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SETD3 reduces KLC4 expression to improve the sensitization of cervical cancer cell to radiotherapy
Biochemical and Biophysical Research Communications 516 (2019) 619e625
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
SETD3 reduces KLC4 expression to improve the sensitization of cervical cancer cell to radiotherapy Qingmei Li a, Yanqin Zhang b, Qiuli Jiang c, * a
Department of the Second Area of Obstetrics, The People's Hospital of Pingyi County, Linyi, 273300, China Department of Nursing, Yulin Traditional Chinese Medicine Hospital, Yulin Shanxi, 719000, China c Department of Gynecology, Hanzhong Central Hospital, Hanzhong Shaanxi, 723000, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 June 2019 Accepted 11 June 2019 Available online 22 June 2019
Resistance to radiotherapy accounts for most therapeutic failures in cervical cancer patients who undergo radical radiation therapy. To indicate the possible molecular mechanism of radioresistance and improve the 5-year survival rate, we focused on how SET domain protein 3 (SETD3) regulated radioresistance in human cervical cancer cells in this study. Our results indicated that SETD3 over-expression markedly increased the radiosensitivity of cervical cancer cells with radioresistance, as evidenced by the further reduced cell viability, proliferation, DNA damage and cell death. In addition, we found that SETD3 down-regulated the expression of kinesin light chain 4 (KLC4), contributing to the radiosensitivity of cervical cancer cells, and the regulatory role of SETD3 could be abolished by KLC4 over-expression. Moreover, nitric oxide (NO) production was significantly reduced by SETD3 over-expression through repressing the expression of inducible NO synthase (iNOS) and endothelial NO synthase (eNOS) in cervical cancer cells. In vivo studies using xenograft animal models also demonstrated that SETD3 overexpression combined with irradiation treatment markedly inhibited tumor growth and induced apoptosis. In summary, our data demonstrated that down-regulated SETD3 expression markedly led to the progression of radioresistance and that promoting SETD3 expression could sensitized cervical cancer cells to radiotherapy, thereby targeting SETD3 might be a potential strategy for the clinical management of cervical cancer to improve the curative effect of radiation in cervical cancer. © 2019 Published by Elsevier Inc.
Keywords: Cervical cancer Radioresistance SETD3 KLC4 NO production
1. Introduction Cervical cancer is the fourth most common malignant disease and one of the major causes of tumor-associated death among females in the world [1,2]. Presently, radiotherapy plays an essential role in the prevention of cervical cancer. Approximately 80% of patients with cervical malignant tumors need radiation therapy. However, resistance to radiotherapy plays a crucial role in treatment failure in patients undergoing radical radiation therapy [3e5]. Nevertheless, the molecular mechanism for radioresistance acquirement and development in cervical cancer still remains unknown. Mechanistically, radiotherapy results in cell cycle arrest and tumor cell death through inducing DNA damage [6]. SET domain protein 3 (SETD3) is a member of the protein lysine
* Corresponding author. E-mail address: [email protected] (Q. Jiang). https://doi.org/10.1016/j.bbrc.2019.06.058 0006-291X/© 2019 Published by Elsevier Inc.
methyltransferase (PKMT) family, which catalyzes the addition of methyl group to lysine residues. Recently, SETD3 was reported to be a positive modulator of DNA-damage-induced apoptosis in colon cancer cells [7,8]. Moreover, SETD3 was suggested to interact with Maf1, PCNA and RNF7, which are crucial signals involved in DNA replication progression, further linking SETD3 to DNA repair [9,10]. SETD3 has been indicated to participate in the oncogenic processes that high SETD3 expression shows oncogenic properties in lymphoma and liver cancer [11,12]. However, there is also study suggesting that lower expression of SETD3 was associated with markedly shorter disease-free survival [13]. Furthermore, SETD3 overexpression reduced cell viability and induced apoptosis in zebrafish models [14]. Thus, SETD3 also exhibits anti-tumorigenic role. Herein, the role of SETD3 in tumor might be depended on its type, and however, the effects of SETD3 on radiation resistance in cervical cancer are little to be reported. In this study, we elucidated that SETD3 expression levels were inversely associated with radioresistance in patients with cervical cancer. Our findings suggested that SETD3 could sensitize tumor to
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radiotherapy through promoting radiation-triggered apoptosis in cervical cancer cells. Moreover, our results suggested that SETD3 elevated the radiosensitivity of human cervical cancer cells by inhibiting KLC4 expression and NO production. These data demonstrated that SETD3 could be a radioresistance biomarker in cervical cancer cells. 2. Materials and methods 2.1. Human specimens Human tissue samples were collected through biopsy from patients before and after radiotherapy in the People's Hospital of Pingyi County (Shandong, China). Initial tissue samples were prepared to determine gene expression, and follow-up tissue specimens (after radiotherapy) underwent a calculation to explore the effectiveness of the radiotherapy. The effectiveness of radiotherapy was calculated following methods as previously indicated by Kitahara et al. [15]. Samples were divided into radiosensitive and radioresistant groups according to the results of calculation. The research was approved by the Ethics Committee of The People's Hospital of Pingyi County (Shandong). All patients provided their informed consent prior to sample collection. 2.2. Cells and treatments Human cervical cancer cell lines of SiHa was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). X-ray resistant SiHa cells (SiHa-XR) were developed using SiHa cells as previously indicated [16]. The cervical cancer parental cell line was exposed to 2 Gy 3 times, 4 Gy 3 times, 6 Gy 3 times, 8 Gy twice and 10 Gy twice. Irradiation was performed at 300 cGy/min using a linear accelerator (SIMEN, Germany). SiHa and SiHa-XR cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO, USA) containing 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA) at 37 C and 5% CO2. Corresponding plasmid and siRNA constructs of SETD3 and KLC4 were designed and synthesized by Western Biomedical Technology (Chongqing, China). Then, cells were transfected with the indicated plasmids or siRNAs with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following manufacturer's protocols. NOS inhibitor of L-NMMA was purchased from Beyotime (Jiangsu, China). Nitrate/Nitrite Assay Kit was also obtained from Beyotime to calculate NO production in cells according to the manufacturer's recommendations. 2.3. Colony formation and cell viability analysis Cells were planted in a well of a six-well plate and cultured under 5% CO2 at 37 C for 2 weeks. Next, the cells were fixed with formalin, stained with 0.1% crystal violet. Tumor cell proliferation was calculated in five microscopic fields. Cell viability was measured with CCK-8 kit (Dojindo, Japan) according to the manufacturer's recommendations. Absorbance was recorded at 450 nm. 2.4. Flow cytometry analysis Cells were washed with phosphate-buffered saline (PBS) and stained with annexin V and propidium iodide (Beyotime) following the manufacturer's instruction using a BD FACSCalibur flow cytometer (BD Biosciences, USA). Apoptosis was defined as the total percentage of cells that were positive both for PI and Annexin V. 2.5. Tumorsphere culture Cells were cultured in stem cell medium consisting of DMEM/
F12 basal medium, N2, and B27 supplements (Invitrogen), human recombinant epidermal growth factor (20 ng/mL), and basic fibroblastic growth factor (20 ng/mL) (Rocky Hill, NJ, USA). As for the tumorsphere formation, cells were incubated at a density of 200 cells/well in 24-well ultra-low-attachment plates and sustained in stem cell media. Tumorspheres that formed within 2 weeks were recorded. For serial tumorsphere formation analysis, the spheres were collected, disaggregated with trypsin/EDTA, filtered via a 40-mm mesh, and then re-plated as described above. Triplicate samples were performed, and the spheres were measured by individuals in a blind fashion. 2.6. Western blotting analysis Cells were lysed using radioimmunoprecipitation assay lysis buffer (Beyotime). Protein concentrations were calculated with a bicinchoninic acid assay (BCA) (Thermo Fisher Scientific, USA). Proteins were then separated on 10% SDS-PAGE and translocated onto PVDF membrane (Millipore, USA), and immunodetected with the primary antibodies, including anti-SETD3 (1:1000 dilutions, Abcam, USA), anti-KLC4 (1:1000 dilutions, Thermo Fisher Scientific) and anti-GAPDH (1:1000 dilutions, Cell Signaling Technology, USA). Then, the membranes were incubated with HRP-conjugated secondary antibodies (Beyotime) for 1 h at room temperature. The blots were then visualized with an enhanced chemiluminescence reagent (Beyotime). Immunoreactive bands were quantitatively analyzed using ImageJ software. 2.7. Comet analysis The comet assay was performed to calculate the level of DNA damage in cells (Trevigen, Gaithersburg, MD, USA). Slides were placed on the electrophoresis slide tray. Electrophoresis was performed by an electric field of 21 V in alkaline electrophoresis solution). Subsequently, the slides were immersed in distilled water and in 70% ethanol. Samples were then dried. Finally, the slides were stained with 1 mg/mL of propidium iodide and captured under a fluorescence microscopy. 2.8. Immunohistochemistry (IHC) Tumor samples were fixed with 4% paraformaldehyde (PFA), embedded in paraffin, and cut into 4-mm-thick sections. After deparaffinizing in xylene and rehydrating using alcohol series, the sections were detected with primary antibody against KI-67 (1:100, Abcam, USA) overnight at 4 C. After incubation with the secondary antibody, the sections were developed using diaminobenzidine and counterstained with hematoxylin. As for cell death analysis in tumor samples, TUNEL Apoptosis Assay Kit (Beyotime) was used following the manufacturer's instruction. 2.9. Quantitative real-time PCR analysis Total RNA of cells was isolated with TRIzol reagent (Invitrogen, USA) and subjected to reverse transcription with Oligo (dT) and MMLV Reverse Transcriptase (Invitrogen). Quantitative PCR amplification was conducted using the ViiA™ 7 Real-Time PCR System (Applied Biosystems, USA) with the SYBR quantitative PCR SuperMix W/ROX (Invitrogen). Samples were normalized to the independent control housekeeping gene GAPDH and were reported following the DDCT method as RNA fold increase: 2DDCT ¼ 2DCT sample DCT reference -2 . The primers used in the study were listed as followings: SETD3 Fw 50 -GTGCATCAAGGGACGCGCTG-30 , Rev 50 GTAGAACCACGGAGCTCATG-3’; KLC4 Fw 50 -CAGTAATGCGCAGATGATGCG-30 , Rev 50 -CCAGAGAAGGGACCACATCA-3’; iNOS Fw 50 -
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AGCAGACTAGTTAGCGGCAT-30 , Rev 50 -GTAGTGTACACGAGGCCAGG3’; eNOS Fw 50 -CGCAGCTCGACGGCGGAACT-30 , Rev 50 -CACAGACCGCGAAGGGAG-3'and GAPDH Fw 50 -TTCAAGTGCAGTAGAGGAGG-30 , Rev 50 -AGCCACAAGCCACGACTAATG-3’. 2.10. Animal experiment All animal experiments were approved by the Animal Care Committee of the People's Hospital of Pingyi County. Animal studies were conducted using 5-week-old female BALB/c nude mice (Vital River Laboratory Animal Technology Co. Ltd, Beijing, China). 1.5 107 SiHa-XR cells stably over-expressing SETD3 or the empty vector were subcutaneously injected into the left anterior axillae of
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nude mice. 3 days following inoculation with the tumor cells, mice were further divided into two groups that one group mice were subjected to irradiation with 8 Gy every 6 days. Tumor volume was measured using vernier calipers every week following the equation: tumor volume¼(length width2)/2. After irradiation for three times, on day 21 after inoculation, all animals were sacrificed and tumor samples were excised for further studies. 2.11. Statistical analysis All experiments were repeated at least three times, and statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA). Student's t-test was used for comparisons
Fig. 1. SETD3 expression is negatively associated with radioresistance in cervical cancer. (A) The indicated cervical cancer cells were subjected to different doses of radiation for 8 h, and then were subjected to colony formation analysis. þp < 0.05 and þþp < 0.01 vs SiHa-XR group. (B) Representative photos of tumorspheres in SiHa-XR and SiHa cells after 6 Gy radiation for 8 h þþp < 0.01. (C) Comet assay and (D) flow cytometry analysis of SiHa-XR and SiHa cells after radiation (6 Gy) for 8 h þþp < 0.01. (E) RT-qPCR and (F) Western blot analysis of SETD3 in SiHa or SiHa-XR cells. þþp < 0.01 vs SiHa group. (G) RT-qPCR analysis of SETD3 in samples from radioresistant cervical cancer patients and radiosensitive cervical cancer patients (n ¼ 9). þþp < 0.01. (H,I) SiHa cells were transfected with SETD3 siRNAs for 24 h, followed by transfection efficacy evaluation using RT-qPCR and Western blot analysis. þp < 0.05 and þþp < 0.01 vs siCon group. Data were the mean ± SEM. ns, no significant difference.
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of group means. The p value < 0.05 was served as significant differences. 3. Results 3.1. SETD3 expression is negatively associated with radioresistance in cervical cancer In this part, the X-ray-resistant SiHa cells were verified. As displayed in Fig. 1A, colony formation analysis indicated that SiHaXR cells showed better survival fraction in comparison to parental SiHa cells after the same irradiation. 6 Gy of irradiation markedly decreased the number and size of tumorspheres in SiHa cells, but was not observed in SiHa-XR cells (Fig. 1B). Comet analysis also indicated that SiHa-XR cells showed decreased DNA damage in comparison to SiHa cells when subjected to the same irradiation (Fig. 1C). Flow cytometry analysis suggested that 6 Gy irradiationinduced significant apoptosis in SiHa cells compared to the controls (0 Gy irradiation), but SiHa-XR cells showed no significant effects (Fig. 1D). These results verified that SiHa-XR was a radioresistant cervical cancer cell line. Then, RT-qPCR and Western blot analysis suggested that SETD3 was markedly down-regulated in SiHa-XR cervical cancer cells compared with the parental SiHa cells (Fig. 1E and F). Clinical analysis also showed the significantly downregulated SETD3 expression in radioresistant samples compared
with samples from radiotherapy-sensitive patients (Fig. 1G). Those experimental and clinical results indicated that reduced SETD3 expression was associated with radioresistance in cervical cancer. Subsequently, we explored the effects of SETD3 on radiosensitivity of human cervical cancer cell by silencing SETD3 expression in SiHa cells (Fig. 1H and I). 3.2. Effects of SETD3 expression on radioresistance in human cervical cancer cells Here, we found that SETD3 knockdown markedly protected SiHa cells from irradiation-triggered cell death, DNA damage and apoptosis (Fig. 2AeC). These findings significantly indicated that SETD3 inhibition led to the progression of radioresistance in human cervical cancer cells. Then, if SETD3 over-expression could promote the sensitivity of radioresistant cervical cancer cells to radiotherapy, SETD3 expression was promoted in SiHa-XR cells (Fig. 2D and E). Colony formation and CCK-8 analysis indicated that SETD3 over-expression markedly elevated irradiation-triggered cell growth suppression and cell death in comparison to the Vec control group in SiHa-XR cells (Fig. 2F and G). Subsequently, the comet and flow cytometry analysis demonstrated that SETD3 over-expression combined with irradiation apparently caused DNA damage and apoptosis in SiHa-XR cells compared with irradiation-single group (Fig. 2H and I).
Fig. 2. Effects of SETD3 expression on radioresistance in human cervical cancer cells. (A) SiHa cells were subjected to radiation at the indicated doses for 8 h, followed by colony formation. þp < 0.05 and þþp < 0.01 vs SiHa-siCon group. SiHa cells transfected with or without siSETD3 for 24 h, followed by radiation (6 Gy) for another 8 h. Then, (B) comet assay and (C) flow cytometry analysis for apoptosis was performed. þp < 0.05 and þþp < 0.01. (D,E) SiHa-XR cells were transfected with SETD3 plasmid for 24 h, followed by transfection efficacy calculation using RT-qPCR and Western blot analysis. þþþp < 0.001 vs Vec group. SiHa-XR cells were subjected to radiation at the indicated concentrations for 8 h. Then, (F) colony formation and (G) CCK-8 analysis were used to determine the cell proliferation. þp < 0.05 and þþp < 0.01 vs Vec group. SiHa-XR cells were transfected with SETD3 plasmid for 24 h, followed by radiation (6 Gy) for another 8 h. Then, (H) comet assay and (I) flow cytometry analysis for apoptosis were conducted. þþp < 0.01. Data were the mean ± SEM.
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3.3. SETD3 up-regulation overcomes radioresistance in cervical cancer in vivo Using xenograft animal models, the effects of SETD3 on radioresistance in cervical cancer were confirmed. As indicated in Fig. 3AeC, irradiation or SETD3 alone treatment showed no influence on the change of tumor size, tumor volume and tumor weight. However, a combination of irradiation and SETD3 overexpression markedly inhibited tumor growth. Immunohistochemical analysis suggested that SETD3 co-treatment with irradiation significantly reduced KI-67 positive levels (a marker for cell proliferation) [17] and enhanced TUNEL (an indicator for apoptosis) [18] expression levels in tumor samples in comparison to the single treatment of SETD3 over-expression or irradiation (Fig. 3DeF). These results indicated that SETD3 over-expression promoted radiosensitivity in human radioresistant cervical cancer cells partly through promoting irradiation-triggered apoptosis. 3.4. SETD3 is negatively correlated with KLC4 expression in human cervical cancer cells KLC4 has been implicated in tumor radioresistance [19]. RTqPCR and Western blot analysis indicated that SiHa-XR cells showed higher KLC4 expression compared to SiHa cells (Fig. 4A). In SiHa cells, SETD3 knockdown markedly increased KLC4 expression both from mRNA and protein levels in SiHa cells (Fig. 4B). Notably, SETD3 over-expression clearly reduced KLC4 mRNA and protein expression levels in SiHa cells (Fig. 4C). In vivo data suggested that SETD3 over-expression combined with irradiation markedly reduced KLC4 expression in tumor samples from mice (Fig. 4D). Then, KLC4 was knocked down to further explore if its reduction could influence the radioresistance of SiHa-XR (Fig. 4E). As shown in Fig. 4F and G, CCK-8 and colony formation results indicated that irradiation-triggered cell death and proliferation inhibition were markedly promoted by siKLC4 in SiHa-XR cells. In SiHa cells,
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siSETD3-restored cell viability and proliferative ability were significantly blocked by KLC4 knockdown (Fig. 4H and I). Then, KLC4 expression was promoted in SiHa-XR cells to investigate if its over-expression could affect oeSETD3-induced DNA damage (Fig. 4J). Comet assay suggested that oeSETD3-induced DNA damage in SiHa-XR cells was clearly rescued by KLC4 over-expression (Fig. 4K). Thus, SETD3 could modulate KLC4 expression to influence the sensitization of cervical cancer cell to radiotherapy. NO production is significantly correlated with radioresistance in cancer [20]. Then, we found that the levels of NO, iNOS, and eNOS were markedly up-regulated in SiHa-XR cells compared with the parental SiHa cells (Fig. 4L). Our results further suggested that inhibiting SETD3 evidently up-regulated NO production, as well as iNOS and eNOS expression in SiHa cells, At the same time, reducing SETD3 expression more significantly elevated irradiation-induced NO, iNOS and eNOS compared to the si-Con group (Fig. 4M). However, SETD3 over-expression markedly reduced NO, eNOS and iNOS levels in SiHa-XR cells in comparison to the Vect group. Additionally, SETD3 over-expression markedly suppressed irradiation-triggered NO, eNOS and iNOS levels in SiHa-XR cells compared with the Vec control group (Fig. 4N). Finally, suppressing NO using L-NMMA, a NOS inhibitor, markedly inhibited siSETD3induced KLC4 expression in SiHa cells (Fig. 4O and P). Therefore, the results above suggested that SETD3 decreased KLC4 expression by suppressing iNOS and eNOS expression to down-regulate NO generation in cervical cancer cells. 4. Discussion Radioresistance is linked with poor clinical outcome in patients with cervical cancer [3,21,22]. However, there is no effective treatment specific for cervical cancer patients. Therefore, investigation of the detailed factors that influence cervical cancer radioresistance is essential for promoting the efficiency of radiation therapy. The present study aimed to identify a radioresistance
Fig. 3. SETD3 up-regulation overcomes radioresistance in cervical cancer in vivo. (A) Representative images of tumor samples. (B) Calculation of tumor volume. (C) Measurements of tumor weight. (D) IHC staining of KI-67 and TUNEL expression. Quantification of (E) KI-67- and (F) TUNEL-positive levels in tumor sections was exhibited. Data were the mean ± SEM. þþp < 0.01.
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Fig. 4. SETD3 is negatively correlated with KLC4 expression in human cervical cancer cells. Western blot and RT-qPCR analysis of KLC4 in (A) SiHa and SiHa-XR cells, (B) SiHa cells with or without SETD3 knockdown and (C) SiHa cells with or without SETD3 over-expression. (D) KLC4 expression in tumor samples from the indicated group of mice using IHC staining. (E) Transfection efficacy calculation of KLC4 knockdown using its specific siRNA by Western blot analysis in SiHa-XR cells. SiHa-XR cells after 24 h of transfection with siKLC4 were subjected to radiation for 8 h, followed by (F) CCK-8 and (G) colony formation analysis. SiHa cells after 24 h of transfection with siSETD3 combined with or without siKLC4 were subjected to radiation for 8 h, followed by (H) CCK-8 and (I) colony formation analysis. þp < 0.05 and þþp < 0.01 vs siCon group; *p < 0.05 vs siSETD3 group. (J) SiHa-XR cells were transfected with KLC4 plasmid for 24 h, followed by transfection efficacy calculation using Western blot analysis. (K) SiHa-XR cells were transfected with KLC4 plasmid for 24 h, followed by radiation (6 Gy) for another 8 h. Then, comet assay was performed. þþp < 0.01 vs Vec group; *p < 0.05 vs oeSETD3 group. (L) RT-qPCR analysis of NO, iNOS and eNOS in SiHa or SiHa-XR cells. (M) SiHa cells transfected with 24 h of siSETD3 were treated with radiation (6 Gy) for 8 h. Then, all cells were harvested for RT-qPCR analysis of NO, iNOS and eNOS. (N) SiHa cells were transfected with 24 h of SETD3 plasmid, followed by 6 Gy of radiation for another 8 h. Then, all cells were harvested for RT-qPCR analysis of NO, iNOS and eNOS. (O,P) SiHa cells were pre-treated with NOS inhibitor (L-NMMA, 10 mM) for 4 h, followed by transfection with siSETD3 for another 24 h. Then, cells were harvested for RT-qPCR and Western blot analysis of KLC4 expression levels. (Q) Proposed model of SETD3 effects on the radiosensitivity in human cervical cancer. Data were the mean ± SEM. þ p < 0.05 and þþp < 0.01.
biomarker and the underlying molecular mechanisms that modulate the response of cervical cancer to radiotherapy. In the present study, we found that SETD3 expression was markedly downregulated in cervical cancer cells with radioresistance, and samples from radioresistant cervical cancer patients in comparison to radiosensitive cervical cancer cells and clinical tissues, respectively. Moreover, the in vitro and in vivo results indicated that SETD3 knockdown markedly up-regulated the radiosensitivity of cervical cancer cells with radioresistance through suppressing irradiationtriggered apoptosis and DNA damage. Essentially, we found that over-expressing SETD3 levels markedly promoted the radiosensitivity in cervical cancer cells with radioresistance, which was partly via the inhibition of KLC4 and NO production (Fig. 4Q). Collectively, these data indicated that SETD4 silence was partially linked to the progression of radioresistance in cervical cancer, and improving SETD3 expression might be a promising therapeutic strategy overcoming radioresistance in patients with cervical cancer. Subsequently, we attempted to explore the molecular mechanism through which SETD3 modulated cervical cancer cell radiosensitivity. Increasing studies have suggested a positive relationship between up-regulated kinesin light chain 4 (KLC4) expression and radioresistance. KLC4 plays an essential role in cell death-associated processes. In lung cancer cell lines, KLC4 deficiency could activate p53, which is involved in the DNA damage
response, demonstrating the potential role of KLC4 in regulating DNA damage response [19]. Irradiation induces DNA damage by various signaling pathways [6,23,24]. Additionally, apoptosis, known as an evolutionarily conserved process, is tightly regulated in various ways to control the pathologies involved in radioresistance in different types of tumors [25,26]. Recently, SETD3 was found to induce apoptosis in colon cancer, and a potential diagnostic marker of resistance to doxorubicin treatment in colon cancer patients, which indicated the potential role of SETD3 in regulating tumor growth with drug resistance [8]. Herein, our results indicated that reduced SETD3 expression led to cervical cancer cell radioresistance, contributing to up-regulated expression of KLC4. Inversely, promoting the expression of SETD3 elevated radiosensitivity in cervical cancer cells, along with enhanced apoptosis and reduced expression of KLC4. These data indicated that SETD3 up-regulation promoted the radiosensitivity of cervical cancer cells through decreasing KLC4 expression. NO is a familiar molecule, which has been extensively studied during tumor progression [20,27]. As reported, increasing NO levels was an effective strategy to protect mice from the effects of total body irradiation [28,29]. In our study, we found that SETD3 inhibited NO production in cervical cancer cells by down-regulating the expression of iNOS and eNOS. Importantly, we found that SETD3 knockdown-induced increase of KLC4 could be eliminated by the prevention of NOS,
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demonstrating that there might be a direct or indirect relationship between NOS production and KLC4 expression involved in cervical cancer progression with radioresistance. And given that NO could act as a radiosensitizer or radioprotector, further studies are still warranted in future to comprehensively reveal the mechanism whereby SETD3 regulates KLC4 expression via NOS pathway. In conclusion (Fig. 4Q), the study indicated that reducing SETD3 expression resulted in the progression of radioresistance, whereas promoting SETD3 expression could markedly overcome radioresistance in human cervical cancer cells. Moreover, we suggested that SETD3 improved rediosensitivity through reducing the expression of KLC4, which was associated with the decrease in NO generation through blocking iNOS and eNOS expression. Based on our results, promoting SETD3 might represent an effective approach for cervical cancer prevention in patients who are resistant to radiotherapy. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.06.058. References [1] C. de Martel, M. Plummer, J. Vignat, et al., Worldwide burden of cancer attributable to HPV by site, country and HPV type, Int. J. Cancer 141 (4) (2017) 664e670. [2] H. Cheng, Inhibiting CD146 by its monoclonal antibody AA98 improves radiosensitivity of cervical cancer cells, Med. Sci. Monit.: Int. Med. J.Exp. Clin. Res. 22 (2016) 3328. [3] B.M. Barney, I.A. Petersen, S.C. Dowdy, et al., Intraoperative Electron Beam Radiotherapy (IOERT) in the management of locally advanced or recurrent cervical cancer, Radiat. Oncol. 8 (1) (2013) 80. [4] M.E.B. Powell, Modern radiotherapy and cervical cancer, Int. J. Gynecol. Cancer 20 (11) (2010) S49eS51. [5] Y. Qing, X.Q. Yang, Z.Y. Zhong, et al., Microarray analysis of DNA damage repair gene expression profiles in cervical cancer cells radioresistant to 252 Cf neutron and X-rays, BMC Canc. 10 (1) (2010) 71. [6] M.E. Lomax, L.K. Folkes, P. O'neill, Biological consequences of radiationinduced DNA damage: relevance to radiotherapy, Clin. Oncol. 25 (10) (2013) 578e585. [7] O. Cohn, M. Feldman, L. Weil, et al., Chromatin associated SETD3 negatively regulates VEGF expression, Sci. Rep. 6 (2016) 37115. [8] E. Abaev-Schneiderman, L. Admoni-Elisha, D. Levy, SETD3 is a positive regulator of DNA-damage-induced apoptosis, Cell Death Dis. 10 (2) (2019) 74. [9] S.E. Cooper, E. Hodimont, C.M. Green, A fluorescent bimolecular complementation screen reveals MAF1, RNF7 and SETD3 as PCNA-associated proteins in human cells, Cell Cycle 14 (15) (2015) 2509e2519. [10] X. Jiang, T. Li, J. Sun, et al., SETD3 negatively regulates VEGF expression during hypoxic pulmonary hypertension in rats, Hypertens. Res. 41 (9) (2018) 691.
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[11] Z. Chen, C.T. Yan, Y. Dou, et al., The role of a newly identified SET domaincontaining protein, SETD3, in oncogenesis, Haematologica 98 (5) (2013) 739e743. [12] X. Cheng, Y. Hao, W. Shu, et al., Cell cycle-dependent degradation of the methyltransferase SETD3 attenuates cell proliferation and liver tumorigenesis, J. Biol. Chem. 292 (22) (2017) 9022e9033. [13] A.S. Pires-Luís, M. Vieira-Coimbra, F.Q. Vieira, et al., Expression of histone methyltransferases as novel biomarkers for renal cell tumor diagnosis and prognostication, Epigenetics 10 (11) (2015) 1033e1043. [14] D.W. Kim, K.B. Kim, J.Y. Kim, et al., Characterization of a novel histone H3K36 methyltransferase setd3 in zebrafish, Biosci. Biotechnol. Biochem. 75 (2) (2011) 289e294. [15] O. Kitahara, T. Katagiri, T. Tsunoda, et al., Classification of sensitivity or resistance of cervical cancers to ionizing radiation according to expression profiles of 62 genes selected by cDNA microarray analysis, Neoplasia 4 (4) (2002) 295e303. [16] Y. Qing, X.Q. Yang, Z.Y. Zhong, et al., Microarray analysis of DNA damage repair gene expression profiles in cervical cancer cells radioresistant to 252 Cf neutron and X-rays, BMC Canc. 10 (1) (2010) 71. [17] A. Urruticoechea, I.E. Smith, M. Dowsett, Proliferation marker Ki-67 in early breast cancer, J. Clin. Oncol. 23 (28) (2005) 7212e7220. [18] M. Colecchia, B. Frigo, C. Del Boca, et al., Detection of apoptosis by the TUNEL technique in clinically localised prostatic cancer before and after combined endocrine therapy, J. Clin. Pathol. 50 (5) (1997) 384e388. [19] J.H. Baek, J. Lee, H.S. Yun, et al., Kinesin light chain-4 depletion induces apoptosis of radioresistant cancer cells by mitochondrial dysfunction via calcium ion influx, Cell Death Dis. 9 (5) (2018) 496. [20] J. Scicinski, B. Oronsky, S. Ning, et al., NO to cancer: the complex and multifaceted role of nitric oxide and the epigenetic nitric oxide donor, RRx-001, Redox biology 6 (2015) 1e8. pez, A. Poitevin, V. Mendoza-Martínez, et al., Cancer-initiating cells [21] J. Lo derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance, BMC Canc. 12 (1) (2012) 48. [22] L. Jing, W. Yuan, D. Ruofan, et al., HOTAIR enhanced aggressive biological behaviors and induced radio-resistance via inhibiting p21 in cervical cancer, Tumor Biol. 36 (5) (2015) 3611e3619. [23] B. Hu, C. Jin, H.B. Li, et al., The DNA-sensing AIM2 inflammasome controls radiation-induced cell death and tissue injury, Science 354 (6313) (2016) 765e768. [24] S. Meylan, S. Incerti, M. Karamitros, et al., Simulation of early DNA damage after the irradiation of a fibroblast cell nucleus using Geant4-DNA, Sci. Rep. 7 (1) (2017) 11923. [25] S. Verghese, T.T. Su, Drosophila Wnt and STAT define apoptosis-resistant epithelial cells for tissue regeneration after irradiation, PLoS Biol. 14 (9) (2016), e1002536. [26] Y. Liu, M. Tian, H. Zhao, et al., IER5 as a promising predictive marker promotes irradiation-induced apoptosis in cervical cancer tissues from patients undergoing chemoradiotherapy, Oncotarget 8 (22) (2017) 36438. [27] S.K. Choudhari, M. Chaudhary, S. Bagde, et al., Nitric oxide and cancer: a review, World J. Surg. Oncol. 11 (1) (2013) 118. [28] J.B. Maxhimer, D.R. Soto-Pantoja, L.A. Ridnour, et al., Radioprotection in normal tissue and delayed tumor growth by blockade of CD47 signaling, Sci. Transl. Med. 1 (3) (2009), 3ra7-3ra7. [29] D.R. Soto-Pantoja, L.A. Ridnour, D.A. Wink, et al., Blockade of CD47 increases survival of mice exposed to lethal total body irradiation, Sci. Rep. 3 (2013) 1038.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
SETD3 reduces KLC4 expression to improve the sensitization of cervical cancer cell to radiotherapy Qingmei Li a, Yanqin Zhang b, Qiuli Jiang c, * a
Department of the Second Area of Obstetrics, The People's Hospital of Pingyi County, Linyi, 273300, China Department of Nursing, Yulin Traditional Chinese Medicine Hospital, Yulin Shanxi, 719000, China c Department of Gynecology, Hanzhong Central Hospital, Hanzhong Shaanxi, 723000, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 June 2019 Accepted 11 June 2019 Available online 22 June 2019
Resistance to radiotherapy accounts for most therapeutic failures in cervical cancer patients who undergo radical radiation therapy. To indicate the possible molecular mechanism of radioresistance and improve the 5-year survival rate, we focused on how SET domain protein 3 (SETD3) regulated radioresistance in human cervical cancer cells in this study. Our results indicated that SETD3 over-expression markedly increased the radiosensitivity of cervical cancer cells with radioresistance, as evidenced by the further reduced cell viability, proliferation, DNA damage and cell death. In addition, we found that SETD3 down-regulated the expression of kinesin light chain 4 (KLC4), contributing to the radiosensitivity of cervical cancer cells, and the regulatory role of SETD3 could be abolished by KLC4 over-expression. Moreover, nitric oxide (NO) production was significantly reduced by SETD3 over-expression through repressing the expression of inducible NO synthase (iNOS) and endothelial NO synthase (eNOS) in cervical cancer cells. In vivo studies using xenograft animal models also demonstrated that SETD3 overexpression combined with irradiation treatment markedly inhibited tumor growth and induced apoptosis. In summary, our data demonstrated that down-regulated SETD3 expression markedly led to the progression of radioresistance and that promoting SETD3 expression could sensitized cervical cancer cells to radiotherapy, thereby targeting SETD3 might be a potential strategy for the clinical management of cervical cancer to improve the curative effect of radiation in cervical cancer. © 2019 Published by Elsevier Inc.
Keywords: Cervical cancer Radioresistance SETD3 KLC4 NO production
1. Introduction Cervical cancer is the fourth most common malignant disease and one of the major causes of tumor-associated death among females in the world [1,2]. Presently, radiotherapy plays an essential role in the prevention of cervical cancer. Approximately 80% of patients with cervical malignant tumors need radiation therapy. However, resistance to radiotherapy plays a crucial role in treatment failure in patients undergoing radical radiation therapy [3e5]. Nevertheless, the molecular mechanism for radioresistance acquirement and development in cervical cancer still remains unknown. Mechanistically, radiotherapy results in cell cycle arrest and tumor cell death through inducing DNA damage [6]. SET domain protein 3 (SETD3) is a member of the protein lysine
* Corresponding author. E-mail address: [email protected] (Q. Jiang). https://doi.org/10.1016/j.bbrc.2019.06.058 0006-291X/© 2019 Published by Elsevier Inc.
methyltransferase (PKMT) family, which catalyzes the addition of methyl group to lysine residues. Recently, SETD3 was reported to be a positive modulator of DNA-damage-induced apoptosis in colon cancer cells [7,8]. Moreover, SETD3 was suggested to interact with Maf1, PCNA and RNF7, which are crucial signals involved in DNA replication progression, further linking SETD3 to DNA repair [9,10]. SETD3 has been indicated to participate in the oncogenic processes that high SETD3 expression shows oncogenic properties in lymphoma and liver cancer [11,12]. However, there is also study suggesting that lower expression of SETD3 was associated with markedly shorter disease-free survival [13]. Furthermore, SETD3 overexpression reduced cell viability and induced apoptosis in zebrafish models [14]. Thus, SETD3 also exhibits anti-tumorigenic role. Herein, the role of SETD3 in tumor might be depended on its type, and however, the effects of SETD3 on radiation resistance in cervical cancer are little to be reported. In this study, we elucidated that SETD3 expression levels were inversely associated with radioresistance in patients with cervical cancer. Our findings suggested that SETD3 could sensitize tumor to
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radiotherapy through promoting radiation-triggered apoptosis in cervical cancer cells. Moreover, our results suggested that SETD3 elevated the radiosensitivity of human cervical cancer cells by inhibiting KLC4 expression and NO production. These data demonstrated that SETD3 could be a radioresistance biomarker in cervical cancer cells. 2. Materials and methods 2.1. Human specimens Human tissue samples were collected through biopsy from patients before and after radiotherapy in the People's Hospital of Pingyi County (Shandong, China). Initial tissue samples were prepared to determine gene expression, and follow-up tissue specimens (after radiotherapy) underwent a calculation to explore the effectiveness of the radiotherapy. The effectiveness of radiotherapy was calculated following methods as previously indicated by Kitahara et al. [15]. Samples were divided into radiosensitive and radioresistant groups according to the results of calculation. The research was approved by the Ethics Committee of The People's Hospital of Pingyi County (Shandong). All patients provided their informed consent prior to sample collection. 2.2. Cells and treatments Human cervical cancer cell lines of SiHa was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). X-ray resistant SiHa cells (SiHa-XR) were developed using SiHa cells as previously indicated [16]. The cervical cancer parental cell line was exposed to 2 Gy 3 times, 4 Gy 3 times, 6 Gy 3 times, 8 Gy twice and 10 Gy twice. Irradiation was performed at 300 cGy/min using a linear accelerator (SIMEN, Germany). SiHa and SiHa-XR cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO, USA) containing 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA) at 37 C and 5% CO2. Corresponding plasmid and siRNA constructs of SETD3 and KLC4 were designed and synthesized by Western Biomedical Technology (Chongqing, China). Then, cells were transfected with the indicated plasmids or siRNAs with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following manufacturer's protocols. NOS inhibitor of L-NMMA was purchased from Beyotime (Jiangsu, China). Nitrate/Nitrite Assay Kit was also obtained from Beyotime to calculate NO production in cells according to the manufacturer's recommendations. 2.3. Colony formation and cell viability analysis Cells were planted in a well of a six-well plate and cultured under 5% CO2 at 37 C for 2 weeks. Next, the cells were fixed with formalin, stained with 0.1% crystal violet. Tumor cell proliferation was calculated in five microscopic fields. Cell viability was measured with CCK-8 kit (Dojindo, Japan) according to the manufacturer's recommendations. Absorbance was recorded at 450 nm. 2.4. Flow cytometry analysis Cells were washed with phosphate-buffered saline (PBS) and stained with annexin V and propidium iodide (Beyotime) following the manufacturer's instruction using a BD FACSCalibur flow cytometer (BD Biosciences, USA). Apoptosis was defined as the total percentage of cells that were positive both for PI and Annexin V. 2.5. Tumorsphere culture Cells were cultured in stem cell medium consisting of DMEM/
F12 basal medium, N2, and B27 supplements (Invitrogen), human recombinant epidermal growth factor (20 ng/mL), and basic fibroblastic growth factor (20 ng/mL) (Rocky Hill, NJ, USA). As for the tumorsphere formation, cells were incubated at a density of 200 cells/well in 24-well ultra-low-attachment plates and sustained in stem cell media. Tumorspheres that formed within 2 weeks were recorded. For serial tumorsphere formation analysis, the spheres were collected, disaggregated with trypsin/EDTA, filtered via a 40-mm mesh, and then re-plated as described above. Triplicate samples were performed, and the spheres were measured by individuals in a blind fashion. 2.6. Western blotting analysis Cells were lysed using radioimmunoprecipitation assay lysis buffer (Beyotime). Protein concentrations were calculated with a bicinchoninic acid assay (BCA) (Thermo Fisher Scientific, USA). Proteins were then separated on 10% SDS-PAGE and translocated onto PVDF membrane (Millipore, USA), and immunodetected with the primary antibodies, including anti-SETD3 (1:1000 dilutions, Abcam, USA), anti-KLC4 (1:1000 dilutions, Thermo Fisher Scientific) and anti-GAPDH (1:1000 dilutions, Cell Signaling Technology, USA). Then, the membranes were incubated with HRP-conjugated secondary antibodies (Beyotime) for 1 h at room temperature. The blots were then visualized with an enhanced chemiluminescence reagent (Beyotime). Immunoreactive bands were quantitatively analyzed using ImageJ software. 2.7. Comet analysis The comet assay was performed to calculate the level of DNA damage in cells (Trevigen, Gaithersburg, MD, USA). Slides were placed on the electrophoresis slide tray. Electrophoresis was performed by an electric field of 21 V in alkaline electrophoresis solution). Subsequently, the slides were immersed in distilled water and in 70% ethanol. Samples were then dried. Finally, the slides were stained with 1 mg/mL of propidium iodide and captured under a fluorescence microscopy. 2.8. Immunohistochemistry (IHC) Tumor samples were fixed with 4% paraformaldehyde (PFA), embedded in paraffin, and cut into 4-mm-thick sections. After deparaffinizing in xylene and rehydrating using alcohol series, the sections were detected with primary antibody against KI-67 (1:100, Abcam, USA) overnight at 4 C. After incubation with the secondary antibody, the sections were developed using diaminobenzidine and counterstained with hematoxylin. As for cell death analysis in tumor samples, TUNEL Apoptosis Assay Kit (Beyotime) was used following the manufacturer's instruction. 2.9. Quantitative real-time PCR analysis Total RNA of cells was isolated with TRIzol reagent (Invitrogen, USA) and subjected to reverse transcription with Oligo (dT) and MMLV Reverse Transcriptase (Invitrogen). Quantitative PCR amplification was conducted using the ViiA™ 7 Real-Time PCR System (Applied Biosystems, USA) with the SYBR quantitative PCR SuperMix W/ROX (Invitrogen). Samples were normalized to the independent control housekeeping gene GAPDH and were reported following the DDCT method as RNA fold increase: 2DDCT ¼ 2DCT sample DCT reference -2 . The primers used in the study were listed as followings: SETD3 Fw 50 -GTGCATCAAGGGACGCGCTG-30 , Rev 50 GTAGAACCACGGAGCTCATG-3’; KLC4 Fw 50 -CAGTAATGCGCAGATGATGCG-30 , Rev 50 -CCAGAGAAGGGACCACATCA-3’; iNOS Fw 50 -
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AGCAGACTAGTTAGCGGCAT-30 , Rev 50 -GTAGTGTACACGAGGCCAGG3’; eNOS Fw 50 -CGCAGCTCGACGGCGGAACT-30 , Rev 50 -CACAGACCGCGAAGGGAG-3'and GAPDH Fw 50 -TTCAAGTGCAGTAGAGGAGG-30 , Rev 50 -AGCCACAAGCCACGACTAATG-3’. 2.10. Animal experiment All animal experiments were approved by the Animal Care Committee of the People's Hospital of Pingyi County. Animal studies were conducted using 5-week-old female BALB/c nude mice (Vital River Laboratory Animal Technology Co. Ltd, Beijing, China). 1.5 107 SiHa-XR cells stably over-expressing SETD3 or the empty vector were subcutaneously injected into the left anterior axillae of
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nude mice. 3 days following inoculation with the tumor cells, mice were further divided into two groups that one group mice were subjected to irradiation with 8 Gy every 6 days. Tumor volume was measured using vernier calipers every week following the equation: tumor volume¼(length width2)/2. After irradiation for three times, on day 21 after inoculation, all animals were sacrificed and tumor samples were excised for further studies. 2.11. Statistical analysis All experiments were repeated at least three times, and statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA). Student's t-test was used for comparisons
Fig. 1. SETD3 expression is negatively associated with radioresistance in cervical cancer. (A) The indicated cervical cancer cells were subjected to different doses of radiation for 8 h, and then were subjected to colony formation analysis. þp < 0.05 and þþp < 0.01 vs SiHa-XR group. (B) Representative photos of tumorspheres in SiHa-XR and SiHa cells after 6 Gy radiation for 8 h þþp < 0.01. (C) Comet assay and (D) flow cytometry analysis of SiHa-XR and SiHa cells after radiation (6 Gy) for 8 h þþp < 0.01. (E) RT-qPCR and (F) Western blot analysis of SETD3 in SiHa or SiHa-XR cells. þþp < 0.01 vs SiHa group. (G) RT-qPCR analysis of SETD3 in samples from radioresistant cervical cancer patients and radiosensitive cervical cancer patients (n ¼ 9). þþp < 0.01. (H,I) SiHa cells were transfected with SETD3 siRNAs for 24 h, followed by transfection efficacy evaluation using RT-qPCR and Western blot analysis. þp < 0.05 and þþp < 0.01 vs siCon group. Data were the mean ± SEM. ns, no significant difference.
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of group means. The p value < 0.05 was served as significant differences. 3. Results 3.1. SETD3 expression is negatively associated with radioresistance in cervical cancer In this part, the X-ray-resistant SiHa cells were verified. As displayed in Fig. 1A, colony formation analysis indicated that SiHaXR cells showed better survival fraction in comparison to parental SiHa cells after the same irradiation. 6 Gy of irradiation markedly decreased the number and size of tumorspheres in SiHa cells, but was not observed in SiHa-XR cells (Fig. 1B). Comet analysis also indicated that SiHa-XR cells showed decreased DNA damage in comparison to SiHa cells when subjected to the same irradiation (Fig. 1C). Flow cytometry analysis suggested that 6 Gy irradiationinduced significant apoptosis in SiHa cells compared to the controls (0 Gy irradiation), but SiHa-XR cells showed no significant effects (Fig. 1D). These results verified that SiHa-XR was a radioresistant cervical cancer cell line. Then, RT-qPCR and Western blot analysis suggested that SETD3 was markedly down-regulated in SiHa-XR cervical cancer cells compared with the parental SiHa cells (Fig. 1E and F). Clinical analysis also showed the significantly downregulated SETD3 expression in radioresistant samples compared
with samples from radiotherapy-sensitive patients (Fig. 1G). Those experimental and clinical results indicated that reduced SETD3 expression was associated with radioresistance in cervical cancer. Subsequently, we explored the effects of SETD3 on radiosensitivity of human cervical cancer cell by silencing SETD3 expression in SiHa cells (Fig. 1H and I). 3.2. Effects of SETD3 expression on radioresistance in human cervical cancer cells Here, we found that SETD3 knockdown markedly protected SiHa cells from irradiation-triggered cell death, DNA damage and apoptosis (Fig. 2AeC). These findings significantly indicated that SETD3 inhibition led to the progression of radioresistance in human cervical cancer cells. Then, if SETD3 over-expression could promote the sensitivity of radioresistant cervical cancer cells to radiotherapy, SETD3 expression was promoted in SiHa-XR cells (Fig. 2D and E). Colony formation and CCK-8 analysis indicated that SETD3 over-expression markedly elevated irradiation-triggered cell growth suppression and cell death in comparison to the Vec control group in SiHa-XR cells (Fig. 2F and G). Subsequently, the comet and flow cytometry analysis demonstrated that SETD3 over-expression combined with irradiation apparently caused DNA damage and apoptosis in SiHa-XR cells compared with irradiation-single group (Fig. 2H and I).
Fig. 2. Effects of SETD3 expression on radioresistance in human cervical cancer cells. (A) SiHa cells were subjected to radiation at the indicated doses for 8 h, followed by colony formation. þp < 0.05 and þþp < 0.01 vs SiHa-siCon group. SiHa cells transfected with or without siSETD3 for 24 h, followed by radiation (6 Gy) for another 8 h. Then, (B) comet assay and (C) flow cytometry analysis for apoptosis was performed. þp < 0.05 and þþp < 0.01. (D,E) SiHa-XR cells were transfected with SETD3 plasmid for 24 h, followed by transfection efficacy calculation using RT-qPCR and Western blot analysis. þþþp < 0.001 vs Vec group. SiHa-XR cells were subjected to radiation at the indicated concentrations for 8 h. Then, (F) colony formation and (G) CCK-8 analysis were used to determine the cell proliferation. þp < 0.05 and þþp < 0.01 vs Vec group. SiHa-XR cells were transfected with SETD3 plasmid for 24 h, followed by radiation (6 Gy) for another 8 h. Then, (H) comet assay and (I) flow cytometry analysis for apoptosis were conducted. þþp < 0.01. Data were the mean ± SEM.
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3.3. SETD3 up-regulation overcomes radioresistance in cervical cancer in vivo Using xenograft animal models, the effects of SETD3 on radioresistance in cervical cancer were confirmed. As indicated in Fig. 3AeC, irradiation or SETD3 alone treatment showed no influence on the change of tumor size, tumor volume and tumor weight. However, a combination of irradiation and SETD3 overexpression markedly inhibited tumor growth. Immunohistochemical analysis suggested that SETD3 co-treatment with irradiation significantly reduced KI-67 positive levels (a marker for cell proliferation) [17] and enhanced TUNEL (an indicator for apoptosis) [18] expression levels in tumor samples in comparison to the single treatment of SETD3 over-expression or irradiation (Fig. 3DeF). These results indicated that SETD3 over-expression promoted radiosensitivity in human radioresistant cervical cancer cells partly through promoting irradiation-triggered apoptosis. 3.4. SETD3 is negatively correlated with KLC4 expression in human cervical cancer cells KLC4 has been implicated in tumor radioresistance [19]. RTqPCR and Western blot analysis indicated that SiHa-XR cells showed higher KLC4 expression compared to SiHa cells (Fig. 4A). In SiHa cells, SETD3 knockdown markedly increased KLC4 expression both from mRNA and protein levels in SiHa cells (Fig. 4B). Notably, SETD3 over-expression clearly reduced KLC4 mRNA and protein expression levels in SiHa cells (Fig. 4C). In vivo data suggested that SETD3 over-expression combined with irradiation markedly reduced KLC4 expression in tumor samples from mice (Fig. 4D). Then, KLC4 was knocked down to further explore if its reduction could influence the radioresistance of SiHa-XR (Fig. 4E). As shown in Fig. 4F and G, CCK-8 and colony formation results indicated that irradiation-triggered cell death and proliferation inhibition were markedly promoted by siKLC4 in SiHa-XR cells. In SiHa cells,
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siSETD3-restored cell viability and proliferative ability were significantly blocked by KLC4 knockdown (Fig. 4H and I). Then, KLC4 expression was promoted in SiHa-XR cells to investigate if its over-expression could affect oeSETD3-induced DNA damage (Fig. 4J). Comet assay suggested that oeSETD3-induced DNA damage in SiHa-XR cells was clearly rescued by KLC4 over-expression (Fig. 4K). Thus, SETD3 could modulate KLC4 expression to influence the sensitization of cervical cancer cell to radiotherapy. NO production is significantly correlated with radioresistance in cancer [20]. Then, we found that the levels of NO, iNOS, and eNOS were markedly up-regulated in SiHa-XR cells compared with the parental SiHa cells (Fig. 4L). Our results further suggested that inhibiting SETD3 evidently up-regulated NO production, as well as iNOS and eNOS expression in SiHa cells, At the same time, reducing SETD3 expression more significantly elevated irradiation-induced NO, iNOS and eNOS compared to the si-Con group (Fig. 4M). However, SETD3 over-expression markedly reduced NO, eNOS and iNOS levels in SiHa-XR cells in comparison to the Vect group. Additionally, SETD3 over-expression markedly suppressed irradiation-triggered NO, eNOS and iNOS levels in SiHa-XR cells compared with the Vec control group (Fig. 4N). Finally, suppressing NO using L-NMMA, a NOS inhibitor, markedly inhibited siSETD3induced KLC4 expression in SiHa cells (Fig. 4O and P). Therefore, the results above suggested that SETD3 decreased KLC4 expression by suppressing iNOS and eNOS expression to down-regulate NO generation in cervical cancer cells. 4. Discussion Radioresistance is linked with poor clinical outcome in patients with cervical cancer [3,21,22]. However, there is no effective treatment specific for cervical cancer patients. Therefore, investigation of the detailed factors that influence cervical cancer radioresistance is essential for promoting the efficiency of radiation therapy. The present study aimed to identify a radioresistance
Fig. 3. SETD3 up-regulation overcomes radioresistance in cervical cancer in vivo. (A) Representative images of tumor samples. (B) Calculation of tumor volume. (C) Measurements of tumor weight. (D) IHC staining of KI-67 and TUNEL expression. Quantification of (E) KI-67- and (F) TUNEL-positive levels in tumor sections was exhibited. Data were the mean ± SEM. þþp < 0.01.
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Fig. 4. SETD3 is negatively correlated with KLC4 expression in human cervical cancer cells. Western blot and RT-qPCR analysis of KLC4 in (A) SiHa and SiHa-XR cells, (B) SiHa cells with or without SETD3 knockdown and (C) SiHa cells with or without SETD3 over-expression. (D) KLC4 expression in tumor samples from the indicated group of mice using IHC staining. (E) Transfection efficacy calculation of KLC4 knockdown using its specific siRNA by Western blot analysis in SiHa-XR cells. SiHa-XR cells after 24 h of transfection with siKLC4 were subjected to radiation for 8 h, followed by (F) CCK-8 and (G) colony formation analysis. SiHa cells after 24 h of transfection with siSETD3 combined with or without siKLC4 were subjected to radiation for 8 h, followed by (H) CCK-8 and (I) colony formation analysis. þp < 0.05 and þþp < 0.01 vs siCon group; *p < 0.05 vs siSETD3 group. (J) SiHa-XR cells were transfected with KLC4 plasmid for 24 h, followed by transfection efficacy calculation using Western blot analysis. (K) SiHa-XR cells were transfected with KLC4 plasmid for 24 h, followed by radiation (6 Gy) for another 8 h. Then, comet assay was performed. þþp < 0.01 vs Vec group; *p < 0.05 vs oeSETD3 group. (L) RT-qPCR analysis of NO, iNOS and eNOS in SiHa or SiHa-XR cells. (M) SiHa cells transfected with 24 h of siSETD3 were treated with radiation (6 Gy) for 8 h. Then, all cells were harvested for RT-qPCR analysis of NO, iNOS and eNOS. (N) SiHa cells were transfected with 24 h of SETD3 plasmid, followed by 6 Gy of radiation for another 8 h. Then, all cells were harvested for RT-qPCR analysis of NO, iNOS and eNOS. (O,P) SiHa cells were pre-treated with NOS inhibitor (L-NMMA, 10 mM) for 4 h, followed by transfection with siSETD3 for another 24 h. Then, cells were harvested for RT-qPCR and Western blot analysis of KLC4 expression levels. (Q) Proposed model of SETD3 effects on the radiosensitivity in human cervical cancer. Data were the mean ± SEM. þ p < 0.05 and þþp < 0.01.
biomarker and the underlying molecular mechanisms that modulate the response of cervical cancer to radiotherapy. In the present study, we found that SETD3 expression was markedly downregulated in cervical cancer cells with radioresistance, and samples from radioresistant cervical cancer patients in comparison to radiosensitive cervical cancer cells and clinical tissues, respectively. Moreover, the in vitro and in vivo results indicated that SETD3 knockdown markedly up-regulated the radiosensitivity of cervical cancer cells with radioresistance through suppressing irradiationtriggered apoptosis and DNA damage. Essentially, we found that over-expressing SETD3 levels markedly promoted the radiosensitivity in cervical cancer cells with radioresistance, which was partly via the inhibition of KLC4 and NO production (Fig. 4Q). Collectively, these data indicated that SETD4 silence was partially linked to the progression of radioresistance in cervical cancer, and improving SETD3 expression might be a promising therapeutic strategy overcoming radioresistance in patients with cervical cancer. Subsequently, we attempted to explore the molecular mechanism through which SETD3 modulated cervical cancer cell radiosensitivity. Increasing studies have suggested a positive relationship between up-regulated kinesin light chain 4 (KLC4) expression and radioresistance. KLC4 plays an essential role in cell death-associated processes. In lung cancer cell lines, KLC4 deficiency could activate p53, which is involved in the DNA damage
response, demonstrating the potential role of KLC4 in regulating DNA damage response [19]. Irradiation induces DNA damage by various signaling pathways [6,23,24]. Additionally, apoptosis, known as an evolutionarily conserved process, is tightly regulated in various ways to control the pathologies involved in radioresistance in different types of tumors [25,26]. Recently, SETD3 was found to induce apoptosis in colon cancer, and a potential diagnostic marker of resistance to doxorubicin treatment in colon cancer patients, which indicated the potential role of SETD3 in regulating tumor growth with drug resistance [8]. Herein, our results indicated that reduced SETD3 expression led to cervical cancer cell radioresistance, contributing to up-regulated expression of KLC4. Inversely, promoting the expression of SETD3 elevated radiosensitivity in cervical cancer cells, along with enhanced apoptosis and reduced expression of KLC4. These data indicated that SETD3 up-regulation promoted the radiosensitivity of cervical cancer cells through decreasing KLC4 expression. NO is a familiar molecule, which has been extensively studied during tumor progression [20,27]. As reported, increasing NO levels was an effective strategy to protect mice from the effects of total body irradiation [28,29]. In our study, we found that SETD3 inhibited NO production in cervical cancer cells by down-regulating the expression of iNOS and eNOS. Importantly, we found that SETD3 knockdown-induced increase of KLC4 could be eliminated by the prevention of NOS,
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demonstrating that there might be a direct or indirect relationship between NOS production and KLC4 expression involved in cervical cancer progression with radioresistance. And given that NO could act as a radiosensitizer or radioprotector, further studies are still warranted in future to comprehensively reveal the mechanism whereby SETD3 regulates KLC4 expression via NOS pathway. In conclusion (Fig. 4Q), the study indicated that reducing SETD3 expression resulted in the progression of radioresistance, whereas promoting SETD3 expression could markedly overcome radioresistance in human cervical cancer cells. Moreover, we suggested that SETD3 improved rediosensitivity through reducing the expression of KLC4, which was associated with the decrease in NO generation through blocking iNOS and eNOS expression. Based on our results, promoting SETD3 might represent an effective approach for cervical cancer prevention in patients who are resistant to radiotherapy. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.06.058. References [1] C. de Martel, M. Plummer, J. Vignat, et al., Worldwide burden of cancer attributable to HPV by site, country and HPV type, Int. J. Cancer 141 (4) (2017) 664e670. [2] H. Cheng, Inhibiting CD146 by its monoclonal antibody AA98 improves radiosensitivity of cervical cancer cells, Med. Sci. Monit.: Int. Med. J.Exp. Clin. Res. 22 (2016) 3328. [3] B.M. Barney, I.A. Petersen, S.C. Dowdy, et al., Intraoperative Electron Beam Radiotherapy (IOERT) in the management of locally advanced or recurrent cervical cancer, Radiat. Oncol. 8 (1) (2013) 80. [4] M.E.B. Powell, Modern radiotherapy and cervical cancer, Int. J. Gynecol. Cancer 20 (11) (2010) S49eS51. [5] Y. Qing, X.Q. Yang, Z.Y. Zhong, et al., Microarray analysis of DNA damage repair gene expression profiles in cervical cancer cells radioresistant to 252 Cf neutron and X-rays, BMC Canc. 10 (1) (2010) 71. [6] M.E. Lomax, L.K. Folkes, P. O'neill, Biological consequences of radiationinduced DNA damage: relevance to radiotherapy, Clin. Oncol. 25 (10) (2013) 578e585. [7] O. Cohn, M. Feldman, L. Weil, et al., Chromatin associated SETD3 negatively regulates VEGF expression, Sci. Rep. 6 (2016) 37115. [8] E. Abaev-Schneiderman, L. Admoni-Elisha, D. Levy, SETD3 is a positive regulator of DNA-damage-induced apoptosis, Cell Death Dis. 10 (2) (2019) 74. [9] S.E. Cooper, E. Hodimont, C.M. Green, A fluorescent bimolecular complementation screen reveals MAF1, RNF7 and SETD3 as PCNA-associated proteins in human cells, Cell Cycle 14 (15) (2015) 2509e2519. [10] X. Jiang, T. Li, J. Sun, et al., SETD3 negatively regulates VEGF expression during hypoxic pulmonary hypertension in rats, Hypertens. Res. 41 (9) (2018) 691.
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