Abstract:

Objective To investigate the expression of aldehyde dehydrogenase 6 family member A1 （ALDH6A1） in clear cell renal cell carcinoma （ccRCC） and its effects on the proliferation， apoptosis， and invasion of renal cancer cells. Methods The Cancer Genome Atlas （TCGA） database was used to analyze the differences in ALDH6A1 mRNA expression levels between ccRCC patients and healthy individuals， as well as the correlation between ALDH6A1 mRNA expression and survival rate of patients. A total of 70 pairs of ccRCC tissues and adjacent tissues were collected from patients who received treatment at General Hospital of the Yangtze River Shipping between January 2021 and October 2024. Reverse transcription-quantitative PCR was performed to detect ALDH6A1 mRNA expression. Immunohistochemistry was used to analyze ALDH6A1 protein expression in ccRCC tissues. The expression of ALDH6A1 protein in different clinicopathological features of ccRCC was analyzed. The renal cancer cell line 786-O was cultured in vitro and divided into blank control group （control group）， ALDH6A1 overexpression group （OE-ALDH6A1 group） and negative control group （OE-NC group）. Cells proliferation was evaluated by CCK-8 assay and EdU assay. Flow cytometry was used to detect cells apoptosis and cells cycle distribution. Transwell chamber assay was used to analyze the migration and invasion abilities of the cells. Western blotting was used to analyze the protein expressions of ALDH6A1， cyclin-dependent kinase 4 （CDK4）， Ki-67， matrix metalloproteinase （MMP）-2， MMP-9， and transcription factor Twist in different cells. Results TCGA database analysis showed that the mRNA expression of ALDH6A1 in ccRCC tissues [15 168 （20 826）] was significantly lower than that in normal tissues [130 368 （296 513）， U=8 946.5， P<0.001]. Kaplan-Meier analysis showed that the 10-year survival rate of patients with high ALDH6A1 mRNA expression （72.13%， n=421） was significantly higher than that of patients with low ALDH6A1 mRNA expression （41.32%， n=112， χ²=8.33， P<0.001）. The relative mRNA expressions of ALDH6A1 in 70 cases of ccRCC tissues and adjacent tissues were 0.58±0.13 and 1.03±0.15， respectively， with a statistically significant difference （t=48.55， P<0.001）. The positive expression rates of ALDH6A1 protein were 32.86% （23/70） and 58.57% （41/70）， respectively， with a statistically significant difference （χ²=9.33， P=0.002）. There were statistically significant differences in the expression of ALDH6A1 in ccRCC tissues among patients with different pathological grades and TNM stages （χ²=4.51， P=0.034； χ²=6.99， P=0.008）. Compared with human normal renal tubular epithelial cells HK-2， the expressions of ALDH6A1 mRNA and protein in human renal cancer cells （786-O， 769-P， ACHN， A498） were all lower （all P<0.05）. Among them， the expression of ALDH6A1 mRNA and protein was the lowest in 786-O cells （all P<0.05）. The absorbance （A） values of 786-O cells in the control group， OE-NC group and OE-ALDH6A1 group were 0.65±0.06， 0.63±0.05 and 0.38±0.04， respectively. The EdU-positive rates were （48.34±5.21）%， （49.56±5.65）% and （27.34±3.28）%， respectively. The apoptosis rates were （2.15±0.43）%， （2.32±0.55）% and （33.46±4.36）%， respectively. The proportions of cells in the G₀/G₁ phase were （22.46±3.56）%， （23.16±3.72）% and （37.82±5.42）%， respectively. The proportions of cells in the S phase were （31.25±3.78）%， （32.89±3.61）% and （26.33±3.87）%， respectively. The proportions of cells in the G₂/M phase were （46.29±5.15）%， （43.95±5.63）% and （35.85±4.21）%， respectively. The numbers of migration cells were 158.46±16.32， 155.62±15.48 and 84.23±9.65， respectively. The numbers of invasion cells were 142.35±15.21， 139.44±13.56 and 72.34±8.61， respectively， all with statistically significant differences （F=52.91， P<0.001； F=40.22， P<0.001； F=300.05， P<0.001； F=24.23， P<0.001； F=4.96， P=0.022； F=7.11， P=0.007； F=53.15， P<0.001； F=52.16， P<0.001）. Compared with the control group and OE-NC group， the A value of cells， the EdU-positive rate， the proportion of cells in the S phase and G₂/M phase， and the number of migration and invasion cells in the OE-ALDH6A1 group were significantly decreased （all P<0.05）， while the apoptosis rate and the proportion of cells in G₀/G₁ phase were significantly increased （all P<0.05）. There were statistically significant differences in the relative expressions of CDK4， Ki-67， MMP-2， MMP-9， and Twist proteins among the control group， the OE-NC group and the OE-ALDH6A1 group （all P<0.001）. Compared with the control group and OE-NC group， the protein expressions of CDK4， Ki-67， MMP-2， MMP-9 and Twist in the OE-ALDH6A1 group were significantly decreased （all P<0.05）. Conclusions ALDH6A1 expression is down-regulated in ccRCC tissues and cells. Patients with high ALDH6A1 mRNA expression have a better prognosis. Overexpression of ALDH6A1 can inhibit the proliferation， migration， and invasion of renal cancer cells， and promote cell apoptosis by regulating the cell cycle and downregulating the expression of proteins related to the epithelial-mesenchymal transition process.

Key words: Carcinoma， renal cell, Cell proliferation, Apoptosis, Neoplasm invasiveness, Aldehyde dehydrogenase 6 family member A1