Lentivirus-mediated Knockdown of HDAC1 Uncovers Its Role in Esophageal Cancer Metastasis and Chemosensitivity

Abstract

Histone deacetylationase 1 (HDAC1) is ubiquitously expressed in various cell lines and tissues and play an important role of regulation gene expression. Overexpression of HDAC1 has been observed in various types of cancers, which indicated that it might be a target for cancer therapy. To test HDAC1 inhibition for cancer treatment, the gene expression of HDAC1 was knockdown mediated by a lentivirus system. Our data showed the gene expression of HDAC1 could be efficiently knockdown by RNAi mediated by lentivirus in esophageal carcinoma EC109 cells. Knockdown of HDAC1 led to significant decrease of cell growth and altered cell cycle distribution. The result of transwell assay showed that the numbers of cells travelled through the micropore membrane was significantly decreased as HDAC1 expression was knockdown. Moreover, HDAC1 knockdown inhibited the migration of EC109 cells as determining by scratch test. Additionally, enhancement of cisplatin-stimulated apoptosis was detected by HDAC1 knockdown. Our data suggested inhibition of HDAC1 expression by lentivirus mediated shRNA might be further applied for esophageal cancer chemotherapy.

Keywords: EC109 cells; chemosensitivity; histone deacetylationase; lentivirus.

Figure 1

Construct of HDAC1 shRNA expression lentivirus. A: diagram of the shuttle vector pGCSIL-GFP. B: The lentivirus package 293T cells were transfected with recombinant vector and helper plasmids. GFP-positive cells were visible 24h after transfection in the package cells.

Figure 2

Knockdown of HDAC1 in EC109 cells. A: EC109 cells were infected with the lentivirus and GFP-positive cells were visible after screening with puromycin. B: The mRNA level of HDAC1 was determined by realtime RT-PCR. The gene expression was normalized to the corresponding GAPDH level and fold changes was calculated by the 2^-△△Ct method according to reference sample and amplification efficiency. C: The protein level of HDAC1 was detected by Western blotting. Western blotting was repeated at least three times. *P< 0.05 by t-test.

Figure 3

Knockdown of HDAC1 inhibited cell growth and cell cycle distribution. A: HDAC1 knockdown EC109 cells and its control cells (NC) were seeded into 96-well plate and cell growth was determined by CCK-8. B: Cells cycle distribution was performed as described in Material and Methods. Experiments were repeated at least three times. *P< 0.05 by t-test. C: Typical cell distribution detected by FACS.

Figure 4

Knockdown of HDAC1 inhibited cell migration and invasion. A: HDAC1 knockdown EC109 cells and its control cells (NC) were plated in Matrigel-coated transwell. The lower side of the transwell membranes were fixed, stained and counted. B: Cell migration of EC109 cells was determined by scratch test. *P< 0.05 by t-test.

Figure 5

Knockdown of HDAC1 enhanced cisplatin-stimulated apoptosis. A: HDAC1 knockdown EC109 cells and its control cells (NC) were seeded into 96-well plate and further treated with cisplatin for 24h. Cell viability was detected by clonogenic assay as described in Material and Methods. B: Similarly treated cells were harvested for Annexin-V/FITC staining and analyzed by FACS. *P< 0.05 by t-test. C: The protein level of active caspase-3 was detected by Western blotting. Western blotting was repeated at least three times.