Dp71 depleted HBE cells displayed increased DNA damage and apoptosis induced by H2O2

Human bronchial epithelium (HBE)-Dp71 anti-sense(AS)cells with stably transfected Dp71 siRNA plasmids were prepared for further exploration of Dp71 biological traits in cells other than PC12. HBE-Dp71AS cells displayed increased DNA damage induced by H2O2. Apoptosis of HBE-Dp71AS cells induced by H2O2 was increased via enhancing caspase 3, caspase 8 and caspase 9. HBE-Dp71AS cells also displayed decreased proliferation and clonogenic formation. RAD51 was proved to be a new binding partner of Dp71 by co-immunoprecipitation (Ip) and immunofluorescence. Reduced RAD51 mRNA and protein levels were observed in HBE-Dp71AS cells. Decreased lamin B1, focal adhesion kinase (FAK), phosphorylated focal adhesion kinase (p-FAK) and phosphorylated protein kinase B (p-AKT) were detected in the HBE-Dp71AS cells, which functioned together with RAD51 as the molecular explanations for the character alterations of HBE-Dp71AS cells. Electronic supplementary material The online version of this article (10.1186/s11658-019-0169-6) contains supplementary material, which is available to authorized users.


Introduction
Dystrophin Dp71 is one of the most widely expressed isoforms of dystrophin, the pathogenic gene of Duchenne muscular dystrophy (DMD), an X-linked recessive disorder [1]. Functioning as one of the most ubiquitously expressed isoforms of dystrophin, Dp71 is a 70-to 75-kDa protein located in all tissues except skeletal muscle [2,3]. Previous research on Dp71 identified its crucial role for cell adhesion, neuronal differentiation and the cell cycle in PC12 cells. Dp71 was proved to be a putative tumor suppressive gene in gastric cancer [4][5][6]. Our preliminary clinical work also identified reduced Dp71 expression in lung cancer. Considering HBE as a usual cell model for pulmonary functional analysis, a shRNA strategy was used to knock down Dp71 in HBE to further clarify its biological significance. HBE-AS cells displayed increased DNA damage under oxidative stress, and decreased proliferation and clone formation capabilities. In a caspase-dependent way, HBE-AS cells displayed an increased apoptosis rate induced by H 2 O 2 . Our further characterization of HBE-AS cells identified RAD51, lamin B1, FAK and AKT to be the molecular explanations for the altered phenotypes of HBE-AS cells.

Construction of Dp71 short hairpin RNA plasmid
According to the open reading frame of the human Dp71 gene (NM_004015), one siRNA sequence (5′-gcactttaattatgacatc-3′) was selected. The scrambled sequence (5′ttctccgaacgtgtcacgt-3′) which has no significant homology with human gene sequences was included as a negative control. Two complementary oligonucleotides for Dp71 (5′gatcccgtctttagctgacctgaataactcgagttattcaggtcagctaaagactttttggat-3′ and 5′-agctatccaaa aagtctttagctgacctgaataactcgagttattcaggtcagctaaagacgg-3′), and for the negative control (5′-gatcccttctccgaacgtgtcacgtctcgagacgtgacacgttcggagaatttttggat-3′ and 5′-agctatc caaaaattctccgaacgtgtcacgtctcgagacgtgacacgttcggagaagg-3′), were synthesized by Invitrogen. Sense or antisense strands are in bold letters and stem loop sequences are in italics. They were annealed to generate double-stranded DNAs and ligated into the linearized shRNA (short hairpin RNA) eukaryotic expression vectors purchased from Genechem (Shanghai, China, containing hU6-MCS-CMV-GFP-SV40-Neomycin elements) to construct Dp71 shRNA or control empty shRNA vectors, which were termed Dp71AS and Dp71 empty shRNA vector (E) respectively. The nucleotide sequences of the plasmids were verified by automated DNA sequencing.
Cell culture and generation of stable transfectants HBE was obtained from the Culture Center, Chinese Academy of Medical Sciences (Shanghai, China). HBE cells were cultured in the same condition as described previously [7]. For stable transfectants, 5 μg of Dp71shRNA plasmid or 5 μg of control empty shRNA plasmid was mixed with 15 μl of Lipofectamine in serum-and antibiotics-free 1640, and the DNA/Lipofectamine mixture was added to the cell culture medium and incubated in the incubator for 4 h. The transfection mixture was removed and cells were maintained in 1640 supplemented with sera. Selection of stable transfectants was initiated with 600 μg/ml of G418 (Invitrogen) 48 h after transfection, a neomycin analog. The stable transfected HBE cells were named HBE-Dp71AS and HBE-Dp71E respectively.

Isolation of cell extracts and western blot analysis
Cultured cells were collected by centrifugation at 1200 rpm for 5 min, and washed twice with PBS. Protein extraction, concentration determination, 10% SDS-PAGE electrophoresis, and membrane incubation with the corresponding primary antibody (rabbit anti-dystrophin, rabbit anti-RAD51 polyclonal antibody purchased from Abcam; rabbit anti-FAK polyclonal antibody, p-FAK polyclonal antibody; rabbit anti-Akt polyclonal antibody, p-Akt polyclonal antibody; rabbit anti-phospho-histone H2AX (γH2AX; Ser 139) antibody (Bioworld Technology, Inc) was performed as described previously. After three washes with TBS-T, horseradish peroxidase-conjugated antirabbit IgG was used as the secondary antibody and developed using the ECL Western blotting analysis system (Amersham-Pharmacia).

Quantitative real-time polymerase chain reaction (QRT-PCR) and RT-PCR
The following primers were used and they produced a 157 bp PCR product for Dp71: 173 bp PCR product for FAK, 146 bp PCR product for lamin B1, 160 bp PCR products

Plate colony formation assay
Clone formation assay was performed as described previously. Clone formation efficiency was calculated according to the formula: (clone number/plated cell number) × 100% [7].

Apoptosis assay
Apoptosis of HBE, HBE-Dp71AS and HBE-Dp71E cells in the log growth phase were induced by 0.2 mM H 2 O 2 (Sigma, St. Louis, USA) for 16 h. The cells then were harvested by trypsinization for flow cytometry. Apoptosis was quantified using the PE Annexin V apoptosis detection kit (BD Pharmingen, San Diego, USA) according to the manufacturer ' s protocol. Cell analyses were made using a FACSCalibur flow cytometer (Becton-Dickinson, Mountain View, CA) and CellQuest software (BD Biosciences). Each assay was repeated 3 times [7].

Measurement of caspase 3, 8, 9 activities
The caspase fluorescent assay kits specific for caspase 3, caspase 8 and caspase 9 (Bio-Vision, San Francisco, USA) were used to detect caspase activation by measuring the cleavage of a synthetic fluorescent substrate. Cell treatment and fold increases in caspase 3, caspase 8 and caspase 9 activities were determined as described previously [8].

Alkaline comet assay for DNA damage
To perform the comet assay, the cell suspension of each cell group was mixed with low melting-point agarose at 37°C, to a final concentration of 0.7%. The mixture (15 μl) was pipetted onto slides pretreated with 0.5% normal-melting-point agarose, to retain the agarose cell suspension. The drop containing the cells was covered with a glass cover slip (24 mm × 24 mm) and left at 4°C for 5 min. The cover slips were gently removed and the slides were then ready for processing. The alkaline comet assay was performed using the basic rationale of Singh et al. The slides were then incubated in the dark for 30 min in cold electrophoresis buffer (300 mM NaOH, 1 mM EDTA, 1% (v/v) DMSO, pH 13) to allow the DNA to unwind before electrophoresis at 25 V for 25 min. After neutralization with 0.5 M Tris-HCl (pH 8.0), the slides were stained with 50 μl ethidium bromide (30 μg/mL, Absin Bioscience Inc., China). Finally, the images were taken by fluorescence microscope and at least 120 randomly selected cells (30 cells from each of the three replicate slides) were analyzed per sample and analyzed using the Comet Assay Software Pect (CASP 1.2.3 beta 1) (http://casplab.com/download). Parameters of tail moment (% DNA in tail × tail length), tail length, and percent of DNA in tail, the most frequently used parameters in the comet assay, were used in this study.

Immunofluorescence and confocal microscopy analysis
The immunofluorescence and confocal microscopy analysis of Dp71, Rad51 and γ-H2AX in HBE was as follows: After the three HBE cells were cultured on glass coverslips for 24 h, cells were treated with 200 μM H 2 O 2 for 30 min as described previously, treated cells and untreated cells were incubated overnight at 4°C with the primary anti-dystrophin, anti-RAD51 and anti-γ-H2AX antibody. Cells were incubated for 10 min at 37°C with 1 mg/ml 49,6-diamidino-2-phenylindole (DAPI) for counterstaining, After washing, coverslips were mounted on microscope slides with VectaShield (Vector Laboratories, Inc., Burlingame, CA, USA) and analyzed in a confocal and multiphoton microscope (TCS-SP5, Leica Microsystems, Heidelberg, Germany), using an oil immersion 636 objective. Co-localization of FITC, TRITC, and DAPI staining was analyzed in single optical sections obtained for two channels throughout the Z axis.

Immunoprecipitation
Total protein extracts in a final volume of 250 ml were incubated overnight at 4°C with 5 μg of rabbit anti-lamin B1, 5 μg of rabbit anti-Dp71 antibody, 5 μg of rabbit anti-FAK, and 5 μg of rabbit anti-RAD51 antibody, previously bound to protein G magnetic beads (Millipore). An irrelevant rabbit polyclonal antibody bound to protein G magnetic beads was performed as a negative control. The immune complexes were precipitated by placing the tube into the magnetic stand (Millipore) and washing 3 times with 500 μl of PBS containing 0.1% Tween 20. Precipitated proteins were separated by SDS-PAGE and analyzed by Western blotting with mouse anti-lamin B1, mouse anti-Dp71 antibody, mouse anti-RAD51 and mouse anti-FAK antibody.

Statistical analyses
All assays were repeated 3 times to ensure reproducibility. Results were displayed as mean ± SE. One-way ANOVA and LSD were used to analyze all experimental data. All statistical analyses were performed with SPSS software (version 17.0; SPSS Inc., Chicago, IL, USA). P < 0.05 was considered as indicating a statistically significant difference. construct (Fig. 1a), while transfection of the control empty vector had little effect on Dp71 expression. These results suggested that Dp71 shRNA constructs potently and specifically inhibited endogenous Dp71 protein expression in HBE cells, and the differences were statistically significant (Fig. 1b). Immunofluorescence also clearly showed reduced Dp71 expression in HBE-Dp71AS cells (Fig. 1c). The stable HBE-Dp71AS and HBE-Dp71E cell lines were used for further functional analyses.

HBE-Dp71 AS cells displayed increased DNA damage induced by H 2 O 2
After the HBE, HBE-Dp71E and HBE-Dp71AS cells had been exposed to 0.2 mM H 2 O 2 for 30 min, the comet assay was used to analyze single strand breaks in these three HBE cell lines. In normal cells, the fluorescence is confined mostly to the nucleus because undamaged DNA cannot migrate. In cells with DNA damage, DNA is denatured by the alkaline solution used for single-strand break detection. The negatively charged DNA fragments are then released from the nucleus and migrate toward the anode. Compared with HBE and HBE-Dp71E cells, HBE-Dp71 AS cells displayed more serious DNA damage ( Fig. 2a and b). Then the foci numbers of histone H2AX phosphorylated at serine 139 (γ-H2AX) were examined for detection of DSB (double strand breaks). As indicated in Fig. 2c, clearly more foci of γ-H2AX were detected in HBE-Dp71 AS cells, compared with HBE, and Dp71E cells. Statistical analysis (Fig. 2d) proved that the differences were significant. Alkaline comet assay and γ-H2AX analysis showed enhanced DNA damage induced by H 2 O 2 in HBE-Dp71 AS cells.   (Fig. 3a), 8% more apoptosis was detected in HBE-Dp71AS cells compared with the HBE-Dp71E and HBE cells; the differences were statistically significant (Fig. 3b). H 2 O 2 can induce the apoptosis via activation of both the extrinsic death receptor apoptosis pathway and the intrinsic apoptotic pathway. Caspase 9 is an initiator of the intrinsic pathway of apoptosis; caspase 8 is an indispensable enzyme of the extrinsic pathway. The activation of both caspase 8 and 9 will ultimately result in the activation of the effector caspase 3. As shown in Fig. 3c, significant fold change of caspase 3 in HBE-Dp71AS was observed compared with HBE and HBE-Dp71E cells after they had been treated with

Dp71 knockdown HBE cells displayed decreased proliferation rate
MTT assays were employed to analyze the proliferation of HBE-Dp71AS, HBE-Dp71E and HBE cell groups. Significant growth inhibition was observed at 48 and 72 h in HBE-Dp71AS cells (Fig. 4a), while there were no significant differences in cell growth between HBE-Dp71E and HBE cells (P > 0.05). Thus the assays indicated that the ablation of Dp71 protein in HBE cells can effectively inhibit their growth.

HBE-Dp71 AS cells displayed reduced clone formation capabilities
The anti-cell proliferation effect of Dp71 was assessed by a clonogenic formation assay. There was a significant reduction in the number and size of foci in HBE-Dp71 AS cells compared with HBE-Dp71E and HBE cells (Fig. 4b). The clone formation efficiency for HBE-Dp71E, HBE and HBE-Dp71AS cells was 85.33 ± 1.72, 88.73 ± 4.67 and 67.53% ± 3.45% respectively. The clone formation efficiency for HBE-Dp71AS cells was 20% less than that in HBE-Dp71E and HBE cells; the differences were statistically significant (Fig. 4c). Together with the results from the MTT assay, it is found that ablation of Dp71 protein can significantly inhibit the growth of HBE cells.

RAD51 interact with Dp71 and Lamin B1 in HBE
RAD51 is a protein that forms discrete nuclear foci and participates in homologous recombination repair upon DNA damage. Interaction between RAD51 and lamin B1 was verified in previous research [9,10]. In our further characterization of Dp71 in HBE cells, it was found that the Dp71 specific antibody pulled down RAD51 successfully, while non-specific IgG failed to pull down RAD51 (Fig. 5a). With RAD51 antibody, Dp71 was successfully precipitated while IgG failed to work (Fig. 5b). Immunofluorescence verified the interaction between RAD51 and Dp71 in the nucleus (Fig. 5d) and cytoplasm (Additional file 1), while the major interaction occurred in the nucleus. Co-Ip results also proved the association between RAD51 and lamin B1. As indicated in Fig. 5b and c, lamin B1 antibody successfully dragged down RAD51, and RAD51 specific antibody precipitated lamin B1. In each assay, however, non-specific IgG failed to precipitate RAD51 and lamin B1. Combined with the proof of RAD51-Dp71 interaction, we proved the existence of Dp71-RAD51-lamin B1 complex in HBE. Immunoblot was carried out to quantify lamin B1 and RAD51 expression in HBE-Dp71AS cells. Compared with parental HBE cells and HBE-Dp71E cells, significant reduction of Dp71, lamin B1, RAD51 mRNA (Fig. 5e, f, and g) and protein (Fig. 5h) was observed. The endogenous RAD51 and lamin B1 expression levels were both reduced after the Dp71 expression was knocked down in HBE cells.

Dp71 depletion resulted in decreased FAK, p-FAK and p-AKT
Dp71 was found associating with most of the β1-integrin complex components (β1-integrin, focal adhesion kinase(FAK), α-actinin, talin and actin) in PC12 cells [11]. Our previous publications proved that FAK is an indispensable component of the cytoplasmic DAPCs in HBE. Via two-way precipitation, Fig. 6a and b show that FAK and Dp71 antibody dragged down each other in the immuno-precipitation process while IgG did not precipitate either protein. The co-Ip assay proved that the cytoplasmic association between Dp71 and FAK is a universal phenomenon in different types of cells. QRT-PCR displayed reduced FAK mRNA in HBE-Dp71AS cells (Fig. 6c, d). Decreased FAK protein and p-FAK expression was also displayed by immuno-blotting in HBE-Dp71AS cells. Although AKT did not display any expression alteration, p-AKT displayed a significant reduction in HBE-Dp71AS cells (Fig. 6e). Knocking down Dp71 in HBE cells resulted in the reduction of cytoplasmic expression of FAK, p-FAK and p-AKT.

Discussion
Being one of the most abundantly expressed dystrophin isoforms, Dp71 is distributed in all tissues except skeletal muscle. The deficiency of Dp71 is involved in mental retardation and retinal dysfunction of DMD patients [2,12,13]. For years, PC12 cells have been used as the traditional cell model to study the biological function of Dp71. In our further characterization of this ubiquitously expressed scaffolding protein, Dp71 was found to contain more functional diversity than expected. Being one of the newly Fig. 5 Dp71 interacts with RAD51 in HBE cells. a Co-Ip revealed that RAD51 and lamin B1 were successfully pulled down by specific Dp71 antibody, while irrelevant IgG failed to pull down these two proteins. b Co-Ip revealed that Dp71 and lamin B1 were successfully pulled down by specific RAD51 antibody, while irrelevant IgG failed to pull down these two proteins. c Co-Ip revealed that Dp71 and RAD51 were successfully pulled down by specific lamin B1 antibody, while irrelevant IgG failed to pull down these two proteins. d Immunofluorescence displayed the co-localization of Dp71 and RAD51 in HBE cells (scale bar: 10 μm). e Significant reduction of Dp71 mRNA was observed in HBE-AS cells. f Significant reduction of RAD51 mRNA was observed in HBE-AS cells. g Significant reduction of lamin B1 mRNA was observed in HBE-AS cells. h Significant reduction of Dp71, RAD51 and lamin B1 protein was observed in HBE-AS cells RAD51 was found to be a new binding partner of Dp71 in our further exploration of HBE-Dp71AS cells. Although cytoplasmic and nuclear interactions between Dp71 and RAD51 were detected in our research, the nuclear Dp71-RAD51 interaction plays a significant role in the increased DNA damage of HBE-Dp71AS cells induced by H 2 O 2 . Co-Ip proved the existence of Dp71-lamin B1-RAD51 protein complex in HBE. RAD51 is proved to be an essential protein for DNA repair by homologous recombination [14,15]. Overexpression of Rad51 in different organisms and cell types increased homologous recombination (HR) and increased resistance to DNA damaging agents. Being an associating protein of Dp71, lamin B1 has been found to be a binding partner of RAD51, which promotes DSB repair and cell survival by maintaining the RAD51 protein stability [9,10]. In HBE-Dp71AS cells, depletion of Dp71 results in Rad51, lamin B1 mRNA and protein reduction. RAD51 suffers both decreased expression and impaired stability, which leads to decreased HR and increased DNA damage stimulated by H 2 O 2, and finally results in increased apoptosis. Via interaction with lamin B1, the nuclear Dp71 is proved to affect the cell growth of PC12 and A549. Decreased lamin B1 expression also slows cell proliferation and induces premature senescence in WI-38 cells [6,16]. In HBE-AS cells, significantly reduced lamin B1 explains the phenotypes of growth inhibition. Increased BRCA1 expression caused by decreased lamin B1 reduction leads to the overactivation of caspase 8 and 3, combined with impaired expression and function of RAD51. The apoptosis rate of HBE-Dp71AS cells increased under H 2 O 2 stimulation.
FAK is a cytosolic non-receptor protein tyrosine kinase that regulates cellular adhesion, motility, proliferation and survival in various types of cells [17][18][19]. FAK is proved to interact with Dp71 in HBE and PC12. Co-Ip also proved the interaction of FAK with Dp71 in HBE in our current research. Decreased FAK mRNA and protein was observed in HBE-Dp71AS cells. FAK is a novel regulator of DNA damage repair in mutant KRAS NSCLC and its pharmacologic inhibition leads to radiosensitizing effects. Specific targeting of focal adhesion kinase in endothelial cells is sufficient to induce tumor-cell sensitization to DNA-damaging therapies and thus inhibit tumor growth in mice. Although FAK-dependent chemo-sensitivity is proved to be related to DNA-damageinduced NF-κB activation, the DNA damage response process FAK participates still needs further exploration. FAK-overexpressed (HL-60/FAK) cells were highly resistant to hydrogen peroxide and ionizing radiation (IR)-induced apoptosis [20,21]. AKT, also known as protein kinase B, is a serine/threonine-specific protein kinase responsible for apoptosis, cell proliferation, transcription and cell migration. Accumulating evidence has implicated AKT as a direct participant in the DNA damage response and repair induced by commonly used genotoxic agents. AKT plays an important regulatory role in activating DNA-PKcs and non-homologous end joining (NHEJ) repair [22,23]. Functioning as an activated downstream target in the FAK pathway, AKT protein expression did not show any change in HBE-AS cells. However, the activated AKT was significantly reduced, which acted together with reduced FAK and RAD51 to attenuate the DNA damage of HBE-Dp71AS cells induced by H 2 O 2 (Fig. 7) . Replication errors after this damage would lead to increased mutations and cancer. The reduced FAK and p-AKT can also explain the decreased proliferation of HBE-Dp71AS cells.