| Objective: To study the effects of ZIP5(SLC39A5) knockdown on inhibiting esophageal cancer growth in vivo.Methods:1 To verify ZIP5 down-regulated in KYSE170 K cells, RT-PCR(reverse transcription polymerase chain reaction) was used to test the relative expression of ZIP5 mRNA in KYSE170 K and KYSE170 S cells.2 To establish xenograft murine model, KYSE 170 K and KYSE 170 S cells were inoculated into the right flank of nude mice(subcutaneous tumor model) with a high dose(5×106), and tumor volumes were measured dynamically with vernier caliper.3 FCM(flow cytometry) was used to exam the percentage of G0/G1 phases and apoptosis in xenografts.4 qRT-PCR(quantitative real-time polymerase chain reaction) was used to determine the relative expression of ZIP5, COX-2, cyclin D1 and E-cadherin mRNA in xenografts, and Immunohistochemistry and Western-blot were used to determine the levels of ZIP5, COX-2 and E-cadherin protein in xenografts.5 Inductively coupled plasma atomic emission spectrometry was used to test the level of Zn in blood and xenografts.Results: 1 The relative expression of ZIP5 mRNA in KYSE170 K was reduced by 79.01% compared with that in KYSE170 S by RT-PCR. 2 Tumor volume growth of xenografts showed that the average tumor size of KYSE170 K group which named test group(614.50 ± 87.12) mm~3 was significantly inhibited compared with KYSE170 S group which named control group(1095.43 ± 266.64) mm~3, and the average weight of xenografts in test group(0.44 ± 0.11) g was reduction compared with that in control group(0.70 ± 0.08) g at the time of sacrifice(all P values<0.05). 3 There were no significant in the percentage of cells in the G0/G1 phase between the test group(48.94% ± 4.46%) and the control group(46.3% ± 2.78%), and the number of apoptosis cells in the test group(32.50% ± 5.24%) was similar to that in the control group(37.16% ± 4.40%). 4 The relative level of ZIP5 mRNA in the test group(0.46 ± 0.33) was reduced compared to the control group(1.06 ± 0.43). The relative level of COX-2 mRNA in the test group(0.64 ± 0.12) was reduced compared to level in the control group(1.03 ± 0.25), the relative level of E-cadherin mRNA in the test group(1.68 ± 0.31) was raised compared to level in the control group(1.00 ± 0.09) by qRT-PCR(all P<0.05). However, the relative level of cyclin D1 mRNA in the test group(1.18 ± 0.94) was similar to level in the control group(1.10 ± 0.69) by qRT-PCR(P>0.05). The scores of ZIP5 protein were 2.55 ± 0.34 in the test group and 5.15 ± 1.74 in the control group. The scores of the COX2 protein in the test group were 1.43 ± 0.69 and 3.23 ± 1.29 in the control group. And the scores of the E-cadherin protein were 10.60 ± 1.33 and 6.70 ± 1.64 in the control group in vivo by immunhistochemical staining. The relative expression level of ZIP5 protein in the test group(0.38 ± 0.02) was reduced compared to level in the control group(0.81 ± 0.13), and the relative expression level of COX-2 protein in the test group(0.13 ± 0.06) was reduced compared to that in the control group(0.33 ± 0.11), the relative expression of E-cadherin protein(0.85 ± 0.05) in test group was raised compared to that in the control group(0.45 ± 0.04) by Western-blot(all P<0.05). 5 The average level of Zn in blood of test group was(2.47 ± 0.27) mg/kg,and the average level of Zn in blood of control group was(2.48 ± 0.28) mg/kg, it was no difference between the test and control groups(P>0.05). The average level of Zn in xenografts of test group was(20.30 ± 3.28) mg/kg, and the average level of Zn in xenografts of control group was(19.60 ± 3.85) mg/kg, it was no difference between the test and control groups(P >0.05).Conclusion:1 Knockdown of ZIP5 inhibits the xenograft growth.2 The mechanism of ZIP5 knockdown inhibiting the xenograft growth might be that ZIP5 knockdown could down-regulate the COX-2, and up-regulate E-cadherin in vivo.3 The levels of Zn in blood and xenograft did not alter resulting from ZIP5 knockdown. |
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