Construction of HDAC6 knockout human liver cancer cell line and detection of its biological functions

  Background  The incidence and mortality rate of liver cancer are increasing year by year. It is particularly important to study the mechanism of hepatocarcinogenesis and its development.

  Objective  To construct the histone deacetylase 6 (HDAC6) gene knockout HepG2 stable expression cell line by using CRISPR/Cas9 genome engineering technology, and detect its effect on the growth of human hepatoma cells.

  Methods  According to the CRISPR/Cas9 target design principles, a single guide RNA (sgRNA) sequence that specifically recognizing the HDAC6 was designed for construction of the eukaryotic recombinant expressional plasmids LentiCRISPR-HDAC6-sgRNA. After sequencing, lentivirus was produced with the recombinant plasmid and infected HepG2 cells. The stable HDAC6 knockout cells were screened through the stress of puromycin, and the knockout effect was detected by sequencing and Western blotting. CCK8 growth curve test was used to detect the effect of HDAC6 gene knockout on cell proliferation, scratch test and Transwell migration test for detecting the effect of HDAC6 gene knockout on cell migration. Western blot was used to identify the effect of HDAC6 gene knockout on the regulation of α-tubulin acetylation. The sensitivity of HepG2 to HDAC6 inhibitor ACY-1215 after HDAC6 gene knockout was detected by cell survival curve experiment, and the effect of HDAC6 gene knockout on HepG2 cell cycle distribution and apoptosis was detected by flow cytometry.

  Results  The stable HDAC6 knockout HepG2 cell line was constructed successfully, HDAC6 knockout could significantly inhibit cell proliferation and migration, enhance the expression of α-tubulin acetylation and reduce the sensitivity of HepG2 cells to ACY-1215. And it could also cause the G1 phase block of HepG2 cells and promote the apoptosis.

  Conclusion  Our experiment demonstrates that knockout of HDAC6 can inhibit HepG2 cell proliferation, migration, increase the acetylation of α-tubulin, adjust the sensitivity of HepG2 cells to ACY-1215, and affect the cell cycle and apoptosis, which provides references on the biological function of HDAC6 in hepatocarcinogenesis.