Comparison of Pre- and Post-translational Expressions of COXIV-1 and MT-ATPase 6 Genes in Colorectal Adenoma-Carcinoma Tissues

Abstract

Objective: Colorectal cancer (CRC) develops from precancerous adenomatous polyps to malignant lesions of adenocarcinoma. Elucidating inhibition mechanisms for this route in patients with a risk of developing CRC is highly important for a potential diagnostic or prognostic marker. Differential expression of nuclear-encoded cytochrome c oxidase subunit 4 (COXIV) seems to contribute to a more unregulated respiration due to loss of ATP inhibition. Majority of energy for tumor transformations are mitochondrial origin. Differences in mitochondrial efficiency may be reflected in the progression of colorectal adenomatous polyps to adenocarcinomas. Here, we evaluate expression levels of COXIV isoform 1 (COXIV-1) and Mitochondrial (MT)-ATP synthase Subunit 6 (ATPase6) in adenomas of tubular, tubulovillous and villous tissues as compared to adenocarcinoma tissues.

Method: Both RT-qPCR and western blot techniques were used to assess COXIV-1 and ATPase6 expression levels in 42 pairs of patients' tissue samples. Protein carbonyl assay was performed to determine levels of oxidized proteins, as a measurement of ROS productions, in the tissue samples.

Results: Differential RNA expression levels of COXIV-1 and ATPase6 from whole tissues were observed. Interestingly, RNA expression levels obtained from mitochondrial for COXIV-1 were significantly decreased in tubulovillous, villous adenomas and adenocarcinoma, but not in the tubular-polyps. Moreover, mitochondrial ATPase6 RNA expression levels decreased progressively from adenopolyps to adenocarcinoma. In mitochondrial protein, expression levels of both genes progressively decreased with a three folds from adenomatous polyps to adenocarcinoma. Whilst the ATPase6 protein expression significantly decreased in adenocarcinoma compared to villous, conversely, the levels of oxidized carbonyl proteins were considerably increased from adenomatous polyps to adenocarcinoma.

Conclusion: Our findings provide evidence that decreased mitochondrial protein expression of COXIV-1 and ATPase6 correlates with increased ROS production during colorectal adenomatous polyps' progression, suggesting the pivotal role of COXIV-1 in energy metabolism of colorectal cells as they progress from polyps to carcinoma.

Keywords: ATP synthase FO Subunit 6; COX subunit 4 isoform 1; Colorectal adenopolyps; Mitochondrial; Reactive oxygen species.

Figure 1:

Comparison of the whole tissue RNA expression levels of COXIV-1 obtained from colorectal adenopolyps (TA: Tubular adenoma; TV: Tubulovillous adenoma; V: Villous adenoma) and adenocarcinoma (CA) patients’ biopsies. Gene expression profile of COXIV-1 RNA was analyzed against Glyceraldehyde-3-phosphate dehydrogenase using RT-qPCR. Relative gene expression is expressed as the ratio of tumor tissues as compared to their paired normal surrounding tissue. Significant differences between groups were defined as^⋆ with a p-value<0.05 (N=42 samples).

Figure 2:

Comparison of the whole tissue RNA expression levels of MT-ATPase6 obtained from colorectal adenopolyps (TA: Tubular adenoma; TV: Tubulovillous adenoma; V: Villous adenoma) and adenocarcinoma (CA) patients’ biopsies. Gene expression profile of ATPase6 RNA was analyzed using RT-qPCR. Relative gene expression is expressed as the ratio of tumor tissues as compared to their paired normal surrounding tissue. Significant differences between groups was defined as^⋆ with a p-value<0.05 (N=42 samples).

Figure 3:

Comparison of the mitochondrial isolated RNA expression levels of COXIV-1 obtained from colorectal adenopolyps (TA: Tubular adenoma; TV: Tubulovillous adenoma; V: Villous adenoma) and adenocarcinoma (CA) patients’ biopsies. Gene expression profile of COXIV-1 RNA was analyzed using RT-qPCR. Relative gene expression is expressed as the ratio of tumor tissues as compared to their paired normal surrounding tissue. Significant differences between groups was defined as^⋆ with a p-value<0.05 (N=42 samples).

Figure 4:

Comparison of the mitochondrial isolated RNA expression levels of ATPase6 obtained from colorectal adenopolyps (TA: Tubular adenoma; TV: Tubulovillous adenoma; V: Villous adenoma) and adenocarcinoma (CA) patients’ biopsies. Gene expression profile of COXIV-1 RNA was analyzed against Glyceraldehyde-3-phosphate dehydrogenase using RT-qPCR. Relative gene expression is expressed as the ratio of tumor tissues as compared to their paired normal surrounding tissue. Significant differences between groups was defined as^⋆ with a p-value <0.05 (N=42 samples).

Figure 5:

Mitochondria ATPase6 and COXIV-1 expression in colorectal tumors and normal tissue. (Upper figure) Lysates were analyzed by Western blot to examine the levels of COXIV-1 protein in normal tissues (N), tubular adenoma tissues (TA), tubulovillous adenoma tissues (TV), villous adenoma tissues (V), and Carcinoma tissues (CA). A control Jurkat cell lysate was also used for protein expression analysis. (Lower figure) Protein expression of ATPase6 analyzed through Western blot using the same parameters above. Each protein was normalized against Superoxide Dismutase 2 (SOD2).

Figure 6:

Comparison Levels of carbonyl reactive proteins in colorectal tissue as measured by spectrophotometry method. Ratios of carbonyl protein in whole tissue samples normalized against adjacent normal tissue samples; TA: Tubular adenoma; TV: Tubulovillous adenoma, V: Villous adenoma and CA: Adenocarcinoma. Significant differences between groups compared to cancer were calculated using ANOVA^⋆ (P<0.05) (n=42 pairs samples). Data are presented as Mean ± S.E.M.

Figure 7:

Comparison levels of carbonyl reactive proteins in colorectal tissue as measured by spectrophotometry method. Ratios of carbonyl protein in whole tissue samples normalized against adjacent normal tissue samples; TA: Tubular adenoma; TV: Tubulovillous adenoma, V: Villous adenoma, and CA: Adenocarcinoma. Significant differences between groups compared to cancer were calculated using ANOVA^⋆ (P<0.05) (n=42 pairs samples). Data are presented as Mean ± S.E.M.