The Role Of Long Noncoding RNA Lnc-RI In Radiosensitivity Of Colorectal Cancer Cells And Its Molecular Mechanism

Colorectal cancer,as one of the common digestive tract malignant tumors,is the third-leading cause of cancer related morbidity in China.Pre-operative radiotherapy is the conventional methods for colorectal cancer therapy,but around 20-40%of colorectal cancer patients exhibit low sensitivity to it or complete resistance.Therefore,it is of scientific significance and clinical value to carry out theresearch on radiosensitivity prediction of colorectal cancer and the related mechanisms.DNA damage repair,especially the DNA double-strands damage repair is the key factor that affect tumor radiosensitivity.In this study,we aim to validate the regulatory function of long non-coding RNA lnc-RI induced by IR to colorectal cancer radiosensitivity,confirm that lnc-RI regulate radiosensitivity of colorectal cancer cells by influencing the DNA damage repair ability,establish gene expression profile of lnc-RI knockdown,conduct bioinformatics analysis of m RNA differential expression,and thus explore the molecular mechanisms of the radiosensitivity of co lorectal cancer cells regulated by lnc-RI.1.By using 60Coγradiation for colorectal cancer cells HCT116、HT29,the CCK-8 assay and clonogenic assay were performed to detect the cell proliferation and cell survival,respectively.The results demonstrated that the radiosensitivity of HCT116 were greater than that of HT29.The relative expression of lnc-RI was detected by q RT-PCR,which certified that the expression of lnc-RI was negatively related to the radiosensitivity in colorectal cancer cells.Several parameters were tested for radiosensitive cells HCT116 and radioresistant cells HT29,such as cell cycle block by flow cytometry,γH2AX expression by Western blot assay,γH2AX foci in the nuclei by indirect immunofluorescence assay.The corresponding results certified that the DNA damage repair ability of HT29 was greater than that of HCT116,indicating that lnc-RI may regulate radiosensitivity of colorectal cancer cells by affecting the abilit y of DNA damage repair.2.Using RNA interference technology,two specific interference sequences（sh RNA-lncRI-1and sh RNA-lncRI-2）for lnc-RI were designed to package lentivirus and infect HCT116 and HT29 cells to create the lnc-RI low-expression cell lines.The HCT116 and HT29 cell of lnc-RI knockdown were exposed to ⁶⁰Coγradiation.The corresponding the cell proliferation and cell survival by CCK-8 assay and clonogenic assay certified that the lnc-RI knockdown expression can increase radiosensitivity of colorectal cancer cells.Besides that,γH2AX expression by Western blot assay,γH2AX foci in the nuclei by indirect immunofluorescence assay proved that lnc-RI knock-down inhibited the DNA double-strands damage repair induced by IR in colorectal cancer cells,thus proving that the lnc-RI knockdown expression can increase the radiosensitivity of colorectal cancer cells by inhibiting the DNA double-strands damage repair ability.3.The gene expression profiling of lnc-RI depletion HT29 cell was performed and analyzed by employing the gene microarrays technology,and thus analyzed the related molecular mechanism of lnc-RI regulated DNA damage repair in genomics.Differential m RNA expression profile analysis showed that the knockdown of lnc-RI expression yielded 2 241 differentially expressed genes,of which 942 genes were up-regulated and 1 299 genes were down-regulated.And the GO database and Pathway signaling analysis demonstrated that lnc-RI was related to DNA replication,cell cycle arrest,p53-apoptotic pathway and DNA damage repair.The down-regulated differentially expressed genes have 24 genes that are related to homologous recombination and non-homologous end joining.Besides that,we found that the related downstream target gene of lnc-RI in DNA damage repair influences DNA damage repair by regulating LIG4 expression,and thus influence the radiosensitivit y of colorectal cancer cells.This study established radiosensitive cell model for colorectal cancer cells and proved not only that long non-coding RNA lnc-RI negatively regulate the radiosensitivity of colorectal cancer cells,but also that lnc-RI regulate radiosensitivit y of colorectal cancer cells by influencing DNA damage repair.Besides that,the study of biofunction of lnc-RI from genomics lays the necessary foundation for further exploring the molecular mechanism of lncRNA regulated radiosensitivity.