| ObjectiveWe inferred that the miRNA-221 on CDKN1C/p57 expression pattern in human colorectal carcinoma (CRC) cell and the regulative effects of miR-221-specific inhibitor on cell proliferation and apoptosis in CRC cells.Methods1. Human colon cancer cell line, including HT-29. Lovo. SW-480.Caco2 and normal human umbilical vein endothelial cells (HUVEC)was proveded by the Shanghai Institutes for Life Sciences Library provides; The gene sequences were searched in GeneBank. and the miRBase database (http://microrna.sanger.ac.uk/) Primer-Express 2.0 software was used to design the primers and probes. The forward primer of internal reference RNU6B was 5'-CTC GCT TCG GCA GCA CA-3', its reverse primer was 5'-AAC GCT TCA CGA ATT TGC GT-3', and the amplified fragment was 96 bp long; The forward primer of miRNA-221 was 5'-CAG CAT ACA TGA TTC CTT GTG A-3',its reverse primer was 5'-CTT TGG TGT TTG AGA TGT TTG G-3',and the amplified fragment was 73 bp long; CDKN1C/p57 mRNA 3', untranslated region (untranslated region, UTR) the forward primer was 5'-TCT AGA GCC CAA AGA GCC C-3', its reverse primer was 5'-TCT AGA GAT TAA ACA TTT TAT ATA AAT GAC-3', and the amplified fragment was 262 bp long; The aforementioned primers were synthesized by Shanghai Sangon Biological Engineering Technology; miR-221 and miR-221 inhibitor 2,-methoxy-modified RNA (anti-miR-221), miR-221 inhibitor of negative control and transfection of negative control were purchased from the Shanghai Jima Drugs manufacture technology company.2. Cell culture and transfection:After cell convention recovery resuspended with RPMI-1640 supplemented with 10%(v/v) fetal bovine serum. The cells were then plated in 25 cm2 culture bottles and incubated in a 5% CO2 humidified atmosphere at 37℃. and then the cells were trypsinized using trypsin-edetic acid when they reached 80% to 90% confluence. Cells aged at passages 4 to 8 were used for experiments. The day before transfection, cells were seeded in antibiotic-free medium at a density of 3×105 cells/well in 6-well plates, when they reached 50% to 70% confluence. MiRNA transfection was performed using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). The extension dyes the reagent density is 50 nmol/L, to settings establishment negative control group, the anti-miR-221 negative control group, the miR-221 extension dyes the group and the anti-miR-221 extension to dye the group, each group in 6-well plates, trades the fresh nutrient fluid for experiments.3. Detection of miRNA-221 expression in the samples by real-time Q-PCR:Uses the stem link law to increase. Total RNA was extracted using Trizol (Invitrogen). The precipitation was dissolved in DEPC-treated water. A nucleic acid protein analyzer (Beckman Coulter) was used to determine the RNA concentration. The purity and integrity of RNA were evaluated in formaldehyde denaturing gel electrophoresis based on two criteria:A260 nm/A280 nm≥1.8 and a band ratio of 28S RNA to 18S RNA≥1.5. by Collection total RNA lug extracted by the above procedure was added into 12μL sterile distilled water and incubated in 72℃for 5 min to open RNA secondary structure. Then it was placed on the ice immediately to prevent RNA from annealing and recovering to its secondary structure. The following reaction solution was prepared in another PCR tube without RNase:dNTP mixture 2.0μL, RNase inhibitor 0.5μL, miRNA-221 reverse transcription primers 0.5μL, RNU6B reverse transcription primers 0.5μL,5x buffer 4.0μL, and M-MLV reverse transcriptase 0.5μL (Promega Company). The reaction solution was add to the solution which contained total RNA, mixed, and incubated at 42℃for 60 min. The obtained cDNA was stored at-20℃. Real-time Q-PCR system of miRNA-221 and RNU6B was constructed:cDNA 5.0μL, Primers 1.0μL, SYBR Green fluorescent dye 10μL, and sterile distilled water 8.0μL. Responds the condition were denaturing at 95℃for 10 min; 40 cycles of 95℃for 15 s,65℃for 30 s,72℃for 30 s; and followed by the extension of 72℃for 10 min. Each sample was detected by PCR in multiple tubes, and the experiment was repeated at least three times (The reagents are purchased from TOYOBO Company. PCR amplifier was ABI PRISM(?) 7300). Take RNU6B as the internal reference and its copy number as the calibration baseline. The value of Ct (cycle threshold) of miRNA-221 in every sample was directly obtained through Light Cycler software. Ct value of RNU6B in the same sample was subtracted from it, thenΔCt of miRNA-221 in this sample was obtained. The result was limited by the different reverse transcription (RT) efficiency of different RNA samples when RNA was detected by real-time Q-PCR, so we corrected withΔCt of normal intestinal mucosa and got -ΔΔCT value. The exact content of miRNA-221 in each sample was calculated by the formula:target gene expression=2-ΔΔCT. The experiment was repeated at least three times.4. Detection of CDKN1C/p57 protein expression by Western-blot:The tissues and cells were rinsed twice with cold PBS buffer and lysed in an ice-cold lysis buffer containing (It contained 150 mmol/L NaCl,50 mmol/L Tris-HCl [pH 7.6], 0.1% SDS,1% NP-40, and protease inhibitor complex).The samples were cleared by centrifugation at 13 OOOg for 10 min; 50μg of protein from the tissue samples was subjected to sodium dodecy sulfate-polyacrylamide get electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene fluoride (PVDF) membrane by in situ electroblotting. The membrane was treated with the blocking buffer (It contained 20 mmol/L Tris-HC1 [pH7.6],150 mmol/L NaCl,0.1% Tween-20, and 5% skim milk powder); the membranes were incubated with primary antibodies against CDKN1C/p57 orβ-actin and then incubated with a horseradish peroxidase-conjugated secondary antibody. (The first antibody and the second antibody were both purchased from Abcam Company). The blot was developed using an ECL detection kit, according to the manufacturer's instructions, and the protein imprinting band was obtained. The experiment was repeated at least three times.5. Detection of cell multiplication with MTT Method:Exponentially growing CRC cells were adjusted to 1.5×104 cells/mL with RPMI1640 , plated in 96-well plates, incubated in a 5% CO2 humidified atmosphere at 37℃. and then incubated for 12 h. abandons the nutrient fluid after the observation cell pastes the wall; Observation the three group of interstitial cell growth speed difference, for example negative control group, anti-miR-221 negative control group and blank control group, The determination two groups cell in not obvious influence situation to carry on experiment. After being transfected with 50 nmol/L miR-221 or anti-miR-221 and incubated for 48 h, 20μL/well MTT (5 g/L) was added to each well. The medium was then removed after a 4-h incubation, and 200μL/well dimethyl sulfoxide (DMSO) was added to dissolve the reduced formazan product. Sub sequently, the plate was read in an enzyme-linked immunity implement at 490nm. Cellular prolif-eration inhibition rate (CPIR) was calculated according to the following formula: CPIR=(1-average A value of the experimental group/average A value of the control group)×100%.6. Cell cycle and apoptosis rate analysis of transfected cells by Flow cytometry:Uses conventional blood serum hungry method, After causing CRC pastes the wall, to adjusted 5×105 cells plated in 24-well plates, Before the experiment, gives non-blood serum nutrient fluid processing cell 24 h, after then the replacement to contain 50 nmol/L miR-221 and the anti-miR-221 complete nutrient fluid processes 48 h; pretreated CRC cells were harvested and washed twice with PBS buffer, fixed with 70% ethanol at -20℃for 30 min and stored at 4℃overnight. The cells were then washed with PBS again, stained with Annexin V-FITC and PI at 4℃for 30 min in darkness. Cell cycle and apoptotic rate were measured using an EPICS XL Flow Cytometer.7. Luciferase assay report carrier construction and activity assay:The human genome DNA in 3'-untranslated region (3'-UTR) of the CDKN1C/p57 gene was amplified by PCR. it purification agarose electrophoresis clones into TA cloning vehicle pMD-T (TaKaRa) when responds the condition were denaturing at 94℃for 2 min; 30 cycles of 94℃for 30 s,55℃for 30 s, and 72℃for 1 min; and 72℃for 10 min Of the PCR products; Some person Will reorganize of CDKN1C/p57 gene 3,-UTR the fragment the pMD-T carrier to carry on the suitable enzyme to cut with through the agarose electrophoresis purification, and cloned into the XbaI site of the pGL3-Control vector (Promega, USA), downstream of the luciferase gene, to generate the vector pGL3-p57. For the luciferase assay, the CRC cells were cultured in 24-well plates and were transfected with 500 ng of either pGL3-p57 or the pGL3 control vector and 50 pmol of miR-221, anti-miR-221 or negative controls. Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. At 24 h after transfection, the firefly luciferase activity was measured using the Dual Luciferase Reporter Assay (Promega). The experiment was repeated at least three times.8. Statistical analysis:All data in the experiment was presented as average±standard deviation (x±s). The data were test by Student-Newman-Keuls q-test. adopt completely random design of the single factor variance analysis, multiple sample mean differences between binary comparison, P< 0.05 indicated statistically significant difference. All the operation was completed with SPSS 13.0 statistical software.Results1. MiR-221 express change after Real-time RT-PCR detected CRC transfection: Examines in the cancer cell miR-221 the cDNA demonstration exponential growth,.and achieving the platform time, the amplification curve is a typical pour S curve to explained that amplification efficiency higher; miRNA-221 PCR product was 73bp long, the corresponding Tm was 84.26±0.56℃, the melting temperature was even, and the shape of the peak was also sharp. To obtains the Ct value which in various samples the goal gene increases to take the miR-221 quantified judgment. Four human CRC cell lines including HT-29 (4.094±0.208), Lovo (1.122±0.138), SW-480 (3.927±0.232), Caco2 (1.831±0.149) respectively. Significant miR-221 overexpression was observed in all four CRC cell lines compared to the miR-221 level in HUVEC (0.223±0.047, P<0.01). The miR-221 expression pattern was detected by real-time RT-PCR following the protocols recommended by commercial corporation. The specific 2,-methoxy-modified RNA oligonucleotide of miR-221 (miRNA inhibitor, anti-miR-221) were designed, synthesized and transfected into CRC cell by liposome. The expression of miRNA-221 was up-regulated obviously compared with anti-miR-221 (2.915±0.442 vs 0.541±0.206). The difference had statistical significance (P<0.01).2. To detect anti-miR-221 proliferation influence in Caco2 cell by the MTT assay:The MTT assay shows NC group(27.2±3.6%),anti-miR-221 NC group(25.8±4.1%),miR-221 transfecting group(16.3±2.9%),anti-miR-221 transfecting group(59.4±4.9%) by CPIR, the miR-221 transfected group and anti-miR-221 transfection group Compared with NC group were differences had statistically significant (P<0.01).3. To detection anti-miR-221 apoptosis of influence in Caco2 cell proliferation cycle by Flow cytometric:MiR-221 was transfected in Caco2 cell will be proliferation active. The G0 time cell number reduces, but the S time cell number proportion elevates MiR-221-specific inhibitor significantly inhibited the expression of miR-221 in Caco2 cell. Moreover, anti-miR-221 could markedly inhibit CRC cell proliferation and induce cell apoptosis (P<0.01)..4. MiR-221 could interact with a target site on the 3,-UTR of CDKN1C/p57 mRNA to inhibit by Luciferase activity analysis:The pGL3-p57 material particle together with miR-221 or anti-miR-221 into Caco2 cells cultured 24 h, MiR-221 could interact with a target site on the 3,-UTR of CDKN1C/p57 mRNA to inhibit CDKN1C/p57 expression by post-transcriptional gene silencing to promote CRC occurrence and progress.MIiR-221-specific inhibitor showed potent inhibitory effect on the growth of CRC cell, which made it a potential anti-tumor candidate for treatment and prevention of CRC. P< 0.01 indicated statistically significant difference. 5. To detection of cell protein after transfection CDKN1C/p57 change by Western-blot:After transfection of miR-221, expression of CDKNlC/p57 in Caco2 cells decreased while the corresponding transfected anti-miR-221 expression of CDKN1C/p57 in the cells increased, the differences were significant (P<0.01)Conclusions1. Our results also demonstrated that miR-221 is dramatically up-regulated in four human CRC-derived cell lines compared with that in HUVEC, and this is in accordance with the results from clinical samples. and so on after transfection of miR-221 up-regulated expression in the CRC cells while show that miR-221 does have to promote the activity of tumor cell proliferation; MiR-221-specific inhibitor showed potent inhibitory effect on the growth of CRC cell. Explained that miR-221 may take the colon cancer gene therapy the new Pharmacology target spot which made it a potential anti-tumor candidate for treatment and prevention of CRC.2. Confirmed that miR-221 was transfection in human colorectal cancer cell lines while CDKN1C/p57 expression by post-transcriptional gene silencing to promote CRC occurrence and progress. MiR-221-specific inhibitor significantly inhibited the expression of miR-221 and up-regulated CDKN1C/p57 protein expression in Caco2 cell. Moreover, anti-miR-221 could markedly inhibit CRC cell proliferation and induce cell apoptosis, we identified miR-221 as a direct regulator of CDKN1C/p57 expression in CRC, demonstrating a new mechanism for CDKN1C/p57 down-regulation in CRC. These findings further emphasize the importance of_miR-221 in CRC carcinogenesis.3. The anti-miR-221 and pGL3-p57 plasmid was increased Fluorescein enzyme activity in the cotransfected Caco2 cells.CDKN1C/p573,-UTR fragment was amplified from genome of human colon and inserted into a luciferase reporter plasmid. It is Conclusion that MiR-221 could interact with a target site on the 3,-UTR of CDKN1C/p57 mRNA to inhibit CDKN1C/p57 expression by post-transcriptional gene silencing (post-transcriptional gene silencing, PTGS) to promote CRC occurrence and progress. |
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