Effect Of Antisense MicroRNA-221on Radiation Sensitivity Of Colorectal Cancer Cells And The Underlying Mechanism

Background and purposeColorectal cancer (CRC) is one of the most common malignant tumors in China and the overall incidence rate shows an upward trend. Radiotherapy has become an important part of a comprehensive treatment plan for colorectal cancer. Therefore, we selected those patients who would benefit the most from the radiotherapy. MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs, with a length of about21-25nucleotides. MiRNAs are able to degrade the target mRNAs or inhibit their translation through binding to the3’-Untranslated Region (3’-UTR) of the target mRNAs, thereby exerting post-transcriptional regulatory effects._MiR-221, a newly discovered miRNA, whose over-expression can enhance the ability of tumor cell invasion, is closely correlated with the occurrence and development of tumors.P57kip2, which is a cyclin-dependent kinase inhibitor, may cause arrest of the cell cycle in the G0/G1phase, and therefore play a negative regulatory role on the cell cycle.MiR-221is capable of promoting the proliferation of colorectal cancer cells by inhibiting the expression of p57kip2, which is a target of miR-221in colorectal cancer cells. However, the expression of miR-221and p57kip2in colorectal cancer cell lines under radiotherapy and whether miR-221may affect the radiosensitivity of colorectal cancer cells by regulating the expression of p57kip2has not been reported.Therefore, in this study, we observed the miR-221and p57kip2mRNA and protein expression changes in CRC Caco2cells and their relationship under different X-ray exposure dose(2Gy、4Gy、6Gy、8Gy).We silence the miR-221expression through antisense oligonucleotides technology and the clonogenic assay was used to measure the surviving cells of CRC Caco2cells and their enhancement of radio sensitivity under different X-ray exposure dose. At the same time, calculation of the mean lethal dose (Do) and quasi-threshold dose (Dq) was measured by single hit multi target model. Therefore, we can preliminary asses the effect of miR-221antisense oligonucleotides (AS-miR-221) on the radiosensitivity of colorectal cancer cells and their underlying mechanisms.Methods(1) Human colorectal cancer cell line Caco2was cultured in RPMI-1640supplemented with10%fetal bovine serum (FBS) in a humidified atmosphere of5%CO2-95%air at37℃. Human colorectal cancer cells, grown to70%confluence, were irradiated with differential doses at3.2Gy/min with6MV X-rays using a Walian600linear accelerator.24hours later, We collected the total RNA and protein according to the instructions. The miR-221�'�p57kip2mRNA level of Caco2cells were detected by Real-time Q-PCR technology and the p57kip2protein changes was detected by Western-blot. Therefore, we can explore the expression rules of miR-221and its target gene p57kip2under radiotherapy and evaluated the role and clinical significance of miR-221/p57kip2signaling pathways in in colorectal cancer radiotherapy. (2) Antisense miR-221sequence (AS-miR-221) were transfected by LipofectamineTM and blank control group and the negative control group were set up. The miR-221and p57kip2mRNA and protein expression were observed by Real-time Q-PCR and Western-blot to verify the transfection efficiency. To evaluate the influence of cell radiosensitivity of Caco2after inhibiting miR-221gene expression, the clonogenic assay was used to measure the surviving cells of CRC Caco2cells and their enhancement of radio sensitivity under different X-ray exposure dose while calculation of the mean lethal dose (Do) and quasi-threshold dose (Dq) was measured by single hit multi target model. The cells were pretreated with p57-siRNA or a negative control (NC)-siRNA for24h, and then transfected with AS-miR-221. Then the cells were exposed to4Gy radiation and colony assay were used to calculated the cell survival fraction. Thus far, we can evaluate the underlying mechanism of As-miR-221on the radiosensitivity of colorectal cancer cells.(3) Results were expressed as means±S.D. Statistical analyses were performed by one-way ANOVA and ANOVA for factorial designs. Student-Newman-Keuls (S-N-K) test was performed for comparison of subgroups. P<0.05was considered significant. SPSS13.0computer program was used for statistical analysis.RESULTS(1) The miR-221and p57kip2expression changes under different X-ray exposure doseThe result displayed that the miR-221and p57kip2cDNA Show exponential growth and reached a platform period. The amplification curve of miR-221and p57kip2showed a set of typical inverted S curve, which revealed a high transfection efficiency. Then the Ct value of purpose gene amplification in the samples was getted through average value to quantitative determined the miR-221and p57kip2mRNA expression. The miR-221expression were2.29±0.21,1.12±0.14,1.93±0.22,0.54±0.14in HT-29、Lovo、SW-480、Caco2, respectively, which showed significant difference compared with blank control group HUVEC cells. We picked up the medium-expression of miR-221, Caco2cells, for further study. Among0-8Gy, the expression level of miR-221improved with the increasing of radiation dose (P<0.01), while there was no significant changes in the expression of p57kip2mRNA (P>0.05). The protein level of p57kip2reduced with the increasing of radiation dose (P<0.01).(2)The influence of AS-miR-221on the radiosensitivity of CRC cellsAS-miR-221and control sequence were transfected into Caco2cells using the LipofectamineTM RNAiMAX Transfection Reagent, according to the manufacturer’s instruction. Real-time Q-PCR was applicated to detect the miR-221expression and the comparative2-△△CT method was used for relative quantification. The miR-221level in the AS-miR-221group, that was lower than the blank control group and the negative control group (0.38±0.07vs1±0.06and1.17±0.14, respectively; P<0.01), which revealed a high transfection efficiency. We then performed a colony assay to measure the radiation sensitivity of the Caco2cell line and evaluated the effect of AS-miR-221. The SF of AS-miR-221transfected cells was53.83±1.23%,29.04±1.50%,9.06±0.54%and3.61±0.14%under2,4,6and8Gy radiations, respectively. However, the SF of control and control sequence transfected cells was84.24±0.72%and80.84±0.82%,68.38±0.71%and65.47±1.22%,35.73±0.73%and36.22±0.78%, and9.22±0.74%and18.45±0.35%, under the equal doses of radiations respectively. The cell death number in AS-miR-221transfection group increased significantly with the increase of irradiation dose (F=264.47, P<0.01), which revealed that AS-miR-221do have obvious radiotherapy sensitization. The Single Hit Multi Target model showed that the Do values were1.42,1.52and0.94, and the Dq values were2.91,2.77and1.81in the control, the control sequence group and the AS-miR-221group, respectively. The Do and Dq values of AS-miR-221group were smaller than the blank control and negative control groups’ which illustrate that the radiotherapy of cells in AS-miR-221group was obviously higher than that of the rest of the two groups.To prove whether the As-miR-221enhance the radiotherapy of CRC cells through regulating the p57kip2expression, we detect the expression of p57kip2gene.The p57kip2mRNA level in the AS-miR-221transfection group was 1.17±0.12, that was no significant difference compared with the blank control group and the negative control group (1.22±0.19and1±0.17, respectively; P>0.05). Westem-blot showed that the gray-scale value of p57kip2protein was1885.56±13.21, significantly higher than that of the blank control and negative control group (959.64±8.64and823.68±7.98, P<0.05). There was no difference among the groups in p57kip2mRNA level while there was significantly difference in p57kip2protein level, showing that miR-221regulated the p57kip2in the post-transcriptional level. The cells were pretreated with p57-siRNA or a negative control (NC)-siRNA for24h, and then transfected with AS-miR-221. Then the mRNA and protein level of p57kip2were detected by Real-time Q-PCR and Western-blot, respectively. The results showed p57-siRNA could obviously inhibited the mRNA and protein expression in Caco2cells. Then the cells were exposed to4Gy radiation and colony assay showed the SF in p57-siRNA pretreatment group (72.30%±1.37%) was significant lower than that in NC-siRNA pretreatment group (53.83%±1.00%).CONCLUSIONS1. Under different exposure dose of X-ray (0-8Gy), the expression level of miR-221improved with the increasing of radiation dose. There was no significant changes in the expression of p57kip2mRNA while the protein level of p57kip2reduced with the increasing of radiation dose. So far, the result shows that the X-ray can up-regulated the miR-221expression in Caco2cells and down-regulated the p57kip2protein level, while they showed a dose-dependent manner.2. AS-miR-221could have a radiation-sensitizing effect through upregulating p57kip2expression, and the anti-proliferation effect of AS-miR-221on colorectal cancer cells could be partly blocked by the siRNA against p57kip2. This suggests that the inhibitory effect of AS-miR-221on colorectal cancer cells is partially mediated by p57kip2and p57kip2-independent of other pathways that may also be involved in the anti-colorectal cancer effect of AS-miR-221. It is consistent with the present understanding of miRNA that there is not a simple "one-to-one" relationship between the miRNA and its targeting mRNA, but a complex network mode.