The Construction Of Coding/Non-Coding Gene Knockout System By CRISPR-Cas9

OBJECTIVE: The CRISPR-Cas9 system was respectively used to knock out coding gene MKL1 and long non-coding RNA HOTAIR.In order to study the function of MKL1 and HOTAIR in cervical cancer,MKL1-Deleted and HOTAIR-Deleted HeLa cell lines were constructed to expand the application of the system.Subsquently,the two kinds of HeLa cell lines were used to examine the effect of MKL1 and HOTAIR on cell proliferation and migration,and to associate them with tumor suppressors to put forward new directions for their functional and mechanical investigation.METHODS: Firstly,Cas9 nuclease was stably expressed in HeLa cells.Secondly,according to the genomic sequences of MKL1 and HOTAIR,the pgRNA library were designed and the expression plasmids were constructed and transfected into Cas9-expressed HeLa cells.Through drug screening,the knockout of MKL1 and HOTAIR was verified by genomic PCR test,western blot analysis and qRT-PCR analysis.The MTT assay and scratch wound healing assay were used to detect respectively cell proliferation and migration.In addition,total RNA was extracted from them and was tested the transcriptional level of tumor suppressor p21,p53,pRB and DLC1.For further study about MKL1 regulating DLC1,the DLC1 promoter sequence was detected by bioinformatics analysis,and expression level of DLC1 was examined after overexpressing MKL1.Luciferase reporting assay and ChIP assay was useding to confirm the control effect of MKL1 to DLC1.Results: HeLa cell line stably expressing Cas9 nuclease was successfully constructed.After transfection of the corresponding pgRNA,Western Blot analysis showed that MKL1 was knocked out,while qRT-PCR analysis showed that lncRNA HOTAIR was down-regulated by 50%.Subsequently,MTT assay showed that MKL1 knockout and HOTAIR down-regulated HeLa cells were less than normal HeLa cells in one week of proliferation,and scratch wound healing assay showed the wound healing rates of two deleted groups were both slower than that of normal HeLa cells.In addition,qRT-PCR analysis showed that at transcriptional level,there was no significant change in p53,DLC1 down-regulated 80%,p21 and pRB were up-regulated 1.5 folds when MKL1 was absent.However,DLC1 and p53 transcription levels both decreased with HOTAIRdown-regulation,while p21 and pRB were up-regulated,significantly.Bioinformatics analysis showed that the promoter of DLC1 upstream 1000 bp contained two CArG box,and DLC1 transcriptional level was up-regulated upon overexpression of MKL1.ChIP assay and luciferase reporting assay indicatied that MKL1 could bind with DLC1 promoter,and any mutation of two CArG box sequence would inhibit the transcription of DLC1.Conclusion: In the CRISPR-Cas9 system,applying pgRNA can knock out MKL1 significantly,while the knockout of lncRNA HOTAIR is not achieved,which only appeared the effect of down-regulation.The reason need explain with further study.In addition,knockout of MKL1 and down-regulation of HOTAIR can both inhibit the proliferation and migration in HeLa cells,and influenced the transcription level of different tumor suppressor genes.It indicated that MKL1 and HOTAIR could participate in the control of cell cycle by regulating tumor suppressors,and it offered new direction for investigation of their mechanism.For example,the study discovered MKL1 could promote DLC1 transcription by targeting CArG box on the promoter of DLC1.These results revealed the value and limitation of utilizing pgRNA-Cas9 system to edit genes.Additionally,the other associations between MKL1 or HOTAIR and tumor repressors remain to be explored in next investigation.