there was a statistical difference of the capacity of proliferation between KYSE170S and KYSE170K.When cultured in deficient zinc by using MTS,the value of OD in KYSE170S in Oh,24h,48h,72h,96h were 0.38±0.05,0.67±0.02,0.95±0.02,1.22±0.16,1.75±0.01,respectively;and the value of OD in KYSE170K in Oh,24h,48h,72h,96h were 0.38±0.01;0.36±0.08;0.72±0.10;0.82±0.01;1.23±0.14,respectively.When compared in same time point,there was a statistical difference of the capacity of proliferation between KYSE170S and KYSE170K.These results revealed that when cultured in deficient zinc and normal concentration of zinc,the capacity of proliferation of KYSE170S was higher than that of KYSE170K.2.2.2 There was no statistical differences of the capacity of proliferation in any time point in KYSE170S or KYSE170K when cultured in deficient zinc and normal concentration of zinc.2.3 The capacity of colony-forming in the cells of KYSE170S and KYSE170K cultured in different concentration of zinc.2.3.1 When cultured in normal concentration of zinc by using colony-forming unit assays,the coloning efficiency of KYSE170S and KYSE170K were 21.5%,16.2%,respectively,and there was a statistical difference.When cultured in deficient zinc by using colony-forming unit assays,the coloning efficiency of KYSE170S and KYSE170K were 23.5%,18.1%,respectively,and there was a statistical difference.These results revealed the coloning efficiency of KYSE170S was higher than that of KYSE170K.2.3.2 There was no statistical difference of the capacity of colony-forming in KYSE170S and KYSE170K when cultured in deficient zinc and normal concentration of zinc.2.4 The ratio of G0/G1 in the cells of KYSE170S and KYSE170K cultured in different concentration of zinc.2.4.1 When cultured in normal concentration of zinc by using FCM,the ratio of G0/G1 in the cells of KYSE170S and KYSE170K were 32.98±1.02,26.24±0.65,respectively and there was a statistical difference.When cultured in deficient zinc by using FCM,ratio of G0/G1 in the cells of KYSE170S and KYSE170K were 43.56±0.69,29.94±0.57,respectively and there was a statistical difference.These results revealed the ratio of G0/G1 of KYSE170S was higher than that of KYSE170K.2.4.2 The ratio of G0/G1 in the cells of KYSE170S when cultured in normal concentration and deficient zinc were 32.98±1.02,43.56±0.69,respectively and there was a statistical difference.The ratio of G0/G1 in the cells of KYSE170K when cultured in normal concentration and deficient zinc were 26.24±0.65,29.94±0.57,respectively and there was a statistical difference.These results revealed the ratio of G0/G1 of KYSE170K became lower when concentration of zinc was higher.2.5 The capacity of migration of KYSE170S and KYSE170K when cultured in different concentration of zinc.2.5.1 When cultured in normal concentration of zinc by using Transwell,the ratio of KYSE170S and KYSE170K were 0.50±0.07,0.44±0.03,respectively and there was a statistical difference.When cultured in deficient zinc by using Transwell,the ratio of KYSE170S and KYSE170K were 0.63±0.01,0.54±0.02,respectively and there was a statistical difference.These results revealed the capacity of migration of KYSE1705 was higher than that of KYSE170K when cultured in different concentration of zinc.2.5.2 When cultured in normal concentration and deficient zinc by using Transwell,the ratio of KYSE170S were 0.50±0.07,0.63±0.01,respectively and there was a statistical difference.When cultured in normal concentration and deficient zinc by using Transwell,the ratio of KYSE170K were 0.44±0.03,0.54±0.02,respectively and there was a statistical difference.These results revealed the capacity of migration of KYSE170S and KYSE170K became lower when concentration of zinc was higher.2.6 The capacity of invasion of KYSE170S and KYSE170K when cultured in different concentration of zinc.2.6.1 When cultured in normal concentration of zinc by using scratch test,ratio of the area of KYSE170S and KYSE170K became into the original 0.43±0.06,0.72±0.14,respectively and there was astatistical difference.When cultured in deficient zinc by using scratch test,ratio of the area of KYSE170S and KYSE170K became into the original 0.38±0.09,0.49±0.09,respectively and there was a statistical difference.These results revealed the capacity of invasion of KYSE170S was higher than that of KYSE170K.2.6.2 When cultured in normal concentration and deficient zinc by using scratch test,ratio of the area of KYSE170S became into the original 0.43±0.06,0.35±0.09,respectively and there was a statistical difference.When cultured in normal concentration and deficient zinc by using scratch test,ratio of the area of KYSE170K became into the original 0.72±0.14,0.49±0.09,respectively and there was a statistical difference.These results revealed the capacity of invasion of KYSE170S and KYSE170K became lower when concentration of zinc was higher.Part two The different concentration of zinc regulates the expression of solute-linked carrier SLC39A5(ZIP5)in occurrence andprogression of esophageal cancer on whole animal levelObjective:Esophageal cancer is a malignant common cancer around the world with a top ranks.It’s reported that the lack of trace element zinc may be associated with the occurrence of esophageal cancer,the gene of SLC39A5(ZIP5)acts as a transporter of the solute-linked carrier family,it is closely related to the level of zinc in the body.In the early stage of our research,we discovered the potential relationship of ZIP5 and occurrence and development of esophageal cancer.In this research,we explore the potential role of trace element zinc and the gene of ZIP5 in esophageal cancer in vivo which may reveal the pathogenesis of esophageal cancer and provide new methods for clinical treatment and prevention.Method:1 We established animal models of esophageal cancer by 4-Nitroquinoline 1-oxide(4NQO)using wild type(WT)mice and ZIP5 knock genotype(KO)mice fed with Deficiency of zinc,Normal zinc,Supplement of zinc.2 We continued the subsequent experiment during and after the establishment.2.1 We used HE staining to detect the rate of tumor formation of two experimental mice fed with different concentration of zinc.2.2 We used the inductively coupled plasma mass spectrometer todetect the level of zinc in hair and blood of two experimental mice fed with different concentration of zinc.2.3 We used the RT-PCR to explore the expression of COX-2,STAT3 and E-cadherin in two experimental mice fed with normal concentration of zinc.Results:1 We established animal models of esophageal cancer.1.1 We established animal models of esophageal cancer by 4-Nitroquinoline 1-oxide(4NQO),the rate of tumor formation of WT mice and KO mice fed with normal concentration of zinc were 45.1%,9.5%,respectively and there was a statistical difference.The rate of tumor formation of WT mice and KO mice fed with supplement of zinc were 59.2%,26.9%,respectively and there was a statistical difference.The rate of tumor formation of WT mice and KO mice fed with deficiency of zinc were 90.6%,46.2%,respectively and there was a statistical difference.These results revealed that the rate of tumor formation of WT mice was higher than KO mice.1.2 There was a statistical difference among the rate of tumor formation of WT mice fed with different concentration of zinc,which is consistent with the results of KO mice.These results revealed that the rate of tumor formation was higher when fed with lower concentration of zinc.2 The results of zinc detection of hair and blood of two experimental mice fed with different concentration of zinc.2.1.1 The level of zinc in hair of WT mice and KO mice were 236.12±39.08,183.99±41.73,respectively and there was no statistical difference when fed with normal concentration of zinc in 12 days.The level of zinc in hair of WT mice and KO mice were 217.38±34.38,131.26±20.62,respectively and there was no statistical difference when fed with deficiency of zinc in 12 days.These results revealed that the level of zinc in hair of WT mice and KO mice was no difference.2.1.2 There was no statistical difference of the level of zinc in hair,blood and liver of WT mice and KO mice when fed with different concentration of zinc in 12 days.2.2.1 The level of zinc in hair of WT mice and KO mice were 144.07±0.58,129.22±5.61,respectively and there was no statistical difference when fed with normal concentration of zinc in 28 weeks.The level of zinc in hair of WT mice and KO mice were 123.76±2.65,107.19±5.26,respectively and there was a statistical difference when fed with deficiency of zinc in 28 weeks.These results revealed that the level of zinc in hair of WT mice was higher than that in KO mice when fed with deficiency of zinc.2.2.2 There was a statistical difference of the level of zinc in hair of WT mice when fed with different concentration of zinc in 28 weeks,which is consistent with the results of KO mice.3 The expression of COX-2,STAT3 and E-cadherin in WT mice and KO mice esophageal cancer.The expression of COX-2 in WT mice and KO mice esophageal cancer were 0.62±0.17,0.19±0.14,respectively and there was a statistical difference by using RT-PCR.The expression of STAT3 in WT mice and KO mice esophageal cancer were 0.20±0.01,0.11±0.04,respectively and there was a statistical difference by using RT-PCR.The expression of E-cadherin in WT mice and KO mice esophageal cancer were 0.04±0.02,0.50±0.28,respectively and there was a statistical difference by using RT-PCR.The expression of COX-2 in WT mice and KO mice esophageal cancer were 9.30±0.29,4.02±0.30,respectively and there was a statistical difference by using immunohistochemistry.The expression of STAT3 in WT mice and KO mice esophageal cancer were 6.35±0.29,1.17±0.24,respectively and there was a statistical difference by using immunohistochemistry.The expression of E-cadherin in WT mice and KO mice esophageal cancer were 2.35±0.29,6.27±0.38,respectively and there was a statistical difference by using immunohistochemistry.These results revealed that the expression of COX-2 and STAT3 was upregulated and the expression of E-cadherin was downregulated in WT mice.Conclusions:1 The capacity of proliferation,migration and invasion of esophageal cancer cells will be stronger when the concentration of zinc is lower;and the low expression of ZIP5 will result in weaker capacity of proliferation,migration and invasion of esophageal cancer cells when the concentration of zinc is stable.2 The rate of tumor formation will be higher when the concentration of zinc is lower in vivo;and the rate of tumor formation in KO mice is lower than in WT mice when the concentration of zinc is stable.3 The low expression of ZIP5 will restrain the expression of COX-2 and STAT3,and promote the expression of E-cadherin and then facilitate the occurrence and progression of esophageal cancer.