TALEN based HPV-E7 editing triggers necrotic cell death in cervical cancer cells

Abstract

Human Papillomavirus E7 and E6 oncoproteins have been considered as suitable candidate anti-viral targets since they cause malignant conversion in cervical cancers. Transcription Activator-Like Effector Nucleases (TALENs) are recent editing tools to knockout genes by inducing double stranded breaks at specific sites in the genome. In here, we have designed specific TALENs to target E7 and analyzed their efficiency in inducing cell death in cervical cancer cells. We found that designed TALENs could yield about 10-12% editing activity as observed from T7E1 and nuclease resistance assays. Down-regulation of E7 and E6 was further evident at the transcript as well as proteins levels indicating that the selected TALENs were effective. TALEN-mediated E7 editing led to cell death as ascertained by cell cycle and Annexin V assays. Annexin profiling suggested that cell death could be due to necrosis as observed by upregulation of necrotic markers such as LDH A, Rip-1, and Cyclophilin A. Necrosis appears to be a better therapeutic response as it could further activate pro-inflammatory cytokines to attract immune cells to eliminate HPV-integrated cells and therefore TALEN editing strategy has the potential to be a promising tool as an adjuvant therapy in cervical cancer along with surgery.

Figure 1

Designing of TALENs targeting the HPV 16 E7 gene. (A) Schematic of TALEN binding site on E7 gene. (B) TALEN predicted activity using SAPTA software. (C) Part of E7 sequence targeted by TALEN.

Figure 2

Selected TALEN pair could effectively target E7 in SiHa cells in vitro. In order to check the designed TALENs were able to target HPV-E7, SiHa cells were transfected with TALENs and endogenous gene disruption of E7 gene was assessed by (A) T7E1 assay indicating a cut product at 258 bp size run on a 2% agarose gel, and (B) Nuclease resistance assay showing a product of 500 bp obtained upon digestion with HpyChIII on E6-E7 gene of size 775 nucleotides. Immunocytochemical analysis showed the presence of 53bp1 in TALEN-treated SiHa cells (D) which was absent in SiHa control cells (C) and HPV^−ve C33A cells (E). Magnification 200X.

Figure 3

TALEN-mediated E7 editing reduced E7 gene expression in SiHa cells. (A) RT-PCR analysis for the transcripts corresponding to E7 and E6 showed a significant reduction in treated (TALEN-transfected) group when compared to controls. (B) Graph represents expression levels of E7 and E6 in control and treated groups. RT-PCR data was further corroborated by western blot analysis which showed a significant reduction in the levels of both E7 and E6 after TALEN treatment (C). Graph depicts the densitometric analysis of western blots of E7 and E6 against β-actin protein levels (D). Immunocytochemical analysis also indicated that E7 protein levels were significantly decreased in TALEN-treated SiHa cells (H–J) when compared to controls (E–G). Further, analysis of E7 expressing cells by flow cytometry also revealed a decrease in the percentage E7 expressing cells in TALEN-treated group (L) than in controls (K). Western blot analysis of pRb and p14ARF suggested that the levels of pRb were increased and that of p14ARF was reduced in TALEN-treated cells when compared to controls (M). The densitometric analysis of the western blots was given in graph (N). Data are expressed as mean ± SD from triplicates of three different experiments. **P < 0.001, ***P < 0.0001.

Figure 4

TALEN-treated cells undergo cell death as assessed by Propidium Iodide staining. FACS analysis of PI stained SiHa cells revealed that most of the TALEN-treated cells showed G1/S arrest. (A) Represents the FACS profile of control cells at 72 hours, (B) TALEN treated SiHa cells at 72 hours, (C) SiHa control at 96 hours, (D)TALEN treated SiHa cells at 96 hours. (E,F) Graphs depicting the percentage of cells in each stages of cell cycle during both time intervals of treatments. Data are expressed as mean ± SD from triplicates of three different experiments. ns = not significant, *P < 0.05, **P < 0.001, ***P < 0.0001.

Figure 5

TALEN-mediated E7 editing in SiHa cells led to cell death by necrosis. Bright field microscopic images of (A) SiHa control and (B) TALEN treated cells. Morphological changes resembling necrosis were observed in SiHa cells (B). Annexin V assay in SiHa control showing 5.2% cell death in quadrant 1 (C), and TALEN treated cells showed 30.1% cell death in quadrant 1 (D) which corresponds to necrotic cell death. (E) Graph representing the percentage of cells in different phases. Lysates of TALEN-treated and untreated SiHa cells were immunoblotted with antibodies to PARP revealed absence of cleavage indicating that cells did not undergo apoptosis (F). But there was a strong expression of RIP-1 which is a marker of necrosis (F). Immunoblotting with antibodies against Cyclophilin A and LDH-A also pointed to that the TALEN-mediated E7 editing induced necrosis in SiHa cells (G). Graphical representation of the proteins levels of RIP-1, CyclophilinA and LDH-A in control and TALEN-treated SiHa Cells (H). Data are expressed as mean ± SD from triplicates of three different experiments. *P < 0.05, **P < 0.001, ***P < 0.0001.

Figure 6

Inhibition of Necrotic cell death in TALEN treated cells by Necrostatin-1. In order to confirm the cell death due to necrosis, we transfected SiHa cells with TALENs and exposed to 50 μM Nec-1 and Annexin V assay was performed using FACS. Annexin V FACS profile of vector alone control (A), Nec-1 alone (B), TALEN-treated (C), TALEN + Nec-1 (D) and H₂O₂-treated positive controls (E). Graph represents the percentage of cells in various phases. Data are expressed as mean ± SD from triplicates of three different experiments. NS-not significant, **P < 0.001, ***P < 0.0001.

Figure 7

Schematic representation of Necrosis induced by TALEN editing of cervical cancer cells and possible activation of immune cells leading to cell death. When HPV genome integrates in the host genome, it leads to the continuous expression of the two oncogenes E6 and E7. These two oncogenes are responsible for transformation and development of tumor. When TALENs targeting E7 gene is administered (A), cells undergo double strand breaks thereby knocking out E7 gene (B). Abrogation of E7 gene leads to upregulation of RiP-1, LDH-A, CypA which in turn trigger necrosis (C). As an extrapolation of this data, pro-inflammatory cytokines could activate the immune cells and induce cell death. Such a strategy would be useful to induce cell death in cases where tumors acquire resistance to apoptosis through several strategies.