Down-regulating HIF-1α by lentivirus-mediated shRNA for therapy of triple negative breast cancer

Abstract

Hypoxia is associated with poor response to treatment in various cancers. Hypoxia inducible factor 1 (HIF-1) is a major transcription factor that mediates adaptation of cancer cells to a hypoxic environment and regulates many genes that are involved in key cellular functions, including cell immortalization, stem cell maintenance, autocrine growth/survival, angiogenesis, invasion/metastasis, and resistance to chemotherapy. HIF-1α has been considered as an attractive therapeutic target for cancer treatment, but there is limited success in this research field. In the present study, we designed a recombinant lentivirus containing HIF-1α siRNA, developed stably transfected cell lines, and tested the anticancer effects of the siRNA on cancer cells in vitro and in vivo. Our results indicated that the stable downregulation of HIF-1α reversed chemoresistance, inhibited proliferation, migration and invasion of cancer cells, and slowed down the tumor growth in breast cancer xenograft models. In conclusion, the recombinant lentivirus containing HIF-1α siRNA provides a new avenue for developing novel therapy for triple negative breast cancer.

Keywords: BCSCs, breast cancer stem cells; EGFR, epidermal growth factor receptor; HIF-1α, hypoxia inducible factor-1α; MDR, multidrug resistance; PARP, poly ADP ribose polymerase; PI3K, phosphatidylinositol 3-kinase; TNBC, triple negative breast cancer; VEGF, va; HIF-1α; apoptosis; gene therapy; recombinant lentivirus; siRNA therapy; stably transfected cell lines; triple negative breast cancer.

Figure 1.

The morphology and proliferation of cells treated with HIF-1α shRNA or NC shRNA cells and untreated MDA-MB-231 in vitro. (A) Flurescence produced by GFP can be seen in HIF-1α shRNA and NC shRNA cells (×200). (B) The decreased expression of HIF-1α in HIF-1α shRNA cells. (C) The relative expression of HIF-1α at protein level is inhibited by 95% in HIF-1α shRNA cells in comparison with NC shRNA cells. (D) Cell proliferation assay by the CCK8 showed inhibited cell growth in HIF-1α shRNA cells. *P < 0.05, **P < 0.01.

Figure 2.

HIF-1α shRNA treated cells line showed decreases in migratory and invasive capacity in vitro. (A) Cell invision capacity of HIF-1α shRNA, NC shRNA cells and MDA-MB-231 was determined by transwell assay at 48 h (×200). (B) Quantitative analysis of the cells migration through the transwell chambers. **P<0.01. (C) Wound healing assays were performed at 24 and 48 h in HIF-1α shRNA, NC shRNA cells and MDA-MB-231 (×40). (D) Quantitative analysis of the healing rate of HIF-1α shRNA, NC shRNA cells and MDA-MB-231. **P<0.01.

Figure 3.

HIF-1α shRNA treatement causes apoptosis in MDA-MB-231. Both early and late apoptosis were increased in HIF-1α shRNA cells examined by flow cytometry (*p < 0.05, **p < 0.01).

Figure 4.

Stable downregulation of HIF-1α reduces the number of cells in G1 phase and increases the cell number in G2/M phase. The number of cells in G1/G0 phase in HIF-1α shRNA group is reduced compared with NC shRNA group and MDA-MB-231 group. Moreover, the number of cells in G2/M phase in HIF-1α shRNA group was increased compared with NC shRNA group and MDA-MB-231 (P < 0.05).

Figure 5.

HIF-1α shRNA inhibits tumor growth in vivo. (A) H & E stain and immunohistochemical assay for xenografts tissues. More necrotic tissue was shown in NC shRNA xenografts by H & E stain for more rapidly proliferation. ER, PR and Her2 of 2 groups are all negative. Ki67 in 2 groups are shown to be 90% and have no significant difference (×100). (B) Measure the xenografts every 4 d post letivirus injection and calculate the volume of xenografts. The growth of tumors was significantly inhibited in HIF-1α shRNA group in comparison with NC shRNA group. (C) The weight of HIF-1α shRNA xenografts showed a fold4- decrease compared with that of the NC shRNA. (D) HIF-1α and VEGF expressions at mRNA level in xenografts of HIF-1α shRNA group were significantly decreased compared to NC shRNA group by quantitive real-time PCR. E: HIF-1α and VEGF expressions at protein level in xenografts of 2 groups using Western blot. F: HIF-1α and VEGF expressions at protein level in HIF-1α shRNA group were significantly decreased compared with the NC shRNA group (*P < 0.05, **P < 0.01).