Global And Quantitative Evaluation Towards Expression Status Of Aldo-keto Reductase Genes In Liver Cancer

The aldo-keto reductase (AKR) superfamily widely exists in prokaryotes and eukaryotes, including 15 human AKRs in which 10 members have enzyme activities. There are several reports that documented AKRs were likely closely correlated with tumorgenesis and tumor progress. AKRs are proposed, therefore, as the cancer biomarkers or the targets of anti-cancer drugs. Whether AKRs are distributed in cancer tissues and cells and how AKRs are involved in pathological process, nevertheless, are still in argument probably due to divergent detection techniques or varied sample sources. Accurate quantification towards the AKR gene expression status in tissues and cells is a key issue to address the conflict views. In this study, we selected hepatocellular carcinoma (HCC) as a subject because HCC was found AKR-associated but its AKR distribution was controversially reported; while we tried to globally measure the AKR gene expression status in the HCC tissues and cell lines using multiple approaches.Four hepatocellular carcinoma cell lines (HepG2, HuH7, BEL7402 and SMMC7721) and one relatively normal hepatic cell line (L-02) as a reference were selected for this study. Total of 55 pairs of hepatic tissues (tumor tissues and the adjacent tissues) were collected from the hospitals. We designed and synthesized 9 pairs of PCR primers for AKRs, and applied them to assess the mRNA abundances in HCC cell lines by Real time PCR. We generated 14 recombinant AKRs and took them as the antigens for generation of the corresponding antibodies. We also achieved 9 specific antibodies of AKRs which are qualified for immuno-based assays. With the AKR antibodies in Western Blot or IHC, we semi-quantified the AKR protein abundances in the HCC cell lines and tissues. We designed and synthesized the unique AKR peptides, and employed MRM/mTRAQ to measure absolute abundances of AKR in the HCC cell line.The results of Real time PCR revealed that this technique can distinguish and quantitate most of the human AKRs; the mRNA level of AKR1C1/1C2 and AKR1C4 can divide the 5 hepatic cell lines into relative normal and cancer groups; most of the human AKRs mRNA abundances were increased significantly in two higher differentiated HCC cell lines, especially HuH7, this suggested that human AKRs probably related with differentiated status of cancer; compared with L-02, the mRNA abundances of AKR subfamily members weren’t complementary with each other.Based upon the semi-quantification with Western blot towards the AKRs protein abundances in cell lines, the protein abundances of most of human AKRs were kept consistent level as compared with L-02 in the 5 hepatic cell lines; the protein profile of HuH7 was obviously different with the other four cell lines; AKR1B1 can distinguish relative normal and cancer cell lines in protein level. Quantification analysis by MRM/mTRAQ showed that this technique can distinguish and quantitate most of the human AKRs in protein level; the results of MRM were consistent with Western blot; only the protein abundancers of AKR1B10 and AKR1C3 matched their mRNA level.The distribution of AKRs in hepatic tissues was evaluated by IHC. The IHC images from 55 pairs of tissues presented that AKR1A1 was positive in 32.7% tumor tissues and in 58.2% adjacent parts, while AKR1B10 were positive in 67.3% tumor tissues and in 41.8% adjacent parts. Statistical analysis further suggested the significant differences of AKR1A1 and AKR1B10 between tumor and the correspondent adjacent regions. Moreover, the up-regulation of AKR1B10 was likely tightly associated with the differentiation states of HCC. IHC data also offered the data indicating the other AKRs without any significant difference between the two tissues regions.For the first time we systematically evaluated the expression status of AKRs in HCC cell lines and tissues. We paid a great attention to clarify the relationship of AKRs with tumorgenesis and progression. Our results demonstrated that none of the AKRs was a specific biomarker for HCC cell lines either at mRNA level or at protein level, while the overall expression status of AKRs was independent from HCC cell lines and tissues. Importantly, some AKRs indeed showed the strong correlations with individual HCC cell lines. We speculate on which the correlation is resulted from cytological rather than tumorgenetic features.