Screening Of MicroRNAs Associated With Melanoma Cells Against T Cell Killing Using CRISPR/Cas9 Library

Melanoma is a very aggressive tumor,and its 5-year survival rate is less than 5%.With the rapid development of treatment methods such as BRAF and MEK inhibitors,immune-targeted therapies and immune checkpoint inhibitors,the survival rate of melanoma patients has been greatly improved.Although immunotherapy has a long history in the treatment of melanoma patients,the immune escape caused by the tumor leads to the failure of immunotherapy.People have not yet learned too much about tumor immune escape,and many human cancers are resistant to immunotherapy.Micro RNA is an endogenous non-coding RNA with a length of about 21 to 23 nt in eukaryotic cells.The aberration of micro RNA expression was frequently observed in human tumors,and it also plays a vital role in the process of immunotherapy.Micro RNA serves as an important biomarker in the diagnosis and prognosis of tumors,and it could affect multiple important immune-related targets for the process of tumor immunity.Micro RNA can cause tumor immune escape by affecting the immunogenicity of tumor cells.It can also regulate the body’s immune response by balancing the tumor microenvironment or regulating the growth and development of immune-related cells.Through the study of micro RNAs regulating tumor immunogenicity and the resistance mechanisms that occur in tumor cells when cytotoxic T cell-mediated killing is performed,it will play an important role in the further research of immunotherapy.ObjectiveCRISPR/Cas9 is a very efficient tool in the field of gene editing.The rapid development of this technology in the medical field has played an important role in discovering more potential tumor immunotherapy targets.CRISPR/Cas9 whole genome screening has been used to search for genes related to tumor cell resistance,tumor cell proliferation and migration.Based on the important role of micro RNAs in tumor immune regulation,we designed CRISPR/Cas9 micro RNA libraries to unbiasedly search for micro RNAs related to cytotoxic T cell killing.Our study may provide new targets for tumor immunotherapy through research on related mechanisms.Method1.We designed a genome-wide library containing all human micro RNA-related sg RNAs,and obtain a CRISPR/Cas9 library with high quality.2.We used a multiple of low viral infections to infect Cas9-expressing A375 cell line,using puromycin to select and obtain micro RNA knockout cell lines.After gradient centrifugation,cytotoxic T cells were obtained by immunomagnetic bead sorting.The A375 Cas9 cell line infected with the library virus was screened by using cytotoxic T cells.After the screening is completed,the genome is extracted to confirm the abundance of the library,and the region targeted by sg RNA is PCR amplified.After the second-generation sequencing is completed,we use MAGe CK VISPR to visualize the sequence quality of the clean sample.After the quality control is qualified,we use the MAGe CK tool to count and standardize the sg RNA of the sample,and use the MAGe CK RRA to calculate the difference in sg RNA enrichment between the experimental group and the control group to obtain the final screening results.3.We constructed a micro RNA18 a knock-out cell line,and obtained a stable knock-out monoclonal strain through expansion culture with limited dilution method,and performed RNA-SEQ analysis on the knock-out cell line.In the process of RNA-SEQ analysis,we first quantified the gene expression level,using Bioconductor DESeq2 to calculate the gene analysis of differential expression between the experimental group and the control group.We compared the overlapped differentially expressed genes between the experimental group and the control group,and searched for the related genes of micro RNA18 a affecting the resistance to cytotoxic T cell killing among the overlapping differential genes.Using GO enrichment analysis and KEGG signaling pathway analysis were used to predict the target genes of micro RNA106 b.Result1.After the relevant process and quality inspection of our CRISPR/Cas9 micro RNA library design,according to the second-generation sequencing results,the designed CRISPR/Cas9 micro RNA library has an accuracy rate of 83.8%,a coveragerate of 100%,a homogeneity of 4,and a coverage factor of 162.We have designed a total of 2196 micro RNA related sg RNAs library,covering almost the latest and the most complete micro RNA.2.We used a multiple of low viral infections to infect Cas9-expressing A375 cell line,using puromycin to select and obtain micro RNA knockout cell lines.After gradient centrifugation,cytotoxic T cells were obtained by immunomagnetic bead sorting.The A375 Cas9 cell line infected with the library virus was screened by using cytotoxic T cells.After the screening is completed,the genome is extracted to confirm the abundance of the library,and the region targeted by sg RNA is PCR amplified.After the second-generation sequencing is completed,we use MAGe CK VISPR to visualize the sequence quality of the clean sample.After RNA-SEQ analysis and target gene prediction,micro RNA18 a may affect the killing of melanoma cells by cytotoxic T cells by targeting THBS1.Finally,through GO enrichment analysis and KEGG signal pathway analysis,the possible role of micro RNA106 b was discussed.3.We successfully constructed a monoclonal cell line of micro RNA18 a and analyzed RNA-SEQ of the knocked out cell line.The RNA-SEQ analysis results of the two monoclonal cell lines knocked out in micro RNA18 a were overlapped.After RNA-SEQ analysis and target gene prediction,we speculate that micro RNA18 a may affect the melanoma EMT process by targeting THBS1,and then affect the killing of melanoma by cytotoxic T cells.We also discussed the possible role of micro RNA106 b through GO enrichment analysis and KEGG analysis.Conclusion1.Successfully constructed a library of 2196 micro RNA-related sg RNA,which almost covers the latest and most complete micro RNA,and laid a solid foundation for the future selection of related micro RNA.2.After knocking down micro RNA18 a,it may affect the killing of melanoma cells by cytotoxic T cells by up-regulating the expression of THBS1 and affecting the EMT process of melanoma.