AKR7A3 Inhibits Proliferation,migration And Invasion Of Gastric Cancer Cells And Its Mechanism

Objective:Gastric carcinoma(GC)is one of the common malignant tumors with high incidence rate and mortality rate.The molecular mechanism of the disease is still unclear.Aldo-keto reductase(AKR)is an NAD(P)H dependent oxidoreductase that is involved in the development of many diseases including cancer.Aldo-keto reductase family 7 member A3(AKR7A3)protein is one member of the human AKR superfamily.In previous studies,we found that AKR7A3 was significantly down regulated in gastric cancer tissues compared with normal gastric mucosa tissues.In this study,we analyzed the clinical significance of AKR7A3 in gastric cancer.We discussed the effect of AKR7A3 on the biological function and behavior of gastric cancer cells,and elucidated the molecular mechanism of AKR7A3 involved in the development of gastric cancer.It provides valuable reference and basis for exploring the molecular mechanism and screening of candidate molecules for targeted therapy of gastric cancer.Methods:1.The expression of AKR7A3 was detected by q RT-PCR,Western blot and immunohistochemistry in gastric cancer tissues and adjacent nonneoplastic tissues,and the correlation between the expression of AKR7A3 with the clinicopathologic data of gastric carcinoma was analyzed.q RT-PCR and Western Blot were used to detect the m RNA and protein expression of AKR7A3 in four different gastric cancer cell lines(AGS,SGC7901,BGC823,MKN45).2.With lipofectamine 2000,SGC7901 and AGS cells were transfected with eukaryotic expression vector pc DNA3.1-AKR7A3 and empty expression vector pc DNA3.1,and MKN45 gastric cancer cells were transfected with AKR7A3 specific sh RNA vector and negative control vector respectively.q RT-PCR and Western blot were used to detect the expression of AKR7A3 in transfected cells and their parents.The effects of AKR7A3 overexpression on SGC7901 and AGS cells proliferation,clone formation,migration and invasion were analyzed by Ed U assay,cell clone formation and transwell migration and invasion experiments.At the same time,the effects of AKR7A3 on the proliferation,clonal formation,migration and invasion of MKN45 cells were analyzed.In vivo,tumorigenicity of SGC7901 cells was detected by subcutaneous tumorigenicity assay in nude mice.3.Immunohistochemical technique was used to detect the expression of β-catenin in gastric cancer tissues and its correlation with the expression of AKR7A3.The expression of total β-catenin,nuclearβ-catenin and their downstream target genes C-myc,Cyclin D1 and Survivin were detected by q RT-PCR,Western blot and laser confocal microscopy.The Cycloheximide(CHX),a protein synthesis inhibitor,was used to block the synthesis of β-catenin and observe the change ofβ-catenin during 0-4 hours.MG132,a 26 S proteasome inhibitor,was used to block protein degradation to observe the degradation pattern ofβ-catenin.The expression of Ubiquitin ligase β-Tr CP in cells with high expression of AKR7A3 and cells with knockdown of AKR7A3 was detected by Western blot.4.The experimental results and data were analyzed by SPSS 26.0and Graph Pad prism 8.0.Results:1.AKR7A3 was down regulated in human GC tissues and cell lines.(1)The level of AKR7A3 was significantly lower in GC tumor tissuses than that in matched adjacent nonneoplastic gastric mucosa tissues,and the down-regulation of AKR7A3 expression was positively correlated with lymph node metastasis and TNM stage of gastric cancer.(2)Although AKR7A3 in MKN45 cells was significantly higher than that in SGC7901 and AGS cells,its expression in all these cells was significantly lower than that in normal gastric tissue.2.AKR7A3 inhibits proliferation,colony formation,migration and invasion of GC cells in vitro as well as tumor growth in vivo.(1)Several clones resistant to G418,SGC7901-pc DNA3.1-AKR7A3,SGC7901-pc DNA3.1,AGS-pc DNA3.1-AKR7A3,AGS-pc DNA3.1,and MKN45-Sh AKR7A3,MKN45-Sh CON were obtained.Moreover,compared with pc DNA3.1 and parental cells,the m RNA and protein level of AKR7A3 significantly increased in the pc DNA3.1-AKR7A3 cells;compared with Sh CON and parental cells,AKR7A3 m RNA and protein levels in MKN45-Sh AKR7A3-1,2 cells were decreased.(2)Exogenous overexpressing of AKR7A3 resulted in inhibition of cell proliferation,colony formation,migration and invasion in SGC7901 and AGS cells.Conversely,knockdown of AKR7A3 promotes cell proliferation,colony formation,migration and invasion in MKN45 cells.(3)Exogenous overexpressing of AKR7A3 in SGC7901 cells resulted in reduction of tumor growth in xenograft mouse model.3.AKR7A3 functions as a negative regulator to Wnt/β-catenin signaling pathway in GC cells.(1)Immunohistochemistry showed that the expression level ofβ-catenin in gastric cancer tissues was higher than that in adjacent nonneoplastic gastric mucosa tissues and the ectopic expression ofβ-catenin in cell nucleus was more likely to occur.Moreover,the ectopic expression of β-catenin was correlated with the low expression of AKR7A3.(2)The expression level of total β-catenin protein and that of the nuclear fraction as well as its target genes protein including C-myc,Cyclin D1 and Survivin in AKR7A3-overexpressing SGC7901 cells were significantly lower than those in empty vector control group and parental cells.On the contrary,compared with the negative vector group and parent cells,depletion of AKR7A3 by sh RNA in MKN45 cells resulted in up-regulation of total and nuclear β-catenin protein and the protein expression of downstream targeted genes.(3)Compared with the corresponding control cells,there was no significant change in the m RNA level of CTNNB1(the gene encodingβ-catenin)was detected in AKR7A3-knockdown MKN45 cells and AKR7A3-overexpressing SGC7901 cells,suggesting that regulation ofβ-catenin by AKR7A3 does not occur at transcriptional level.But the stability of β-catenin protein was significantly decreased in AKR7A3-overexpressing cells after blocking the syntheses of β-catenin with CHX,a protein synthetase inhibitor,and the addition of 26 S proteasome inhibitor MG132 could alleviate the down regulation of β-catenin induced by overexpression of AKR7A3,indicating that AKR7A3 regulatesβ-catenin at the protein degradation level.Furthermore,the expression of E3 ubiquitin ligase β-Tr CP increased in cells with high expression of AKR7A3,but significantly decreased in cells with knockdown of AKR7A3.These data further confirmed that AKR7A3 play a role by promoting the ubiquitin-proteasome degradation of β-catenin.Conclusions:(1)the expression of AKR7A3 in gastric cancer tissues were lower than that in the adjacent nonneoplastic tissues.The down-regulation of AKR7A3 expression was positively correlated with lymph node metastasis and TNM stage in patients with gastric cancer.(2)AKR7A3 might play a role in inhibiting proliferation,migration and invasion of gastric cancer cells.(3)AKR7A3 might promote the ubiquitination degradation ofβ-catenin.