The Influence And Mechanism Initially Of Long Non-coding RNA-UCA1 On The Radiosensitivity Of Colorectal Cancer Cells

Objective:To investigate the effect and mechanism of long non-coding RNA-UCA1 on the radiosensitivity of colorectal cancer via screening lncRNAs related to radiosensitivity of CRC cells by high-throughput sequencing.Methods:(1) After radioresistant colorectal cancer cell lines CCL244 and radiosensitive colorectal cancer cell lines HCT116 exposed to 6Gy X-ray, High throughput sequencing was used to screen the differentially expressed long non-coding RNAs.(2) Real-time quantitative PCR was used to detect the expression of LncRNA-UCA1 in colorectal cancer cells and tissues.(3) SiRNA interference sequences were designed and synthesized and then screened the most effective siRNA.(4) After siRNA interference sequences were transfected to cells and then combined with 6Gy X-ray, MTT, colony-forming assay, cell apoptosis assays, cell cycle distribution analysis, cell wound healing assay and western blot assay was performed to detect cell viability, cell proliferation, cell apoptosis ratio, cell cycle distribution, migration and the expression of apoptosis related protein of Caspase 3, Bcl-2, migration associated protein MMP-2, MMP-9 and EMT associated protein ZEB1, Vimentin.Results:(1) LncRNA profiling showed that the change of lncRNA-UCA1 expression level was the most obvious in the first 15 lncRNAs with increased expression level in radioresistant cell lines CCL244, while there was no statistically significant difference in radiosensitive cell lines HCT116.(2) The relative expression of lncRNA-UCA1 in 32 cases of CRC tissues and adjacent normal tissues was 33.24 ± 0.26,(P<0.001). There are about 78.13% of CRC tissues with high level expression. The expression level of lncRNA-UCA1 was higher after accepted neoadjuvant radiotherapy and chemotherapy than before in four pairs of clinical samples. Meanwhile, the relative expression of lncRNA-UCA1 was overexpressed in HCT116, CCL244, SW480 and LOVO cells compared to normal intestinal epithelial cells FHC.(3) Three siRNA interference sequences were transfected to cells separately using Liposome-based transfection method. SiRNA-2 showed the highest efficiency(57%) of transfection compare to other two siRNAs.(4) After down-regulation of lncRNA-UCA1 combined with 6Gy X-ray in CCL244 cells, the cell viability was declined. In addition, the mean lethal dose(D0) were 1.01 Gy and 1.69 Gy respectively. The quasi-threshold dose(Dq) was 1.91 Gy and2.71 Gy, the sensitizing enhancement ratio(SER) was 1.67 and all these results illustrated that down-regulation of lncRNA-UCA1 enhanced the radiosensitivity of CCL244 cells. Also, the cell apoptosis ratio was significantly increased, meanwhile the apoptosis related protein of Caspase 3 and Bcl-2 was separately up-regulated and down-regulated. Moreover, the percentage of cells in G2/M phase was decreased. The migration of CCL244 cells was significantly inhibited[(16.06±1.30)% vs(35.92±1.63)%, P<0.05], meanwhile the migration associated protein MMP-2 and MMP-9, EMT associated protein ZEB1 and Vimentin were also decreased.Conclusions: LncRNA-UCA1 was differently expressed in radioresistant colorectal cancer CCL244 cells before and after irradiation with X-ray. Down-regulation of lncRNA-UCA1 could enhance the radiosensitivity of CCL244 cells.