摘要
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,miR338-3p 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 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,miR338-3p 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.
基金
This work was supported by a grant from the National Natural Science Foundation of China （No. 81101896）.
