Screening And Functional Study Of Radiation-related LncRNA Based On CRISPRa System

Research BackgroundLong noncoding RNA(lncRNA)is a type of non-coding RNA with a length of more than 200 nucleotides,which regulates gene expression through various mechanisms in biological processes.Ionizing radiation(IR)is a general term for radiation that can cause the ionization of substances,and it can affect the body through direct,indirect and bystander effects.Recently,it has been found that lncRNA play a regulatory role in the process of damage and repair caused by IR.Currently,the function of lncRNA in IR regulation is rarely studied,Due to the complexity of the structure and mechanism of action of lncRNA.CRISPR library screening technology allows systematic analysis and validation of a number of genes related to screening conditions through large-scale screening.Therefore,lncRNA genes involved in IR regulation can be screened on a large scale using this technique,and their functions and mechanisms in radiation damage and repair can be further explored.The CRISPR/Cas9-mediated homologous directed repair mechanism can accomplish the premature termination of lncRNA transcription by inserting a screening marker and transcription termination signal(ploy A signal)into the target lncRNA,which is an important tool for lncRNA loss-of-function study.The study is divided into two parts,the first part is the screening and functional study of radiation-related lncRNA based on CRISPRa system,the second part is construction of long noncoding RNA SNHG1 knockout cell line using CRISPR/Cas9 system and its function detection.Research Objectives1.In A549 cells,to screen radiation-related functional lncRNA from 4766 lncRNAs in the National Center for Biotechnology Information(NCBI)database,based on the CRISPR gene activation library.And,to verify and analyze the function and mechanism of some lncRNAs in the process of radiation damage.2.To construct lncRNA small nucleolar RNA host gene 1(SNHG1)knockout A549 cell line using CRISPR/Cas9-mediated homology-directed repair mechanism and to detect biological function of SNHG1.Research Methods1.Screening and functional study of radiation-related lncRNA based on CRISPRa system.(1)The CRISPR lncRNA activation lentivirus library was constructed,and the successfully constructed A549-d Cas9-VPR transcription-activated cells were infected lentivirus library.The radiation-related lncRNAs were screened by conventional cell proliferation and cell colony formation assay two ways.(2)Cells screened on day 0 as background interference removal and cells screened on day 14 were collected,cellular genomicDNA was extracted,sgRNA sequences were amplified.After that,high-throughput sequencing and bioinformatics analysis were conducted to identify candidate radiation-related lncRNAs.(3)Cell colony formation assay was performed to verify the radiosensitivity of candidate lncRNAs,CCK-8 assay and flow cytometry to detect the effects of candidate lncRNAs on cell proliferation and apoptosis,and transcriptome sequencing to analyze the potential molecular mechanisms of candidate lncRNAs.2.Construction of long noncoding RNA SNHG1 knockout cell line using CRISPR/Cas9 system and its function detection.(1)Small guide RNA(sgRNA)targeting the SNHG1 gene was designed and cloned into the Lenti CRISPR v2 plasmid,named Cas9-sgSNHG1 plasmid.The5’homology arm(5’Arm)and 3’ homology arm(3’Arm)sequences were amplified from A549 cell genome DNA and p EGFP-Blast-poly A sequence was amplified from p EGFP-Blast plasmid.The three sequences were linked by overlap PCR into5’Arm-p EGFP-blast-polya-3’Arm.Gbison clone to construct SNHG1 homologous recombinant template plasmid.(2)SNHG1 homologous recombinant template and Cas9-sgSNHG1 plasmid were co-transfected into A549 cells to construct SNHG1 stable knockout A549 cell line.The effect of SNHG1 knockout on proliferation and the ability of clone formation of A549 cells was determined by CCK-8 assay and cell colony formation assay.Research Results1.Screening and functional study of radiation-related lncRNA based on CRISPRa system.(1)1035 radiation-resistant lncRNAs and 461 radiation-sensitive lncRNAs were obtained by cell proliferation screening.1329 radiation-resistant lncRNAs and 177radiation-sensitive lncRNAs were obtained by cell colony formation assay.After comprehensive analysis,5 radiation-sensitive lncRNAs and 2 radiation-resistant lncRNAs were selected for radiation sensitivity verification.(2)The analysis of cell proliferation and cell colony formation assay results showed that overexpression of LINC02606,LOC101928782,LOC401312 and IPO9-AS1 could enhance the sensitivity of A549 and other cells to IR.(3)The effect of candidate lncRNAs on apoptosis under radiation conditions was analyzed by flow cytometry,and the results showed that increased radiation-induced apoptosis in LOC401312 and IPO9-AS1 overexpression cells.2.Construction of long noncoding RNA SNHG1 knockout cell line using CRISPR/Cas9 system and its function detection.(1)Cas9-sgSNHG1 and SNHG1 homologous recombinant template plasmids were constructed,and A549 cell lines with high knockout efficiency were screened.(2)The knockout of SNHG1 inhibited the cell proliferation and the ability of clone formation of A549 cells.Research Conclusions1.Screening and functional study of radiation-related lncRNA based on CRISPRa system.(1)In this study,A series of potential lncRNAs affecting radiosensitivity were screened using the CRISPRa system,and the radiosensitivity validation results showed that LINC02606,LOC101928782,LOC401312 and IPO9-AS1 had radiation sensitization effect.(2)Functional studies revealed that LOC401312 and IPO9-AS1 promoted IR-induced apoptosis,suggesting that these two lncRNAs could possibly affect the radiosensitivity of A549 and other cells through regulation of apoptosis pathway,and the specific mechanism needs to be further studied.2.Construction of long noncoding RNA SNHG1 knockout cell line using CRISPR/Cas9 system and its function detection.(1)SNHG1 knockout A549 cell lines were constructed,and the function of SNHG1 oncogene was inhibited.(2)The CRISPR/Cas9-mediated homologous targeted repair technology can effectively inhibit the expression of lncRNAs,which can be widely used for functional studies of lncRNAs.