ABSTRACT
The novel proteasome subunit Adrm1 located on the 20q13 amplicon was differentially expressed in colorectal cancer by semiquantitative RT-PCR. Adrm1 mRNA was overexpressed in 46.2% (18/39) colorectal cancer tissues compared to their matched normal mucosa and significantly correlated with lymph node metastasis of colorectal cancer (P=0.037). Knockdown of Adrm1 by shRNA in human colon carcinoma RKO cells inhibited their anchorage-independent growth, cell migration as well as cell proliferation through inducing apoptosis and cell cycle arrest at the G1 phase. In addition, stable RNA interference of Adrm1 gene synergistic with 5-Fu treatment suppressed RKO cell growth in vitro. Collectively, these data suggested that Adrm1 is potentially oncogenic and may play an important role in colon tumorigenesis. Regiment with combined application of Adrm1 RNA interference and chemotherapy may emerge as a novel therapeutic strategy for Adrm1 overexpressed colorectal cancer.
Subject(s)
Cell Movement/genetics , Chromosomes, Human, Pair 20/genetics , Colorectal Neoplasms/pathology , Membrane Glycoproteins/metabolism , RNA Interference , Blotting, Western , Cell Line, Tumor , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Gene Expression , Humans , In Situ Nick-End Labeling , Intracellular Signaling Peptides and Proteins , Membrane Glycoproteins/genetics , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , RNA, Messenger/analysis , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
Colorectal cancer is one of the most common causes of cancer-related deaths throughout the world. Recently, we reported that proteasome subunit PSMA7 located on 20q13 amplicon was overexpressed and associated with liver metastasis of colorectal cancer. The results indicate that PSMA7 may play an important role in the colorectal cancer progression and provide a unique target site for the development of therapeutic drugs. However, it is unknown how aberrant PSMA7 activation critically regulates the metastatic behavior of colorectal cancer cells. To investigate the role of PSMA7 in the progression of colorectal cancer, we employed the RNA interference technology to knock down the PSMA7 gene in human colon cancer cell line RKO and analyzed its effect and explored the involved mechanisms. Depletion of PSMA7 by shRNA in RKO cells inhibited their anchorage-independent growth and cell invasion and migration. Moreover, PSMA7 depletion was able to strongly suppress the in vivo tumorigenic ability of RKO cells. These effects may be induced by inhibiting CD44 expression directly or indirectly. Genetic or pharmacological inhibition of PSMA7 may therefore be a beneficial strategy in the treatment of colorectal cancer patients.
Subject(s)
Cell Movement , Colorectal Neoplasms/prevention & control , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , RNA, Small Interfering/pharmacology , Animals , Apoptosis , Blotting, Western , Cell Adhesion , Cell Cycle , Cell Proliferation , Colony-Forming Units Assay , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Female , Genetic Therapy , Humans , Immunoenzyme Techniques , Mice , Mice, Inbred BALB C , Mice, Nude , Proteasome Endopeptidase Complex/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
OBJECTIVE: To investigate the effects of the novel proteasome subunit Adrm1 knockdown by RNA interference on proliferation of colorectal cancer cells. METHODS: The shRNA eukaryotic expression vector against Adrm1 was constructed and transfected into colon cancer RKO cells. The Adrm1-shRNA stable transfected clones were selected. Experimental cells were divided into 3 groups: the experimental group containing stable Adrm1-shRNA transfected cells, the control group containing only RKO colon cancer cells and stable empty vector transfected control group. The Adrm1 protein expression level was analyzed by Western blot. The colony-forming ability of the three groups was assessed by soft agar assay. The cell proliferation and apoptosis were analyzed by methyl thiazolyl tetrazolium (MTT) method and in situ end labeling (TUNEL) assay. Cell cycle changes were assayed by flow cytometry. RESULTS: Adrm1-shRNA effectively suppressed Adrm1 expression in the experimental group. Silencing of Adrm1 in RKO cells significantly inhibited their anchorage-independent growth, only occasional individual colonies were formed. The apoptosis rate of experimental group was (12.4 +/- 1.1)%, significantly higher than that of the stable empty vector transfected control group. The proportion of G(0)/G(1) and S/G(2) phase cells in the experimental group was (41.2 +/- 1.1)% and (58.8 +/- 1.1)%, respectively. The cells were arrested at G(1) phase. In addition, Adrm1 RNA interference combined with 5-Fu treatment significantly suppressed colorectal cancer cell growth in vitro. CONCLUSION: Silencing of Adrm1 by RNA interference can significantly suppress proliferation of RKO cells through inducing apoptosis and arresting the cell cycle. The combined application of Adrm1 RNA interference and chemotherapy may become as a novel therapeutic strategy for Adrm1 overexpressed colorectal cancer.
Subject(s)
Cell Proliferation , Colorectal Neoplasms/pathology , Membrane Glycoproteins/genetics , RNA Interference , RNA, Small Interfering/genetics , Antimetabolites, Antineoplastic/pharmacology , Apoptosis , Cell Cycle , Cell Line, Tumor , Colorectal Neoplasms/metabolism , Drug Resistance, Neoplasm , Fluorouracil/pharmacology , Genetic Vectors , Humans , Intracellular Signaling Peptides and Proteins , Membrane Glycoproteins/metabolism , Plasmids , TransfectionABSTRACT
The proteasome subunit PSMA7 located on the 20q13 amplicon was found to be differentially expressed in colorectal cancer by semiquantitative RT-PCR. PSMA7 mRNA was overexpressed in 37.5% (12/32) colorectal cancer tissues while it was either of a low level or not expressed in matched normal mucosa. The aim of this study was to examine the PSMA7 protein expression in 62 colorectal cancer primary sites, 34 lymph node metastatic sites and 13 liver metastatic sites by immunohistochemistry and clarify the correlations of this expression with the clinicopathological parameters. PSMA7 high expression was detected in 38.8% (24/62) colorectal cancer primary sites, 52.9% (18/34) lymph node metastatic sites and 100% (13/13) liver metastatic sites but not in the normal colorectal tissues. The PSMA7 high expression was significantly correlated with liver metastasis (P=0.028). Survival was significantly lower in patients with a PSMA7 high expression than in those with a PSMA7 low expression (P=0.0012). Moreover, in multivariate analysis, the PSMA7 expression demonstrated an independent prognostic factor (P=0.004, relative risk 5.057; 95% confidence interval, 1.682-15.201). These results indicated that PSMA7 may play an important role in colorectal cancer progression and provide a unique target site for the development of therapeutic drugs. The evaluation of PSMA7 expression in primary colorectal cancer at the time of surgery may be a valuable tool for defining patients with a high risk of developing liver metastasis.
Subject(s)
Biomarkers, Tumor/metabolism , Colorectal Neoplasms/pathology , Liver Neoplasms/diagnosis , Liver Neoplasms/secondary , Proteasome Endopeptidase Complex/metabolism , Adult , Aged , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Chromosomes, Human, Pair 20/genetics , Colorectal Neoplasms/enzymology , Female , Gene Expression , Humans , Male , Middle Aged , Proteasome Endopeptidase Complex/analysis , Proteasome Endopeptidase Complex/genetics , RNA, Messenger/analysis , RNA, Messenger/metabolism , Up-RegulationABSTRACT
OBJECTIVE: To investigate the correlation between the proteasome subunit PSMA7 expression in colorectal cancer and its role in liver metastasis. METHODS: To identify the PSMA7 protein expression in 62 primary site colorectal cancers, 34 lymph node metastatic sites and 13 liver metastatic sites by immunohistochemistry and clarify the correlation of its expression with the clinicopathological parameters. RESULTS: High expression of PSMA7 was detected in 38.7% (24/62) of primary site colorectal cancer, 52.9% (18/34) of lymph node metastatic sites and 100% (13/13) liver metastatic sites but not in the normal colorectal tissue. High expression of PSMA7 was significantly correlated with liver metastasis (P = 0.028). The survival rate was significantly lower in patients with high expression of PSMA7 than in those with low expression of PSMA7 (P = 0.0008). As well, in multivariate analysis, PSMA7 expression demonstrated to be an independent prognostic factor (P = 0.004, relative risk 5.057; 95% confidence interval, 1.682-15.201). CONCLUSION: PSMA7 may play an important role in the colorectal cancer progression. Evaluation of PSMA7 expression in primary colorectal cancer at the time of surgery might be a valuable test in defining patients with a high risk of developing liver metastasis.