Expression And Function Of Proteasome Subunit PSMA7 In Colorectal Cancer

The proteasome subunit PSMA7 located on the 20q13.33 is one of the 7 proteasomeα-subunits. PSMA7 was reported to be overexpressed in several malignant tumors, and play important roles in regulation of some important cancer-associatedproteins. However, the role of PSMA7 in the development of cancer is still unknown. Objective:1. To investigate the expression of PSMA7 in colorectal cancer and clarify thecorrelations between of this expression and the clincopathologic factors.2. To construct the shorthairpin RNA (shRNA) expressing plasmids which target to PSMA7 and evaluate its effectiveness of RNA interference.3. To explore the roles and possible mechanisms of PSMA7 in the progression of colorectal cancer.Material and methods:1. RT-PCR to evaluate the level of PSMA7 mRNA was carried out in 32 cases colorectal cancerous tissues and matched normal colorectal mucosa. Immunohistiochemistry to evaluate the level of PSMA7 protein in the colorectal cancer was performed in tissues from 62 primary sites, 34 metastatic lymph nodes and 13 metastatic liver lesions as well as matched normal colorectal mucosa. The correlations between the level of PSMA7 protein in the primary sites of colorectal cancer and the clincopathologic factors were analyzed.2. The shRNA expressing plasmids which target to PSMA7 was constructed and transfected to human colorectal cancer line RKO by cationic liposome, Lipofectamine 2000. The effectiveness of RNA interference was evaluated by Western Blot.3. Comparative studies between PSMA7 shRNA transfected RKO cell and control cells were carried out, including cell morphology, proliferation, apoptosis, cell cycle, anchorage-independent growth, invasion and migration, actin cytoskeleton and the growth of human colorectal RKO xenograft in nude mice. Expression of MMPs and TIMPs were evaluated by RT-PCR, expression of CD44 was evaluated by RT-PCR andWestern Blot.Results:1. PSMA7 was found to be overexpressed in colorectal cancer when compared tomatched normal mucosa. Moreover, expression of PSMA7 in primary tumor was significantly correlated with liver metastasis. High expression of PSMA7 was associated with poor survival, and multivariate analysis revealed expression of PSMA7 to be an independent prognosis factor.2. The shRNA expressing plasmids which target to PSMA7 was constructed and transfected to human colorectal cancer line RKO by cationic liposome, Lipofectamine 2000, successfully. The stable PSMA7 shRNA transfected clone was selected and efficient inhibition of PSMA7 expression was confirmed by Western Blot.3. Comparative studies between PSMA7 shRNA transfected RKO cell and control cells revealed that knockdown of PSMA7 affected the cell morphology, inhibited the anchorage-independent growth, suppressed cell invasion and migration, affected the organization of actin cytoskeleton as well as suppressed the tumorigenicity in vivo without significant influence on the proliferation, apoptosis and cell cycle transition. PSMA7 shRNA transfection resulted in significant inhibition of CD44 expression without obvious effect on the expression of MMPs and TIMPs. Conclusions:PSMA7 was found to be overexpressed in colorectal cancer when compared to matched normal mucosa. Moreover, expression of PSMA7 in primary tumor was significantly correlated with liver metastasis and was an independent prognosis factor. RNAi was successfully carried out by construction of PSMA7 shRNA expressing plasmid and transfection to RKO cell by liposome, and the functional study of PSMA7 revealed that: knockdown of PSMA7 affected the cell morphology, inhibited the anchorage-independent growth, suppressed cell invasion and migration, affected the organization of actin cytoskeleton as well as suppressed the tumorigenicity in vivo. These results indicated that PSMA7 may play an important role in the progression of colorectal cancer. The initiative study on molecular mechanism of PSMA7 revealed that PSMA7 shRNA transfection inhibited the expression of CD44, regulation of CD44 expression may contribute to the effects of PSMA7.