Expression Of ABAT In Clear Cell Renal Cell Carcinoma And Its Effect On The Proliferation,Migration And Metabolism Of Renal Carcinoma Cell Lines

Objective:1.To detect the expression mRNA and protein levels of ABAT in clear cell renal cell carcinoma and normal kidney tissues,then to explore the relationship between ABAT expression and the occurrence,development and prognosis of renal cell carcinoma;2.To study the effect of expression of ABAT on the proliferation,migration and metabolism of renal carcinoma cells.Methods:1.The expression of ABAT was predicted in clear cell renal cell carcinoma by data mining.2.Using qRT-PCR to detect the expression of ABAT mRNA in 50 pairs of clear cell renal cell carcinoma and its matched adjacent normal kidney tissues.3.The expression of ABAT protein in 25 pairs of clear cell renal cell carcinoma and corresponding adjacent normal tissue were analyzed by immunohistochemistry.4.The clinical data of TCGA database were used to analyze the effect of ABAT expression on the clinical prognosis of patients with clear cell renal cell carcinoma.5.The ACHN and 786-0 cells were transfected with ABAT overexpression vector lentivirus to construct stable ABAT overexpressing ACHN and 786-0 cell lines.6.The NC and ABAT stable overexpressing of ACHN and 786-0 cell lines were identified by qRT-PCR and Western Blot.7.The effect of overexpression of ABAT on proliferation of ACHN and 786-0 cells was detected by WST-1 method.8.Plate colony formation assay was conducted to analyze the effect of ABAT overexpression on colony formation of ACHN and 786-0 cells.9.The effects of ABAT overexpression on the migration of ACHN and 786-0 cells were analyzed by Transwell assays.10.The effects of ABAT overexpression on the metabolism of ACHN and 786-0 cells were analyzed by NADP/NADPH and lactate assays.Results:1.Based on the Oncomine database,the expression level of ABAT mRNA was down-regulated in clear cell renal cell carcinoma compared with the level in the normal tissue.In the TCGA database,the ABAT gene mRNA expression levels of 72 normal kidney tissue were compared with 539 clear cell renal cell carcinoma tissue,the ABAT mRNA expression level was significantly lower in clear cell renal cell carcinoma than in normal kidney tissues(p<0.001).The results of qRT-PCR revealed that ABAT mRNA was lower in clear cell renal cell carcinoma than in adjacent normal kidney tissues(p<0.001).2.The results of immunohistochemistry showed that 16(16/25)cases ABAT proteins were highly expressed in adjacent normal kidney tissues,18(18/25)cases were low-expressed in clear cell renal cell carcinoma.ABAT protein was lower in clear cell renal cell carcinoma tissue than in normal kidney tissues(p<0.05).3.The expression of ABAT in 50 patients with clear cell renal cellcarcinoma was not related to age,gender,tumor size,clinical stage and pathological grade.4.The analysis conducted by GEPIA showed that ABAT mRNA expression levels were associated with overall survival(p<0.01)and disease-free survival(p<0.01)in patients with ccRCC.5.The result of Western Blot experiments showed that ABAT protein in human kidney cells(293T,HKC)expressing higher than renal carcinoma cell lines(A498,ACHN,786-0,CAKi-1).6.Identification of the overexpressing stability of ACHN ABAT,ACHN NC,and 786-0 ABAT,786-0 NC cell lines.The qRT-PCR results showed that ABAT mRNA levels in ABAT group were higher than in the NC group both in ACHN and 786-0.(p<0.001).Western Blot results also demonstrated that ABAT protein expression level in ABAT group was higher than that in the NC group.7.As we can see in the WST-1 assay s,the proliferation ability of ACHN and 786-0 renal carcinoma cells in ABAT group were weaker than that of NC group.In the 786-0 cell line,the OD values of NC group and ABAT group were(0.830±0.065)vs(0.576±0.019)(p<0.001)at 48h,and at 72h were(1.222±0.031)vs(0.943±0.040)(p<0.001),the differences were statistically significant.In the ACHN cell line,the OD values of NC group and ABAT group were(0.34±0.019)vs(0.257±0.018)respectively at 48h(p<0.001),(0.822±0.020)vs(0.676±0.011)at 72h(p<0.001).The results of this experiment indicated that overexpression of ABAT inhibited the proliferation of ACHN and 786-0 cells.8.The plate colony formation assay results:In the 500 cells/well cell group of 786-0 cell line,the colony numbers of cell in NC group and ABAT group were(99.67±6.11)and(78.00±6.08)respectively,the number of clones in the ABAT group was significantly lower than that in the NC group(p<0.05).And in 100 cells/well cell group of 786-0 cell line,the colony numberof cell in NC group was(41.33±3.79)and in ABAT group was(30.67±1.53),as seen in the result of 500 cells/well cell group(p<0.05).In the 500 cells/well cell group of ACHN cell line,the colony number of cell in NC group and ABAT group were(191.67±4.93)and(170.33±4.04)respectively,there was statistically significant between the number of clones in the ABAT and NC group,p<0.01;the colony numbers in NC and ABAT 100 cell/well group were(59±3.61)vs(42.67±2.08)for ACHN cell line,p<0.01.The results showed that overexpression of ABAT inhibited the ability of colony forming ability in ACHN and 786-0 cells.9.The Transwell assay:In the ACHN cells,the number of cell in the ABAT vs NC group were(33.333±4.444)vs(45.833±5.419)respectively,(p<0.001).In the 786-0 cell line,the number of cell migration in the ABAT group and the NC group were(82.667±5.508)vs(122.333±9.292)respectively,(p<0.01).The Transwell assay results show that overexpression of ABAT can attenuate the migration of ACHN and 786-0.10.NADP/NADPH assay and lactate assay:NADP/NADPH assay results:In the 786-0 cell line,NADP/NADPH was increased in the ABAT group than NC group(p<0.01).In the ACHN cell line,NADP/NADPH was also increased in the ABAT group than NC group(p<0.01).Lactate assay results:In 786-0 cells,the ABAT overexpression group had a 15.65%decrease compared with the NC group(p<0.01).In ACHN cells,ABAT overexpression was associated with a decrease of approximately 13.20%in lactate assay relative to the NC group(p<0.01).Conclusion:1.The expression of ABAT mRNA and protein in clear cell renal cell carcinoma tissues were lower than that in the normal kidney tissues,and the expression level was correlated with the prognosis of the patients.2.ABAT gene overexpression has a significant inhibitory effect on the proliferation,colony formation and migration of renal carcinoma cells,and is related to the metabolism of renal carcinoma cells.