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 ...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 differ展开更多
AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The re...AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The recombinant viral vector encoding the pre-miR338-3p or miR-338-3p-inhibitor and the two packaging plasmids psPAX2 and pMD2.G were cotransfected into human embryonic kidney 293T cells to package lentivirus.The supernatant containing the lentivirus particles was harvested to determine the viral titer,and this supernatant was then used to transduce CRCderived cell line,SW-620.Flow cytometry was utilized for sorting the green fluorescent protein(GFP) + cells to establish the SW-620 cell line stably expressing premiR-338-3p or miR-338-3p-inhibitor.Moreover,the expression of miR-338-3p was determined by real-time reverse transcriptase polymerase chain reaction,andWestern blotting was used to detect the expression of the smoothened(SMO,the possible target of miR-3383p) protein in SW-620 cells.Furthermore,the status of CRC cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry,respectively.RESULTS:Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLVTHM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully.GFP was expressed after the SW-620 cells were transduced by the lentivirus.Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased(relative expression 3.91 ± 0.51 vs 2.36 ± 0.44,P < 0.01).Furthermore,overexpression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells,which showed obviously suppressed proliferation ability [cellular proliferation inhibition rate(CPIR) 61.9% ± 5.2% vs 41.6% ± 4.8%,P < 0.01].Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased(relative expression 0.92 ± 0.29 vs 2.36 ± 0.44,P < 0.展开更多
基金This work was supported by a grant from the National Natural Science Foundation of China （No. 81101896）.
文摘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 differ
基金Supported by National Natural Science Foundation of China,No.81101896
文摘AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The recombinant viral vector encoding the pre-miR338-3p or miR-338-3p-inhibitor and the two packaging plasmids psPAX2 and pMD2.G were cotransfected into human embryonic kidney 293T cells to package lentivirus.The supernatant containing the lentivirus particles was harvested to determine the viral titer,and this supernatant was then used to transduce CRCderived cell line,SW-620.Flow cytometry was utilized for sorting the green fluorescent protein(GFP) + cells to establish the SW-620 cell line stably expressing premiR-338-3p or miR-338-3p-inhibitor.Moreover,the expression of miR-338-3p was determined by real-time reverse transcriptase polymerase chain reaction,andWestern blotting was used to detect the expression of the smoothened(SMO,the possible target of miR-3383p) protein in SW-620 cells.Furthermore,the status of CRC cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry,respectively.RESULTS:Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLVTHM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully.GFP was expressed after the SW-620 cells were transduced by the lentivirus.Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased(relative expression 3.91 ± 0.51 vs 2.36 ± 0.44,P < 0.01).Furthermore,overexpression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells,which showed obviously suppressed proliferation ability [cellular proliferation inhibition rate(CPIR) 61.9% ± 5.2% vs 41.6% ± 4.8%,P < 0.01].Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased(relative expression 0.92 ± 0.29 vs 2.36 ± 0.44,P < 0.
