ABSTRACT
<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>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.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Colorectal Neoplasms , Genetics , Pathology , Disease Progression , Gene Expression Regulation, Neoplastic , Genetics , Physiology , MicroRNAs , Genetics , PrognosisABSTRACT
Hypermethylation in the promoter region is an important epigenetic mechanism for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferases are considered to be the main cause of promoter hypermethylation. In order to explore the roles of two methyltransferase members (DNMT1 and DNMT3b) in the cholangiocarcinoma tumorigenesis, antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene was constructed respectively, and were co-transfected into the human cholangiocarcinoma cell line QBC-939 to observe their biological effects on the cell growth and proliferation ability, apoptosis, cell cycle alteration, and the tumorigenesis ability in the subcutaneous tissue of nude mouse. The results demonstrated that co-transfection with antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene and single transfection with antisense eukaryotic expression plasmid of DNMT1 gene can suppress the growth and proliferation of QBC-939, block the cell cycle at G1 phase, increase the apoptosis rate, minimize the tumor size in the subcutaneous tissue of nude mouse. The suppressing biological effect of co-transfection is stronger than single transfection with antisense DNMT1. Meanwhile, single transfection with antisense eukaryotic expression plasmid of DNMT3b gene has no effects on the biological characteristics of QBC-939. This study suggests that DNMT1 gene plays a key role in DNA methylation and DNMT3b gene may act as an accessory to support its function in inactivation of tumor suppressor genes. Combination DNMT1 and DNMT3b will increase their biological effects and have the synergistic effect on suppressing the growth of human cholangiocarcinoma cell line QBC-939.
Subject(s)
Apoptosis , Biliary Tract Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Cholangiocarcinoma/metabolism , DNA (Cytosine-5-)-Methyltransferases/biosynthesis , DNA (Cytosine-5-)-Methyltransferases/genetics , Down-Regulation , Gene Expression Regulation, Neoplastic , Genetic Vectors , Mice, Nude , Neoplasm TransplantationABSTRACT
Hypermethylation of the promoter region is one of the major mechanism of tumor suppressor gene inactivation. In order to provide a research tool for the study on the function of MBD1gene in DNA methylation and tumorigenesis, antisense MBD1 gene eukaryotic expression plasmid was constructed and transfected into human biliary tract carcinoma cell line QBC-939 to observe its effect on the expression of MBD1 mRNA and protein by using RT-PCR and FCM respectively. Following the transfection, the mRNA level of MBD1 gene decreased from 0. 912±0. 022 to 0. 215±0.017, and the protein level of MBD1 gene also decreased from (80.19±5.05) % to (35.11±4.05)%. There were very significant differences in the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukary otic expression plasmid transfection group (P＜0.01). It was suggested that transfection with the antisense MBD1 gene eukaryotic expression plasmid can significantly reduce the expression level of MBD1 gene in QBC-939, and this study may provide a valid tool for the investigation of the function of MBD1 gene and its role in biliary tract carcinoma.
ABSTRACT
Hypermethylation of the promoter region is one of the major mechanism of tumor suppressor gene inactivation. In order to provide a research tool for the study on the function of MBD1 gene in DNA methylation and tumorigenesis, antisense MBD1 gene eukaryotic expression plasmid was constructed and transfected into human biliary tract carcinoma cell line QBC-939 to observe its effect on the expression of MBD1 mRNA and protein by using RT-PCR and FCM respectively. Following the transfection, the mRNA level of MBD1 gene decreased from 0. 912 +/- 0.022 to 0.215 +/- 0. 017, and the protein level of MBD1 gene also decreased from (80.19 +/- 5.05) % to (35.11 +/- 4.05) %. There were very significant differences in the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukaryotic expression plasmid transfection group (P < 0.01). It was suggested that transfection with the antisense MBD1 gene eukaryotic expression plasmid can significantly reduce the expression level of MBD1 gene in QBC-939, and this study may provide a valid tool for the investigation of the function of MBD1 gene and its role in biliary tract carcinoma.
Subject(s)
Biliary Tract Neoplasms/metabolism , Biliary Tract Neoplasms/pathology , Cell Line, Tumor , DNA Methylation , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Eukaryotic Cells/metabolism , Gene Expression Regulation, Neoplastic , Genetic Vectors , Oligonucleotides, Antisense/genetics , Plasmids/genetics , Transcription Factors/biosynthesis , Transcription Factors/genetics , TransfectionABSTRACT
Objective To study the effect of transfection of antisense MBD1 gene eukaryotic expression vector on the expression of MBD1 gene in human cholangiocarcinoma cell line QBC-939.Methods The(constructed) antisense MBD1 gene eukaryotic expression vector was transfected into the human(cholangiocarcinoma) cell line QBC-939 using lipofectamine transfection reagents,and positive cell clones were obtained using G418 selection after transfection.The constructed recombinant vector was transfected into(QBC-939) cells successfully and was confirmed by amplifying the exogenous neo~R gene with PCR method.The expression level of MBD1 gene mRNA and protein was detected by RT-PCR and FCM methods respectively.Results Following the transfection,the MBD1 gene mRNA level in human cholangiocarcinoma cell line QBC-939 decreased from 0.912?0.022 to 0.215?0.017,and the MBD1 gene protein level also(decreased) from(80.19?5.05)% to(35.11?4.05)%.There were very significant differences on the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukaryotic expression vector transfection group(P
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Objective To investigate the repressing effects of cytosine deaminase(CD) and herpes simplex(virus) thymidine kinase(HSV-tk) double suicide genes coexpression system on human cholangiocarcinoma QBC939 cells and QBC939 cells transplantated tumor in nude mice.Methods CD and HSV-tk double suicide genes were transfered into QBC939 cells using liposomes.After G418 selection,the positive clones of QBC939/CD+tk cells were picked up and cultured.The expression of CD and HSV-tk genes was(confirmed) by RT-PCR.In vitro,the QBC939/CD+tk cells were treated with 5-Fc and/or GCV,and the cytoxicity efficacy was evaluated by microculture tetrajolium test(MTT) method.The QBC939/CD+tk cells were inoculated subcutaneously into nude mice,and when the tumors were palpable,5-Fc and GCV were(injected) intraperitoneally,and the volumes of transplantated tumors were measured before and after medication.Results Double suicide genes were stably expressed in QBC939/CD+tk cells.The repressing capability of combination of 5-Fc and GCV on QBC939/CD+tk cells was more effective than that of using either 5-Fc or GCV alone.The increase of cell-repressing was assaciated with increase the concentration of the prodrug.The repressing effect of combination of the 2 prodrugs on early period of transplantation tumor was obvious,even complete abolition of tumor was noted,and moreover a marked local bystander effect was observed.(Conclusions) In vitro and in vivo,the cell-repressing efficacy of double suicide gene system on cholongiocarcinoma cells and the tramsplanted tumor of nude mice was significant,and the bystander effect was obvious.
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Objective To investigate the effects of the tumor suppressor gene PTEN in growing inhibition and down-regulating mTOR in cholangiocarcinoma QBC939 cells in vitro.Methods QBC939 cells were transfected with plasmids wild-type PTEN and C124S-PTEN in vitro.After transfection,the expression of the PTEN and phosphorylation of AKT and mTOR was detected by Western blot.Flow cytometry was used to analyze apoptosis and cell cycle of the transfected cells.Results Compared with the control,the expression of phosphorylation AKT was decreased and mTOR were down-regulated respectively when transfected with the wild-type PTEN.However,after transfection with mutation-type PTEN,the level of PTEN in the cells by increased,but phosphorylation AKT level and mTOR expression had no significant change.Conclusions PTEN can be actived by phosphorylated AKT.Actived AKT decreased the mTOR which led to tumor cells apoptosis and regulation of the tumor cell cycle.In the pathway of signal transmission of PI3K/AKT/PTEN/mTOR,PTEN and mTOR are closely related through phosphorylation of AKT.