| ObjectiveWe inferred that miRNA-221 was up-regulated in colorectal cancer and exerted its carcinogenesis through inhibiting CDKN1C/p57 expression. In order to verify this hypothesis, we observed the differential expression of miRNA-221 and CDKN1C/p57 in colorectal cancer and adjacent non-tumorous tissues in this study and expected to provide new ideas for the research on the pathogenesis, diagnosis, and treatments of colorectal cancer.Methods1 Tissue origin:34 samples of colorectal cancer and control para-cancerous tissues were harvested from patients who underwent their operation in our hospital from September 2008 to May 2009, and were confirmed by the postoperative pathological examination. Among them,20 cases were male, and 14 cases were female; They were 32～74 years old, with the average age of 52.5±11.8 years; 15 cases were colon cancer, and 19 cases were rectal cancer. Para-cancerous tissues were harvested from the intestinal mucosae that were 5 cm away from the cancer, while normal controls were the intestinal mucosae that were over 10 cm away from the cancer. All cases did not receive radiotherapy or chemotherapy before operation. The samples were snap frozen in the liquid nitrogen after collection and stored at-80℃2 Design and synthesis of primers:The gene sequences were searched in GeneBank. 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; The forward primer of CDKN1C/p57 was'-CGT TCT TCT CGG GTG GA-3', its reverse primer was 5'-CTG TAC TCA CTT GGC TCA-3', and the amplified fragment was 500 bp long; The forward primer of internal reference GAPDH was 5'-ACC ACA GTC CAT GCC ATC AC-3', its reverse primer was 5'-TCC ACC ACC CTG TTG CTG TA-3', and the amplified fragment was 452 bp long. The aforementioned primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.3 Detection of CDKN1C/p57 mRNA expression by semi-quantitative RT-PCR: Total RNA was extracted with routine Trizol reagent (Invitrogen Company). The precipitation was dissolved in DEPC-treated water. Nucleic acid protein analyzer (Beckman Coulter, USA) was used to determine RNA concentration. The purity and integrity of RNA were identified by two aspects:A260 nm/A280 nm was≥1.8, and the band ratio of 28S RNA to 18S RNA was≥1.5 in formaldehyde denaturing gel electrophoresis.3μg total RNA was obtained as the template, and then RT-PCR was performed according to the specification of RNA PCR Kit Ver 3.0 of TaKaRa Company. The parameters of PCR were 94℃for 2 min; 30 cycles of 94℃for 30 s, 55℃for 30 s,72℃for 1 min; 72℃for 10 min.1.5% agarose gel electrophoresis (It contained 0.5 g/L ethidium bromide) was performed with 10μL PCR amplified products. Ultraviolet gel imaging system (Pharmacia Master Image, USA) was used to semi-quantitatively analyze CDKN1C/p57 mRNA expression level. mRNA relative content=integral absorbance value of target gene band/integral absorbance value of internal reference GAPDH. The above experiment was repeated at least three times.4 Detection of miRNA-221 expression in the samples by real-time Q-PCR:lug total RNA 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. The parameters of PCR 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). 5 Quantitative calibration, judgment, and analysis of PCR products: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, then ACt 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 with ACt 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.6 Detection of CDKN1C/p57 protein expression by Western-blot:The tissue mass was rinsed with precooled PBS twice, placed in ice-cold lysate (It contained 150 mmol/L NaCl,50 mmol/L Tris-HCl [pH 7.6],0.1% SDS,1% NP-40, and protease inhibitor complex), ground, homogenated, smashed by ultrasonication for 10 minutes, centrifuged at 13,000 g for 10 min, and the total protein of the lysate was harvested. 50μg total protein was used to perform SDS-PAGE and transferred to a PVDF membrane by in situ electroblotting. The membrane was treated with the blocking buffer (It contained 20 mmol/L Tris-HCl [pH7.6],150 mmol/L NaCl,0.1% Tween-20, and 5% skim milk powder), then incubated respectively with rabbit anti-human CDKN1C/p57 (diluted by 1:2000) and internal inference GAPDH (diluted by 1:1000) polyclonal antibody. After the membrane was rinsed, it reacted with horseradish peroxidase coupled with goat anti-rabbit second antibody (diluted by 1:5000) (The first antibody and the second antibody were both purchased from Abcam Company). It was developed by the chemiluminescence reagents of enhanced chemiluminescence (ECL) and the protein imprinting band was obtained. The above experiment was repeated at least three times.7 Statistical process:All data in the experiment was presented as average± standard deviation (x±s). The data were test by t-test of paired design multiple comparison. P<0.05 indicated statistically significant difference. All the operation was completed with SPSS 13.0 statistical software.Results1 CDKN1C/p57 mRNA expression:The experiment detected CDKN1C/p57 mRNA expression in 34 samples by semi-quantitative RT-PCR firstly. The experimental results showed that CDKN1C/p57 mRNA expression in 4 colorectal cancer samples decreased significantly compared with non-tumorous tissues. These cases would be excluded before the next experiment, while the remaining 30 samples (CDKN1C/p57 mRNA expression had not significant difference between colorectal cancer and non-tumorous tissues) were included into the later research.2 Specificity of PCR products:PCR products of different sequences or length melt at different temperatures, so different peaks can be observed. Only when a specific PCR product generates, the single peak can be observed at the melting curve. Tm mainly depends on the fragment length, sequence composition, GC content, Mg2+ concentration in the reaction, and so on. In this study, 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.3 The expression of miRNA-221 in 30 colorectal cancer samples was up-regulated obviously compared with non-tumorous tissues (2.041±1.401 vs 0.806±0.341). The difference had statistical significance (P<0.01).4 CDKN1C/p57 protein expression:CDKN1C/p57 protein expression in 30 colorectal cancer samples decreased obviously compared with non-tumorous tissues (3.019±1.708 vs 0.972±0.316). The difference had statistical significance (P<0.01). The expression of miRNA-221 has a negative correlation with the expression of CDKN1C/p57 (r2=0.9156, P<0.01).5 The expression of miRNA-221 has relationships with the TNM stage and invasive depth (P<0.05).Conclusions1 The data proved that expression of CDKN1C/p57 mRNA content was low in about 11.8%(4/34) of colorectal cancers, which accorded with the hypothesis of loss of imprinting of the maternal gene allele methylation in tumor cells before the experiment, and caused the down-regulation of CDKN1C/p57 mRNA expression at transcriptional level. However, down-regulation of the expression in protein level was probably not caused by post transcriptional gene silencing of miRNA-221.2 Real-time Q-PCR and Western-blot detection of the remaining samples demonstrated that miRNA-221 expression was up-regulated in colorectal cancer, so it could be inferred that it combined with CDKN1C/p57 mRNA in an incomplete complementary pairing pattern.3 Expression of CDKN1C/p57 in protein level obviously decreased in colorectal cancer tissues, and it could be inferred that tumor cells probably gained the proliferative capacity through miRNA-221 mediated post-transcriptional silencing of CDKN1C/p57.4 MicroRNA-221 has the potential value in pathogenesis, diagnosis, and treatments of colorectal cancer.
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