Knocking Down Nucleolin Expression Enhances the Radiosensitivity of Non-Small Cell Lung Cancer by Influencing DNA-PKcs Activity

Over the past twenty years, the worldwide incidence of non-small cell lung cancer (NSCLC) and associated mortality have increased steadily each year (Silvia et al., 2014). Although significant advancements in the treatment of NSCLC, only minimal increases in survival rates have been produced (Fernandes et al., 2012). Therefore, new treatment modalities that improve survival rates are urgently needed. A clearer understanding of the biology of NSCLC at a molecular level will aide materially in the development of these modalities, including ways to accurately predict the prognosis of patients with certain clinical-pathological characteristics and identification of new targets for radiation therapy. Improving tumor radiosensitivity is an effective


Introduction
Over the past twenty years, the worldwide incidence of non-small cell lung cancer (NSCLC) and associated mortality have increased steadily each year (Silvia et al., 2014).Although significant advancements in the treatment of NSCLC, only minimal increases in survival rates have been produced (Fernandes et al., 2012).Therefore, new treatment modalities that improve survival rates are urgently needed.A clearer understanding of the biology of NSCLC at a molecular level will aide materially in the development of these modalities, including ways to accurately predict the prognosis of patients with certain clinical-pathological characteristics and identification of new targets for radiation therapy.
Improving tumor radiosensitivity is an effective UTRs of p53 mRNA.C23 binds to the 5'-and 3'-UTR interaction region that is critical for the recruitment of RPL26 to p53 mRNA after DNA damage (Chen et al., 2012).C23 interacted with Rad50 and was recruited to DNA double-strand breaks (DSBs) in an MRE11-NBS1-RAD50 complex-dependent manner (Goldstein et al., 2013).Notably, C23 knockdown exhibited repreesion of both ATM-dependent phosphorylation following exposure to γ-ray and activated cell cycle checkpoint subsequently.Furthemore, the knockdown of C23 gene may induce the deficiency of the catalytic subunit of autophosphorylation of DNA-dependent protein kinase (DNA-PKcs) (Kobayashi et al., 2012).The results demonstrated that C23 was involved in DNA DSBs repair.Moreover, the relationship between C23 expression and DNA-PKcs activity remains unclear in NSCLC.
In this study, we assume that the inhibition of C23 expression can improve the radiosensitivity of NSCLC.C23 may be affected by DNA-PKcs activity to inhibit DNA damage repair.We expect to provide new targets for radiation sensitivity for lung cancer.

Cell culture
Based on the mRNA expression profiles of human lung cancer cell lines used in previous studies, human lung adenocarcinoma (NSCLC) cell line A549 and human large cell lung carcinoma cell line NCI-H460 (obtained from the Institute of Cancer Prevention and Treatment, Harbin Medical University) was chosen for this analysis.Cells were cultured in a 5% CO 2 incubator at 37ºC in RPMI 1640 (Hyclone, Beijing, China) supplemented with 10% bovine serum.

Clonogenic survival assay
The effectiveness of the combination of C23_siRNA and ionizing radiation was determined using clonogenic survival assays.Cells were initially transfected with C23_ siRNA.After 24 hours, cells were planted on a 6-well plate in a limiting dilution assay (100, 200, 400, 1000, 5000, 10000 cells per well) and then the plate was treated with 0, 2, 4, 6, 8, 10 Gy of ionizing radiation.Briefly, the cells were irradiated with 6 MeV photons/100 cm focus-surface distance at a dose rate of 4.0 Gy/min at room temperature in T-25 flasks.After treatment, the cells were trypsinized and counted.The cells were then washed with PBS and returned to the incubator to allow for macroscopic colony development.The colonies were counted after 14 days, and the plating efficiency and surviving fraction for each treatment were calculated based on the survival of nonirradiated cells transfected with C23_siRNA alone.

Quantification of apoptosis
To quantify apoptotic cells were identified using fluorescein isothiocyanate (FITC)-labeled recombinant chicken Annexin V (ZomanBio, Beijing, China) in combination with propidium iodide (PI).Briefly, 10 5 cells were resuspended in 500 μL of Ringer's solution, incubated for 30 minutes at 4ºC in the dark with 1 μg Annexin V-FITC and 1 μg PI and analyzed on a Coulter EPICS XL flow cytometer with System XL II software.

Cell proliferation assay
Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) detection kit (Zomanbio, Beijing, China).Cells were seeded at a concentration of 2×10 3 cells per well in 96-well plates.After 24 hours of growth and achieving a confluence of about 70-80%, cells were treated with siRNAs.At 24, 48 and 72 hours after transfection, 10 μl of CCK-8 solution was applied to each well followed by a 3 hour incubation at 37ºC.Absorbance values for all wells were then determined at 450 nm in on a microplate reader (Elx800, BioTec).

Cell cycle analysis
Adherent cells (1×10 6 /ml) were collected by trypsinization, washed with PBS for 10 minutes and centrifuged at 120xg.The cells were resuspended in a hypertonic solution containing 50 μg/mL PI and 10 μg/ mL RNaseA (Beyotime, Shanghai, China).Fluorescenceactivated cell sorting (FACS) was performed using a Coulter EPICS XL flow cytometer (Coulter) and System XL II software.
Immunofluorescence and the quantification of Cells were cultured on six-well slides and exposed to 4 Gy/min of X-ray irradiation.At 15, 30, 60, 120 and 360 minutes after X-ray irradiation, the cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature.The cells were then permeabilized with 0.25% Triton in PBS for 15 minutes, blocked with 3% BSA for 1 hour at room temperature and incubated with phospho-specific H2AX (1:100; Cell Signaling) and phospho-specific S2056 DNA-PKcs (1:100; Epitomics).Immune complexes were visualized after incubation with the FITC-conjugated secondary antibody.The nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Invitrogen), the coverslips were mounted in fluorescence mounting medium, and the fluorescence was visualized using a confocal laser scanning microscope equipped with the LSM5 PASCAL system (Carl Zeiss).

Real-time PCR assays
Total RNA was extracted with TRIzol (Invitrogen) according to the manufacturer's instructions.Reverse transcription was performed with oligo dT primers (Roche, Switzerland).Real-time PCR was carried out in an Applied Biosystems 7500 System with Power SYBR Green PCR Master Mix (Roche, Switzerland).Relative levels of gene expression were determined with glyceraldehyde 3-phosphate dehydrogenase (β-actin) as the control.

Statistical analysis
All data were presented as mean ± standard deviation and calculated using SPSS13.0statistical software.Selection of one-way ANOVA or independent samples t-test was based on experimental setup, and P<0.05 was considered statistically significant.All experiments were performed in triplicate.

C23 expression was negatively correlated with the radiosensitivity of NSCLC cell lines
We detected the expression of C23 mRNA in A549 and H460 cell lines, and found that cell survival curves shifted to the left accompanied with the high grade of C23 expression (Figure 1A, 1B).As a result, we considered that the expression of C23 was negatively correlated with the radiosensitivity of NSCLC cell lines.

Down-regulation of C23 enhanced radiosensitization of NSCLC cells
The influence of C23 on NSCLC cell radiosensitivity was first examined.Transfection of A549 cells with C23_ siR significantly decreased C23 expression at 24 and 48 hour time points (Figure 2A, 2B) (P<0.05).Furthermore, treatment with C23_siR shifted the A549 survival curve to the left with a radiation dose enhancement factor of 1.61, as measured by clonogenic survival assays, suggesting that C23 knockdown increased the radiosensitivity of A549 cells (Figure 2C).
Further research was taken in order to investigate whether inhibition of C23 increased the radiation sensitivity.The absorbance values of the A549 cells at 24, 48 and 72 hours post-transfection with C23_siR were notably lower than the control or NC group, as measured by the CCK-8 assay.Moreover, the cell proliferation inhibition rate in C23_siR-treated cells showed a notable increase (i.e.proliferation decreased) compared with untreated cells (Figure 3C).Flow cytometry results showed that the amount of apoptotic cells at 24 hours post-transfection with C23_siR was greater both C23_siR and radiation given than C23_siR or radiation given alone.
Collectively, these data indicated that C23 knockdown promoted radiation-induced apoptosis in A549 cells (Figure 3A).Typically, cells are most radiosensitive in the G2/M phase and most radioresistant in the S phase.Using flow cytometry, the cell cycle was detected at 24 hours after receiving C23_siR treatment.In C23_siR-treated cells, the G2/M fraction was increased and the S phase cells was decreased compared with the control or NC cells (Figure 3B), suggesting that C23 knockdown may put cells in a more radiosensitive stage of the cell cycle.
DNA repair has been associated with tumor radiosensitivity.C23 has been proved to play a direct and/ or indirect role in DNA repair.Therefore we assessed the effect of C23 on DNA repair using immunostaining.The foci that form at DNA DSBs were visualized in treated and untreated cells with antibodies against γ-H2AX.The induction of DNA DSBs and DNA repair was studied by detecting H2AX serine 139 phosphorylation at 15, 30, 60, 120 and 360 minutes after irradiation and by quantifying the formation of γ-H2AX foci at 15, 30, 60, 120 and 360 minutes.The formation of γ-H2AX foci in A549 cells was detected between 15 minutes and 360 minutes after irradiation.Compared with control and NC cells, γ-H2AX levels increased 15 minutes to 360 minutes after irradiation in C23_siR-treated cells (Figure 3D).
These results suggested that C23 knockdown increased radiosensitivity of NSCLC cells through promoting radiation-induced apoptosis, adjusting redistribution within the cell cycle, and inhibiting DNA damage repair.

C23_siR suppresses DNA damage repair of NSCLC cells through inhibiting DNA-PKcs activity
To further establish C23 activity in DNA damage repair, expression of the DNA damage gene DNA-PKcs in treated and untreated cells was examined.The results showed that the expression of C23 and DNA damage repair genes including DNA-PKcs and γ-H2AX first increased, then reduced from 30 min to 360 min after X-ray irradiation (Figure 4C).Therefore, we considered that C23 was involved in DNA damage repair after radiotherapy.RT-PCR showed that DNA-PKcs mRNA was downregulated in C23 knockdown cells (Figure 4A, 4B, 5A, 5B).Compared with control and NC cells, DNA-PKcs protein expression and phosphorylation of DNA-PKcs sites S2056 and T2609 were decreased at 30, 60, 120 and 360 minutes after irradiation (Figure 4C).These results suggested that C23 knockdown inhibited DNA damage repair by suppressing DNA-PKcs activity and therefore improved radiation sensitivity in NSCLC cells.

Discussion
We reported that inhibition of C23 increased the radiation sensitivity of A549 cell lines.Radiosensitivity of tumor is associated with the apoptosis, cell division cycle , radiation damage repair of tumor cells, etc.Studies have found C23 excessive expression in human various tumor cells (Guo et al., 2014) and its resistant role in apoptosis (Schokoroy et al., 2013).In this study, we found that the inhibition of C23 expression may promote radiationinduced apoptosis.C23_siR-treated cells were found to have more cells in G2/M fraction and less in S phase of the cell cycle, which indicated that C23 knock-down put cells in a more radiosensitive stage of the cell cycle.
In order to safeguard the genome against mutation or DNA loss, cells respond to stress by transiently delaying proliferation and mobilizing repair factors (Daniely et al., 2002).Radiation-induced expression of C23 protein and mRNA were increased in our research and involved in DNA damage repair.A number of papers revealed that both heat shock and DNA-damaging agents induced a relocalization of C23 from the nucleolus to the nucleoplasm.These studies revealed that C23 subsequently bound replication protein A, which resulted in a DNA replication blockade (Daniely et al., 2000).C23 was sequestered within the nucleolus and its release which was triggered by recognition of DNA damage, functioned to help the cell regulate the complex response to DNA damage.Finally, the release of C23 might lead to cell cycle arrest and activation of DNA repair (De et al., 2006).These findings were consistent with the hypothesis that C23 was involved in DNA DSBs repair.C23 may contribute to genome stability in two ways: by modulating the activities of several DNA repair enzymes through physical interaction and by enhancing the translation of some of these protein after genotoxic stress (Yang et al., 2002).H2AX phosphorylation is crosslinking with damaged chromatin and damage repair proteins gathering (Celeste et al., 2003).H2AX phosphorylation has a positive correlation with the accumulated number of γ-H2AX foci and DSBs (Sone et al., 2014).Our immunofluorescence results showed that down-regulation of C23 expression could increase the quantification of γ-H2AX foci and decrease radiation-induced DNA damage repair.C23 knockdown showed decrease in radiation-induced chromatin accumulation of DNA damage repair factors, which agreed with the delayed kinetics of γ-H2AX focus.We found that DNA-PKcs phosphorylation at the S2056 cluster decreased significantly more than untreated and NC cells in immunofluorescence experiments (Figure 4D).C23 knockdown may reduce radiation-induced accumulation of DNA-PKcs .
DNA-PKcs, a key protein in the NHEJ pathway, consists of a 465 kDa catalytic subunit (DNA-PKcs) and a heterodimeric regulatory complex (Ku) that comprises a 70 kDa subunit (Ku70) and an 86 kDa subunit (Ku80) (Xing et al., 2015).Studies have shown that DNA-PKcs can affect cellular radiosensitivity (Costantini et al., 2007) and recognized that.The inherent radiation sensitivity of cancer cells is in greatly influenced by the ability of DNA DSBs repair (Dikomey et al., 1998).Expression of DNA-PKcs and Ku70 decreased from 30 to 360 minutes after irradiation in C23 knockdown cells.These results suggested that DNA-PKcs protein kinase activity decreased following C23 knockdown.A large number of phosphorylation sites were clustered in different regions of DNA-PKcs (Davis et al., 2013).Two prominent phosphorylation clusters which had been identified to be phosphorylated and autophosphorylation in response to irradiation are the T2609 and S2056 (Chen et al., 2005) clusters.Both the S2056 and T2609 clusters deactivation at the same time could increase the radiosensitivity (Nagasawa et al., 2011).We found that DNA-PKcs phosphorylation sites at the S2056 and T2609 significantly suppressed after irradiation in C23 knockdown cells (Figure 5C).The activation for phosphorylation site at the S2056 and T2609 was critical for DNA DSBs repair.If lose damage repair, DSBs can lead to chromosome fracture, loss and ultimately cause cell death; therefore, inhibiting DNA DSBs repair can increase the irradiation sensitivity of cancer cells.Our research was the first study which found that C23 could regulate the function of DNA-PKcs.C23 knockdown reduced the radiation damage repair and increased the radiosensitivity in A549 and H460 cells through inhibiting DNA-PKcs phosphorylation activity at the S2056 and T2609 cluster.
In this study, we suggested that C23 knockdown inhibited DNA damage repair by suppressing DNA-PKcs activity and therefore improved radiation sensitivity in NSCLC cells.However, we found that only a portion function of C23 in the field of DNA damage repair after irradiation.Further study in the role of C23 in DNA damage repair could provide a new target for radiosensitivity improvement in NSCLC patients (Goldshmit et al., 2014).

Figure 2 .Figure 1 .
Figure 2. C23_siRNA Inhibited C23 Expression and Enhanced the Radiosensitivity of A549 Cells.(A) RT-PCR analysis of C23 mRNA expression in untreated and treated A549 cells.C23 mRNA levels were significantly lower in the C23_siR group.β-actin mRNA was used as an internal control.*P<0.05versus control or NC group.(B) Western blot analysis of C23 protein expression in untreated and treated A549 cells.C23 protein levels were notablely lower in the C23_siR group.β-actin protein was used as an internal control.P<0.05 versus control or NC group.(C) A549 cells were transfected with C23_siR, NC or control for 24 hours and subsequently irradiated with 2-10 Gy of X-rays.After 14 days, the cells were fixed and stained.The surviving fractions were calculated based on colony counts and plating efficiency.Treatment with C23_siR shifted the A549 survival curve to the left with a radiation dose enhancement factor of 1.61

Figure 4 .
Figure 4. Down Regulation of C23 Expression Reduced the Activity of DNA-PKcs, DNA-PKcs S2056 and DNA-PKcs T2609 in A549 Cells.(A) RT-PCR analysis of C23 mRNA expression in untreated and treated A549 cells after radiation for 1 hours.C23 mRNA levels were notably lower in the RT plus C23_siR and C23_siR groups.β-actin gene was used as an internal control.*P<0.05versus control or NC group.(B) RT-PCR analysis of DNA-PKcs mRNA expression in untreated and treated A549 cells after X-ray irradiation for 1 hours.DNA-PKcs mRNA levels were notablely lower in the RT plus C23_siR and C23_siR groups.β-actin gene was used as an internal control.*P<0.05versus control or NC group.(C) A549 cells were transfected with C23_siR and then exposed to 4 Gy of radiation.C23, DNA-PKcs, DNA-PKcs S2056, DNA-PKcs T2609 levels were notably decreased in the C23_siR group.(D) Cells were transfected by C23_siR, NC or control for 24 h, and treated by irradiation with 4 Gy.Immunofluorescent images of representative A549 cells stained for DNA-PKcs S2056 (Rhodamine, green) and DNA (DAPI, blue) 1 hours after irradiation.We found that the foci of phospho-specific S2056 DNA-PKcs decreased significantly in C23 knockdown cells than control and NC cells.Scale bar: 10 µm

Figure 5 .
Figure 5. Knockdown C23 Expression Reduced the Activity of DNA-PKcs, DNA-PKcs S2056 and DNA-PKcs T2609 in H460 Cells.(A) RT-PCR analysis of C23 mRNA expression in H460 cells after radiation for 1 hours.C23 mRNA levels were notably lower in the RT plus C23_siR and C23_siR groups.β-actin gene was used as an internal control.*P<0.05versus control or NC group.(B) RT-PCR analysis of DNA-PKcs mRNA expression in H460 cells after X-ray irradiation for 1 hours.DNA-PKcs mRNA levels were notablely lower in the RT plus C23_siR and C23_siR groups.β-actin gene was used as an internal control.*P<0.05versus control or NC group.(C) H460 cells were transfected with C23_siR and then exposed to 4 Gy of radiation.C23, DNA-PKcs, DNA-PKcs S2056, DNA-PKcs T2609 levels were notably decreased in the C23_siR group