Knockout Esophageal Squamous Cell Carcinoma Cell Line EC109 DMRT2 Gene By CRISPR/Cas9 Technique And Study The Biological Functions

BackgroundIn China, esophageal cancer is one of the highest incidences of cancer, the main pathology type is still squamous cell carcinoma. Although currently there are a lot of progress in the epidemiology, early diagnosis, comprehensive treatment and prevention of esophageal cancer, but its mortality remains high. Malignant tumor results from both multi-stage progressive development, genetic mutations and environmental factors. Driven by endogenous and exogenous gene mutations of cancer-promoting factors, many tumor cells gradually progress to clinically visible pathological morphology. The activation of oncogenes and inactivation of tumor suppressor genes play an important role in tumorigenesis and progression. In our previous study, using comparative analysis of 47,000 transcripts and variants expression of the CDNA chip of 10 pairs esophageal squamous cell carcinoma tissue and normal tissue we found several upregulated expressed genes: DMRT2, CTTN, MCM10 and SCYA26. DMRT2 gene belongs DM genes family(double-sex and mab-3 relatated transcription factor), which expresses a kind of transcription factor, the main of its function is the regulation of sex determination and differentiation.However, some researches found that in non-small cell lung cancer, breast cancer and renal clear cell carcinoma, expression of DMRT2 gene decreases. In order to investigate the biological function of DMRT2 in esophageal squamous cell carcinoma, we make use of CRISPR / Cas9 technology to kock out and knock down esophageal squamous carcinoma cell line Ec109 DMRT2 gene in this study and inquiry DMRT2 role in esophageal squamous cell by a series of functional experiments. ObjectiveTo build DMRT2 knockout/knockdown esophageal cancer cell lines by CRISPER/Cas9 technique, and investigate the functional role of DMRT2 in cancer cells. Methods(1) Culture five esophageal squamous carcinoma cell lines; Westernblot screened DMRT2 high/low expression cell lines;(2) In DMRT2 exon, designed sg RNA and reporter( containing sg RNA sequence for subsequent screening knock-out/knock-down cells). Construct recombinant plasmid PGL3-U6-sg RNA and pm Cherry- EGFP-reporter. Verify the recombinant plasmids connection. sg RNA/reporter/cas9 plasmid transfected JH293 cells by LIP2000, screening the endogenous activity sg RNA. Furthermore, EGFP plasmid transfected esophageal squamous cells by LIP2000, screening the best transfection proprotion(3) The endogenous activity sg RNA, reporter and Cas9 plasmid(provided by Professor Du of the Sino-British Molecular Laboratory, Zhengzhou University) were co-transfected high expression DMRT2 esophageal squamous carcinoma EC109 cells, flow cytometry sorted single positive cells in 96-well plate, monoclonal cell culture.(4) Extracted monoclonal cell genome, and designed primers to sg RNA upstream and downstream, then pcr the fragment, linked to T-SIMPLE, transformed DH5?,and send to gene sequencing, while Westernblot verified DMRT2 ptotein expression of the monoclonal cells.(5) used the Incu Cyte ZOOM instrument, did cell functional experiments, including cell proliferation assay, cell wound scratch assay, cell invasion assay. In addition, using flow cytometry to detect cell apoptosis. Results(1) semi-quantitative Western Blot analysis showed high DMRT2 expression esophageal squamous cell carcinoma cell lines are EC109, EC9706, K30, HKESC1; while DMRT2 low expression cell line is K510.(2) Designed and screened endogenous activity sg RNA3: accggagctggaagaggacgtctg. Flow cytometer sorted single cell and cultured monoclonal cell. By gene sequencing and Westernblot verification, finally we get one DMRT2 knockout cell line, 4 knockdown cell lines.(3) Selected 1-8(-/-),4-8(+/-)cell lines,wild-type EC109 to do functional experiments. The results are as follows:(1) cell proliferation assay: the cell proliferation ability of EC109 1-8(- /-) and 4-8(+/-) are higher than the wild-type EC109.(2) wound scratch migration assay: observed the migration rate of those cells after scratches, at early stage EC109 cells migrate faster than the others', while after 24 hours the migration rate of EC109 1-8(-/-) and 4-8(+/-)cells faster than EC109.(3)invasion experiment: the three groups' invasion rate have no significant difference.(4) Flow cytometry detect cell apoptosis proportion: we previously induce cell apoptosis by using 0.2mmol/l hydrogen peroxide for 12 hours, then use the apoptosis kit detect the cell apoptosis ratio, and found that the early cell apoptosis ratio of EC109 1-8(-/-)and 4-8(+/-)are higher than EC109 cells. Conclusion1. DMRT2 play a role in tumor proliferation and migration, and suppress tumor cells proliferation and migration.2. DMRT2 also play a role in early apoptosis of tumor cells, and suppress the incidents of early apoptosis.