The Differential Expression And Functional Study Of ATP5J In Colorectal Cancer

ATP5J, also known as coupling factor6(CF6), is one of the subunits of F1F0-ATP synthase which synthesizes cellular ATP from ADP and inorganic phosphate. ATP synthase is composed of a water-soluble protein complex of F1and a hydrophobic transmembrane portion Fo. ATP5J is a component of F0and responsible for the connection of F0with F1. Recently we found that ATP5J was over-expressed in tissue samples from patients with colorectal cancer. It has been reported that some subunits of ATP synthase are abnormally expressed in cancer cells. However, the expression of ATP5J gene in colorectal cancer and its cilinical significance and function have not been reported.Aim:We want to comfirm the over-expression of ATP5J in colorectal cancer, and investigate the clinical significance and function of over-expression of ATP5J on cell biological behaviours, such as cell morphology, cell growth, survival status, cell migration, as well as anti-cancer therapy sensitivity and cancer prognosis.Methods:Firstly, we use RT-PCR method, western blot analysis and immunohistochemical staining to analyze the expression of ATP5J mRNA and ATP5J protein in fresh tumor tissues, adjacent normal tissues and paraffin sections from colorectal cancer patients, as well as five colorectal cancer cell lines (DLD1, RKO, SW620, SW480, and Colo320). Secondly, the clinical pathological features and follow-up data of the relevant colorectal cancer patients were collected to analyze the correlation between ATP5J expression and clinicalpathological characteristics and survival status as well. Thirdly, we down-regulated ATP5J expression by stable transfection with ATP5J shRNA plasmid and up-regulated ATP5J expression by stable transfection with pcDNA3.1(+)/ATP5J plasmid in DLD1cells. After that, we use MTT assay, cell clongenic assay, scratch wound-healing assay, cell migration assay and flow cytometry assay to study the differences in cell morphology, cell growth, cell survival, cell migration and anti-cancer therapy sensitivity in DLD1cells after the change of ATP5J expression, for evaluating the functions of ATP5J in colorectal cancer.Results:The expression of ATP5J in clinical colorectal caner tissues was higher than that in the adjacent normal tissues, both on mRNA level and protein level. And that was also significantly higher in metastatic lymph nodes than in primary cancer tissues (P<0.05). There was a correlation between ATP5J expression and tumor differentiation (P<0.05), but not gender, age, T stage, lymph node metastasis, even survival status (P>0.05). Meanwhile, ATP5J was over-expressed in colon cancer cell lines (DLD1, RKO, SW620, SW480, and Colo320) but not in normal human fibroblast cell lines (NHFB)(P<0.05). Further studies demonstrated that down-regulation of ATP5J expression by small RNA interference would decrease the migration ability of cancer cells, and increase the sensitivity of cancer cells to5-Fu treatment. Meanwhile, up-regulation of ATP5J expression by stable transfection with pcDNA3.1(+)/ATP5J plasmid would increase the migration ability of cancer cells, and decrease the sensitivity of cancer cells to5-Fu treatment (P<0.05). However, the results of MTT assay, cell cycle by flow cytometry, or clone formation by clonogenic assay revealed no differences in cell survival (P>0.05).Conclusions:ATP5J was over-expressed in patients with colorectal cancer, and there was a correlation between ATP5J expression and differentiation of tumors, but not survival status. Cell experiments showed that ATP5J might be involved in cell migration and5-Fu sensitivity in colorectal cancer.