Kai Sun1, Guiyuan Su2, Haijun Deng2, Jingqing Dong2, Shangtong Lei2, Guoxin Li2. 1. Department of General Surgery, Nanfang Hospital of Southern Medical University, Guangzhou, Guangdong 510515, China. Email: sunkai9602@sina.com. 2. Department of General Surgery, Nanfang Hospital of Southern Medical University, Guangzhou, Guangdong 510515, China.
Abstract
BACKGROUND: miR-338-3p is a recently discovered miRNA and is involved in cell differentiation. However, few data are yet available on the aberrant expression of miR-338-3p in human colorectal carcinoma (CRC). This work aimed to investigate the relationship between miR-338-3p expression pattern and clinicopathological features of human CRC and the possible regulative mechanisms. METHODS: The 40 CRC, adjacent nontumorous tissues and 2 human CRC-derived cell lines (SW-480 and SW-620) were collected, respectively, and the total RNA and protein were isolated routinely. The miR-338-3p expression pattern was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Northern blotting. Smoothened (SMO, possible target of miR-338-3p) mRNA and corresponding protein expression pattern were detected by semiquantitative RT-PCR and Western blotting. miR-338-3p expression patterns were compared between nontumor mucosa and CRC samples, graded by progression-related factors. Disease outcome was calculated by Kaplan-Meier survival analysis to determine whether miR-338-3p was related to disease-free survival (DFS) and overall survival (OS) of patients. Moreover, SMO 3'-UTR fragment was PCR amplified from genome DNA of human colon and inserted into a luciferase reporter plasmid. The luciferase reporter plasmid construct was then transfected into CRC cells together with pre-miR-338-3p or anti-miR-338-3p and the luciferase activity in the transfected cells was detected. RESULTS: The expression of miR-338-3p was significantly downregulated in CRCs than those in the adjacent nontumorous tissues, and the value was negatively related to advanced TNM stage and local invasion (P < 0.01). Furthermore, miR-338-3p value was decreased markedly in SW-620 cell line relative to SW-480 (P < 0.01). Low expression of miR-338-3p was associated with unfavorable outcome in DFS but not in OS independent of clinical covariates. Moreover, RT-PCR and Western blotting analysis demonstrated that there was no significant difference in SMO mRNA expression between the corresponding CRCs and nontumorous tissues, whereas SMO protein markedly increased in CRCs (P < 0.01). A significant increase in luciferase activity was detected in CRC cells, which were cotransfected with the luciferase reporter plasmid construct and anti-miR-338-3p (P < 0.01). CONCLUSIONS: miR-338-3p is expressed differentially in CRC and associated with progression and prognosis of CRC. SMO might be a possible target of miR-338-3p, which made it a potential antitumor candidate for treatment and prevention of CRC.
BACKGROUND: miR-338-3p is a recently discovered miRNA and is involved in cell differentiation. However, few data are yet available on the aberrant expression of miR-338-3p in humancolorectal carcinoma (CRC). This work aimed to investigate the relationship between miR-338-3p expression pattern and clinicopathological features of human CRC and the possible regulative mechanisms. METHODS: The 40 CRC, adjacent nontumorous tissues and 2 human CRC-derived cell lines (SW-480 and SW-620) were collected, respectively, and the total RNA and protein were isolated routinely. The miR-338-3p expression pattern was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Northern blotting. Smoothened (SMO, possible target of miR-338-3p) mRNA and corresponding protein expression pattern were detected by semiquantitative RT-PCR and Western blotting. miR-338-3p expression patterns were compared between nontumor mucosa and CRC samples, graded by progression-related factors. Disease outcome was calculated by Kaplan-Meier survival analysis to determine whether miR-338-3p was related to disease-free survival (DFS) and overall survival (OS) of patients. Moreover, SMO 3'-UTR fragment was PCR amplified from genome DNA of humancolon and inserted into a luciferase reporter plasmid. The luciferase reporter plasmid construct was then transfected into CRC cells together with pre-miR-338-3p or anti-miR-338-3p and the luciferase activity in the transfected cells was detected. RESULTS: The expression of miR-338-3p was significantly downregulated in CRCs than those in the adjacent nontumorous tissues, and the value was negatively related to advanced TNM stage and local invasion (P < 0.01). Furthermore, miR-338-3p value was decreased markedly in SW-620 cell line relative to SW-480 (P < 0.01). Low expression of miR-338-3p was associated with unfavorable outcome in DFS but not in OS independent of clinical covariates. Moreover, RT-PCR and Western blotting analysis demonstrated that there was no significant difference in SMO mRNA expression between the corresponding CRCs and nontumorous tissues, whereas SMO protein markedly increased in CRCs (P < 0.01). A significant increase in luciferase activity was detected in CRC cells, which were cotransfected with the luciferase reporter plasmid construct and anti-miR-338-3p (P < 0.01). CONCLUSIONS: miR-338-3p is expressed differentially in CRC and associated with progression and prognosis of CRC. SMO might be a possible target of miR-338-3p, which made it a potential antitumor candidate for treatment and prevention of CRC.