Analysis Of Expression Differences Of MiRNA Sequencing Based On CLIC1 Gene Knockout In Gastric Cancer Cells

Objective: 1.To compare the differential expression of miRNA in the genomes of three groups of cells(gastric cancer cell line mgc-803,highly metastatic gastric cancer cell line mgc-803-l,mgc-803-clic1-ko);2.The differential expression of exosome miRNA in cell lines mgc-803 l and CLIC1 was compared with that in cell lines mgc-803-loe and CLIC1 knocked out cell lines MGC-803-CLIC1-KO.Methods: 1.In the early stage,the research group transplanted tumor cells on the plantar of nude mice,repeatedly extracted tumor cells from the popliteal lymph nodes of nude mice,and established the highly metastatic gastric cancer cell line mgc-803-l with significantly higher invasion and migration capacity than the parental cells.2.According to zhang feng’s Human Ge CKOv2 library,the sg RNA sequence of CLIC1 gene was searched,constructed on the CRISPR/Cas9 vector GV392 plasmid,and transfected into 293 T cells.Sg RNA with the highest DNA cutting activity was determined by Sanger sequencing;MGC-803-L cells were transfected with vector plasmid by limited dilution method,and the cells screened by puromycin for 48 h were inoculated in96-well plates.Monoclonal cell lines were obtained by limited dilution method.WB validated the expression of CLIC1 protein,extracted genomic DNA from the completely unexpressed monoclonal cell lines,Sanger sequencing and TA cloning to analyze the genotypes of the monoclonal cells that knocked out CLIC1,and the mgc-803-lko cells that knocked out the CLIC1 gene were the most stable.3.The genomes of MGC-803 parent cells,MGC-803-L and MGC-803-LKO cells were extracted for miRNA sequencing and rt-pcr to verify the differentially expressed mirnas.4.The above three groups of cells were expanded for culture,and mycoplasma scavenger was added to culture for one week to kill mycoplasma.Then 100 ml of cell supernatant was taken and exosomes were extracted by ultra-high speed gradient centrifugation.Mi RNA was extracted from exosomes for miRNA sequencing and verified by PCR for differentially expressed miRNA,and then combined with miRNA at the cell level.Results: 1.After the elimination of CLIC1 gene by CRISPR/Cas9 technique,Western Blot results of mgc-803-ko cells and mgc-803-l cells showed that the expression of CLIC1 gene was significantly reduced(p < 0.001).2.High metastatic gastric cancer cell line MGC-803-L,MGC-803 L-CLIC1-KO cell genome sequencing of micrornas,the two groups compare the MGC-803-L and MGC-803 L-CLIC1-KO sequencing results of two groups of cells,a total of 69 differentially expressed genes,randomly selected high expression quantity of seven micrornas,custom-made primers,cellular level to test whether the miRNA expression differences consistent with the results of sequencing,the selected two groups of micrornas,Hsa-mir-122-5p and hsa-mir-146a-5p,respectively.3.The sequencing results of mirnas from the superexosomes of the MGC-803,MGC-803-L,and mgc-803l-clic1-ko groups were compared,and a total of 145 differentially expressed miRNAs were found.Fifteen mirnas with high expression levels and large differences were randomly selected for validation(hsa-mir-100-5p,hsa-mir-182-5p,hsa-mir-183-5p,hsa-mir-10a-5p,hsa-mir-23b-3p,hsa-mir-148b-3p,hsa-mir-22-3p,hsa-mir-486-5p,hsa-mir-193b-3p,hsa-mir-122-5p,hsa-mir-122-5p,hsa-mir-122-5p,hsa-mir-3p)Mir-142-5p,hsa-mir-331-3p,hsa-mir-145-5p,hsa-mir-134-3p)miRNA primers were designed to extract cell supernatant exosomes,and the trend of the above-mentioned miRNA was verified by rt-pcr.The results showed that the expression trend of four groups of miRNA was consistent with the sequencing results,namely: hsa-mir-22-3p,hsa-mir-122-5p,hsa-mir-142-5p,and hsa-mir-146-5p,respectively.4.Based on the results of miRNA sequencing at the cell level and exosome level,the differentially expressed mirnas were further studied.Conclusions:1.Successfully constructed CLIC1 knocked out high-cell line mgc-803-l,laying the foundation for further research on the role and mechanism of CLIC1 gene in gastric cancer.2.Hsa-mir-22-3p,hsa-mir-122-5p,hsa-mir-142-5p and hsa-mir-146-5p may be potential markers for gastric cancer cells.