TALEN-mediated MicroRNA-21 Gene Knockout In HeLa And OCI-Ly3 Cancer Cell Lines

Objective 1. Apply TALEN-mediated gene depletion strategy to knockout(KO) micro RNA-21(mi R-21) gene in He La human cervical carcinoma cells and OCI-Ly3 human diffuse large B-cell lymphoma(DLBCL) cells. Investigate the feasibility of establishing stable micro RNA knockout mammalian cancer cell lines by using TALEN. 2. Compare the phenotype and genotype difference between wild type(WT) and mi R-21 KO cells. Study the role of mi R-21 in human tumorigenesis and try to reveal new diagnostic or therapeutic targets. Methods 1.Three pairs of TALENs were designed and made to cut the seed sequence of mi R-21(TALEN pair1,P1), the loop sequence of the pre-mi R-21(TALEN pair2,P2) and a downstream sequence of pre-mi R-21(TALEN pair3,P3).2.He La cervical carcinoma cells and OCI-Ly3 diffuse large B-cell lymphoma cells were transfected with TALENs. Surveyor assay was employed to estimate the TALEN-induced mutation efficiency. 3.Single-cell-derived clones were screened for mi R-21 deletion by genomic DNA PCR. 4.Deep sequencing and real time RT-PCR assays were used to confirm the knockout of mi R-21. 5.Proliferation inhibition of mi R-21 deleted He La cells was detected by both MTS assay and monolayer colony formation assay. Soft-agar colony formation assay was performed to test the reduction of tumorigenesis. Cisplatin sensitivity was determined by MTS assay and Annexin-V/PI double staining analysis.6.Mi R-21 targeting gene profiling was analyzed by m RNA deep sequencing. And some up-regulated mi R-21 target genes were verified by real time RT-PCR and Western blot. Finally,NOD/SCID/IL2 R gamma null(NSG) mice were xenotransplanted to characterize tumorigenic properties of mi R-21 knockout He La cells.Results 1.Successful construction of three pairs of TALENs targeting different mi R-21 locus(TALEN pair1 to seed sequence of mi R-21; TALEN pair2 to loop sequence of pre-mi R-21 and TALEN pair3 to a downstream sequence of pre-mi R-21)were confirmed by restriction enzyme analysis and DNA sequencing. 2. Surveyor mutation detection assay showed in He La cells mutation rate of TALEN P1 is about 5.1%, P2 is about 7.0% and P3 is about 7.2%.Furthermore, when combining two pairs together in one time cell transfection, the mutation rate increased to 8.4% and 14.0% with P1 plus P3 and P2 plus P3. 3. To disrupt or delete mi R-21 sequence, we performed“double-digestion” by co-transfecting He La cells with TALEN P1 and TALEN P3. Single cell derived clones were screened by genomic DNA PCR. We found 11 clones carrying a deletion from 92 clones screened. We then performed quantitative RT-PCR to measure the mi R-21 level in these clones and further characterized clones #38, #39, #75, and #92 by TA cloning and sequencing. The He La cells used appeared to have 3 copies of the mi R-21 gene. The amount of mi R-21 in clone #75 is dramatically decreased and is below 0.1% of the level expressed in the parental line. 4. Micro RNA deep sequencing and the expression of a luciferase reporter with a mi R-21 target sequence confirmed the KD of mi R-21 in He La#38 and the extremely low level of mi R-21 in He La #75. 5. In order to obtain complete mi R-21 knockout clones, we performed a second round of TALEN-triggered gene disruption on clone #38, which has two mi R-21 deletion alleles and one functional mi R-21 allele. We screened 164 single cell derived clones by PCR and sequencing, and found 3 mi R-21 KO clones:#3837,#3838 and #38124. 6. Both MTS assay and monolayer colony formation assay indicated the loss of mi R-21 decreases the He La cells proliferation. Soft-agar colony formation assay showed the loss of mi R-21 decreases the ability of anchorage-dependent growth. 7. MTS assay and Annexin- V/PI double staining analysis after Cisplatin treatment showed He La cells become more sensitive to this drug after mi R-21 KO. 8. Deep sequencing of m RNA to compare the gene profile between WT and #75、#3837 by Sylamer analysis indicated that loss of mi R-21 resulted in a global increase of m RNAs carrying a mi R-21 target sequence. 9. Gene expression changes were determined by comparing each of the knockout clones(#75 and #3837) with the parental line; 165 genes were found commonly up-regulated by more than 2 fold. There are more than 230 human genes carrying mi R-21 target sequences in their 3’ UTR and considered as mi R-21 target genes in the Target Scan or mi Records database; 10 of those genes were in the up-regulated gene list.We selected 4 of those genes : ACTA2, RECK, TAGLN, and TGFB2,and analyzed the RNA levels by real-time RT-PCR;all four RNAs were verified to be up-regulated in the knockout clones. 10. Western bolt to detect the expression of two well-characterized mi R-21 regulated genes PDCD4 and PTEN showed their expression level significantly increased in mi R-21 KO clones:#75, #3837 and #3838. 11. Xenotransplantation into immunodeficient NSG mice show: Although the tumor size was not significantly different, the xenograft derived from He La #75 appeared to be less proliferative than the xenograft derived from WT or #39 : the cell density was lower, the cells and nuclei were smaller, the nucleoli were less prominent, and there were fewer mitotic figures. 12. In order to obtain mi R-21 null OCI-Ly3 clones to study mi R-21 function in DLBCL, we co-transfected OCI-Ly3 cells with TALEN P1 and P3 by nucleofector.Single-cell-derived clones were screened by genomic DNA PCR.Four mi R-21 KO clones :#33,#64,#104 and #126,were isolated after two-round screening. Conclusion To investigate mi R-21 function in human cancer cells, we constructed 3 pairs of mi R-21 targeting TALENs and used them to delete the mi R-21 sequences in He La cells and OCI-Ly3 cells. By analyzing singlecell-derived mi R-21 knockout clones, we found He La cells lacking mi R-21 were less proliferative and more sensitive to genotoxic agents. We also compared the gene expression profiles of the mi R-21 knockout clones and the parental line by RNA deep sequencing. Genes and pathways that are involved in signaling, cell adhesion and motility, extracellular matrix, and angiogenesis and blood circulation were significantly affected by the loss of mi R-21. Our study demonstrates that the function of a micro RNA gene can be studied in human cancer cells using TALEN-induced gene disruption, while also revealing mi R-21 regulated pathways that may be critical in cancer.