Our data showed that, in contrast to HCV 1a and 2a, the folding of HCV 1b envelope proteins E2 (sites N1, N2, N10) and E1 (sites N1, N5) was disrupted, however that. The supernatant was centrifuged through the 30% sucrose cushion at 23000g for 16 h at 4 °C (Beckman Coulter Optima L-100XP centrifuge, 80Ti rotor).

Kelch proteins are implicated in the pathogenesis of many human diseases, including cancer. Nasopharyngeal carcinoma (NPC) is a rare malignancy in most countries, but prevalent in southern China and certain areas of Southeast Asia. In this study, we identified Kelch Domain Containing 4 (KLHDC4), an orphan member of the kelch repeat superfamily, as a prognosis marker for NPC. We examined the expression of KLHDC4 in 168 NPC cases by immunohistochemical staining and found a substantially higher level of KLHDC4 in NPC biopsies compared to adjacent normal nasopharyngeal mucosa. KLHDC4 expression was significantly related to the T classification ( P.

Introduction Nasopharyngeal carcinoma, a malignant tumor arising from the epithelium of the nasopharynx [], shows a remarkable racial and geographical distribution: it is rare in most parts of the world but occurs with high frequency in Southeast Asia, particularly in southern China, with an incidence of more than 20 cases per 100,000 population per year []. Interplay among several factors, including genetic susceptibility, Epstein-Barr virus (EBV) infection and dietary exposure to nitrosamines and nitrites during childhood, are believed to contribute to the etiology of NPC [, ]. Although 5-year survival rates of 80% can be achieved with the typical treatment of high-dose radiotherapy plus adjunctive chemotherapy, recurrence and metastasis still occur and are the main causes of treatment failure [, ]. Therefore, identifying molecular biomarkers for NPC prognosis and progression and developing an innovative targeted therapy are urgently needed.

Apoptosis, which is essential for maintaining tissue homeostasis by eliminating unnecessary or deleterious cells, is a cell suicide program that is under precise, multi-stage regulation. There are essentially two distinct pathways for triggering apoptosis: the death receptor pathway mediated by procaspase-8 and the mitochondrial pathway involving procaspase-9 []. Through diverse stimuli, activated caspase-8, -9 can further initiate the function of downstream effectors (principally caspase-3, -6, -7 and PARP), which in turn cleave a variety of substrates to give rise to internucleosomal DNA fragmentation and characteristic morphological changes []. Suppression of apoptosis is a recognized hallmark of cancer, and because the apoptotic program can be manipulated to produce massive cell death, the genes controlling apoptosis are ideal drug targets for cancer treatment [, ]. Indeed, many proapoptotic compounds and proteins have been reported to be effective in killing NPC [–], suggesting that targeting apoptosis pathways is a promising strategy for NPC treatment. The kelch superfamily is one of the largest evolutionarily conserved families, and the ~100 known family members have been implicated in diverse cellular functions, including cell migration, cytoskeletal arrangement, protein degradation and gene expression [, ].

The kelch repeat domain, which is present in all kelch proteins, is composed of five to seven tandem repeats of the kelch motif, forming a β-propeller structure that functions as a scaffold for protein-protein interactions. Kelch protein dysregulation has been reported to be associated with multiple human diseases, including cancer, skeletal muscle development and neurological disorders []. Moreover, recent reports have demonstrated that the kelch repeat proteins associate with the complex-type ubiquitin E3 ligases called the Cullin-RING ubiquitin ligases (CRLs) and play a role in substrate recognition of this complex [].

However, the physiological roles and underlying molecular mechanisms of most kelch repeat proteins remain elusive. Here, we provide evidence that KLHDC4, an uncharacterized kelch repeat containing protein, is substantially overexpressed in NPC. High level of KLHDC4 associates with poor prognosis in NPC patients. Moreover, CRISPR/Cas9-mediated knockout of KLHDC4 in NPC cells results in a decrease in tumor cell growth and migration and is accompanied with increased apoptosis both in vitro and in vivo. Reagents The reagents used were as follows: mouse monoclonal antibodies against Flag (F3165, Sigma-Aldrich), GAPDH (KC-5G4, Kangcheng Biotech); rabbit monoclonal antibody against HA (3724, Cell Signaling Technology); rabbit polyclonal antibodies against KLHDC4 (HPA041665, Sigma-Aldrich), cleaved caspase-3 (9664, Cell Signaling Technology), cleaved PARP (5625, Cell Signaling Technology), Ki-67 (ab15580, Abcam). Horseradish peroxidase-conjugated (HRP) goat anti-mouse/rabbit secondary antibodies were purchased from Promega. Noble agar (214220) was purchased from BD.

All other chemical reagents were obtained from Sigma-Aldrich, unless otherwise indicated. Cell cultures The NPC cell line CNE2 was kindly provided by Dr.

Chao-Nan Qian, Sun Yat-sen University Cancer Center (SYSUCC), China. Two normal nasopharyngeal epithelial cell lines NPEC-N2-Bmi1, NPEC-N5-Tert and two NPC cell lines SUNE1, SUNE2 were kindly provided by Dr. Mu-Sheng Zeng, Sun Yat-sen University Cancer Center, China.

The other cell lines, NP69, CNE1, HK1, HNE1, HONE1 were obtained from SYSUCC. All cell lines were thawed from early passage stocks and passaged for less than 6 months. The normal nasopharyngeal epithelial cell lines, NP69, NPEC-N2-Bmi1 and NPEC-N5-Tert, which were immortalized by SV40, Bmi-1 and Tert, respectively, were grown in keratinocyte/serum-free medium (Invitrogen).

Human NPC cell lines (CNE1, CNE2, SUNE1, SUNE2, HK1, HNE1, HONE1) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Biological Industries). Cells were grown in a humidified 5% CO 2 incubator at 37°C and passaged using standard cell culture techniques. DNA mismatch-specific endonuclease (T7E1) assay 293T cells in 6-well dishes were cultured to 50–60% confluence as mentioned above. The cells were transfected with 1 μg of KLHDC4 single-guide RNA (sgRNA) plasmid (ligated into the pX330 vector; sgRNA#1: 5’-GGCGGACAGCTGTGGGTCTT-3’; sgRNA#2: 5’-CACAGCTGTCCGCCACCTTG-3’; both sgRNAs targeting the exon 5 of KLHDC4 gene were selected from a published database [] of predicted high-specificity protospacer adjacent motif target sites in the human exome) and 5 μl of Lipofectamine 2000 per well. As a control, cells were also tranfected with the pX330 empty vector. The cells were harvested at 72 hours post-transfection, and genomic DNA was extracted (Qiagen DNeasy Blood & Tissue Kit). A region of exon 5 of the KLHDC4 gene was amplified with genomic DNA-specific primers (forward: 5’-TGACTGAGGACGTGCTTTCC-3’; reverse: 5’-CCACAGGAGAAGAGCTGCAA-3’).

The homoduplex PCR products were denatured and rehybridized using stepdown annealing conditions to generate homo- and heteroduplexes. The mixture of duplexes was treated with T7E1 endonuclease for 20 minutes at 37°C (New England Biolabs); the reaction was stopped using 1.5 μl of 0.25 M EDTA, and the products were analyzed on a 3% agarose gel. Establishment of a KLHDC4 knockout cell line CNE2 cells were cultured in 6-well dishes to 70–80% confluence and then cotransfected with 1 μg of KLHDC4 sgRNA#2 plasmid plus 1 μg of pSpCas9(BB)-2A-GFP plasmid and 5 μl of Lipofectamine 2000 per well. GFP was used as a fluorescent marker to sort the transfected cells. At 48 hours post-transfection, the cells were sorted into 96-well plates using fluorescence-activated cell sorting (FACS) with a Beckman-Coulter MoFlo XDP device. Single cells were validated as KLHDC4 knocked-out clone by western blotting and Sanger sequencing and then expanded as the KO cell line.

Transwell invasion assay Cells were trypsinized and pelleted by centrifugation. After washing twice in phosphate buffered saline (PBS buffer), the cells were resuspended in serum-free DMEM medium at a density of 4 × 10 5 cells/ml, and 200 μl of the cell suspension was seeded onto the basement Matrigel-coated membrane matrix (BD Biosiences). Fetal bovine serum was added to the lower chamber as a chemoattractant. After 20 hours, the noninvading cells were gently removed with a cotton swab. Invasive cells located on the lower side of the chamber were fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature prior to Crystal Violet (, Beyotime) staining. Three independent visual fields were examined via microscopic observation, and the number of cells was determined.

Flow cytometry Cells were stained with annexin V-FITC and PI (KGA108, KeyGEN) and evaluated for apoptosis by flow cytometry according to the manufacturer’s protocol. Download Inazuma Eleven 3 Sekai E No Chousen The Ogre English. Briefly, 1 × 10 6 cells were washed twice with PBS and stained with 5 μl annexin V-FITC and 10 μl PI in 1 × binding buffer for 15 minutes at room temperature in the dark.

Apoptotic cells were determined using a Beckman-Coulter Flow Cytometry FC500. Both early (annexin V-positive/PI-negative) and late (annexin V-positive/PI-positive) apoptotic cells were included when assessing cell death. Western blotting Cells were lysed in NETN buffer (20 mM Tris-HCl at pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing 50 mM β-glycerophosphate (14405, Merck), 1 μg/ml pepstatin A (P5318, Sigma-Aldrich) and 10 μM leupeptin (L2884, Sigma-Aldrich). The lysate protein concentration was measured using the BCA protein assay kit (Pierce); after normalization to equal amounts, proteins were separated by 12% SDS-PAGE, transferred to PVDF membranes and probed with the indicated primary antibodies. The blots were then incubated with species-specific HRP-conjugated secondary antibodies, and the immunoreactive bands were visualized by enhanced chemiluminescence (ECL, Pierce).

In vivo animal studies All mice were handled according to the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University Cancer Center (Reference number: 00090462).

Female BALB/c nude mice (Hunan Slac Jingda Laboratory Animal Co., Ltd., Hunan, China) aged 6 weeks were used for tumor xenografts. All the animals were housed in standard cages (4 animals per cage) under specific pathogen-free conditions. Rodent laboratory chow and tap water were provided ad libitum and maintained under controlled conditions with a temperature of 24 ± 1°C, humidity of 50 ± 10%, and a 12: 12h light/dark cycle. Food and water were freely available throughout the experiments. The nude mice were randomly divided into two groups (8 mice per group), CNE2 control group and KLHDC4 KO group, respectively. Cells (5 × 10 5 in 0.2 ml PBS) were injected subcutaneously into the left dorsal flank of nude mice.

Mice were monitored every 12 hours for the first 3 days after inoculation of tumor cell lines, then daily thereafter. Tumor sizes were measured every 3–4 days. Mice were killed 4 weeks post-injection via cervical dislocation, and tumors from the two groups were extracted and weighed. The perpendicular diameters of the tumors were measured using a caliper, and the tumor volume were calculated using the formula: tumor volume (V) = π/6 × large diameter × smaller diameter 2. The following are general humane endpoints for animals that require euthanasia in this study: 20% decrease in normal body weight; the inability to reach food or water for more than 24 hours; a tumor burden greater than 10% body weight or a tumor that exceeds 20 mm in any one dimension.

All efforts were made to minimize animal suffering. Patient selection and immunohistochemical (IHC) staining We evaluated the prognostic relevance of KLHDC4 expression in a cohort of 168 NPC patients who had undergone definitive treatment with curative intent at our institute from 2005 to 2013. The present study was reviewed and approved by the Institutional Review Board of Sun Yat-sen University Cancer Center, and all participants provided written informed consent. The NPC clinical samples were fixed in 10% formalin and embedded in paraffin, and we deparaffinized and rehydrated sections of the embedded specimens. For IHC staining, deparaffinized slides were retrieved with the appropriate antigen, and endogenous peroxidase activity was blocked with 1% H 2O 2 in PBS for 30 min. The slides were then exposed to an anti-KLHDC4 antibody at 4°C overnight.

Immunostaining was performed using Envision System with diaminobenzidine (Dako). A semi-quantitative scoring criterion was used for the IHC results, whereby both the staining intensity and positive areas were recorded. Kingpin Life Of Crime Cd Crack. A staining index (values 0–7), obtained as the intensity of KLHDC4-positive staining (negative, 0; weak, 1; moderate, 2; or strong, 3 scores) and the proportion of immunopositive cells of interest (4.5). Statistical analyses The SPSS software version 19.0 and GraphPad Prism 5 software were used to perform the statistical analyses. Correlation of the KLHDC4 staining intensity to clinicopathological characteristics was measured using Pearson’s Chi-Square or Fisher’s exact test. Cumulative survival was calculated by Kaplan-Meier analysis, and comparison between groups was achieved using the log-rank test. Each experiment was performed three times in triplicate.

The significance of variances between groups was determined by the t-test. All statistical tests were two-sided, and P. KLHDC4 is overexpressed in NPC tissue and cell lines To reveal a potential role for KLHDC4 in NPC, we performed immunohistochemical (IHC) analysis of KLHDC4 expression in patient-derived NPC biopsies. The KLHDC4 protein was detected in the cytoplasm of both normal nasopharyngeal epithelia and carcinoma cells. Among all primary tumor samples examined, 84 samples had both tumor and normal epithelial tissues on the same slides. The KLHDC4 expression levels in both tumors and adjacent normal nasopharyngeal tissues were scored and positive index was calculated.

The positive rate of KLHDC4 in tumor tissues is 68.9% (51/84), and that in corresponding adjoining normal nasopharyngeal epithelia is 40.5% (30/84; χ 2 = 12.027, P. Elevated KLHDC4 expression correlates with poor prognosis in NPC patients To investigate whether KLHDC4 expression can serve as a novel prognostic marker for NPC patients, we examined the correlation of KLHDC4 expression with clinicopathological factors including age, sex and pathological stage in 168 NPC patients. The staining intensity and distribution varied among the samples (), and a cutoff value according to the ROC curve was used to separate patients into two groups: a low KLHDC4 group (score ≤4.5, N = 103) and a high KLHDC4 group (score >4.5, N = 65). As shown in, KLHDC4 expression was significantly correlated with T classification ( P = 0.016), N classification ( P = 0.043) and total staging ( P = 0.002).

No difference was noted in KLHDC4 expression when stratified by gender ( P = 1.000), age ( P = 0.754), or M stage ( P = 0.300). The Kaplan-Meier survival curves showed that the patients with high KLHDC4 expression had a significantly poorer overall survival (OS) and metastasis-free survival (MFS) when compared with patients with low KLHDC4 expression, as defined by the log-rank test ( P = 0.005,; P = 0.012, ). Knockout of KLHDC4 reduces NPC cell growth To gain insight into the role of KLHDC4 in the development and progression of NPC, we generated KLHDC4 knockout cells using CRISRP/Cas9-mediated gene editing technology in CNE2, a typical NPC cell line that is primarily used in NPC research. We constructed two sgRNAs targeting different regions in one of the first few exons of the human KLHDC4 gene without obvious potential off-target effects according to bioinformatic analysis. The sgRNAs were transfected with the Cas9 expression construct into human 293T cells. Compared to empty gRNA vector, sgRNA#2 exhibited efficient cleavage activity as assessed by the T7E1 assay (). Therefore, sgRNA#2 was selected for generating KLHDC4 knockout cells.

Western blotting showed the complete absence of the KLHDC4 protein in the KO cells (). We also extracted genomic DNA from these cells for PCR amplification of the sgRNA#2 target region and subcloned the PCR products into the pMD18-T vector for Sanger sequencing. Analysis of 10 independent clones revealed 3 distinct indels surrounding the protospacer adjacent motif (PAM), further validating the KO status of these cells (). Knockout of KLHDC4 reduces NPC cell growth. Next, we attempted to elucidate biological significance of KLHDC4 in NPC progression.

During cell culture we noticed that KLHDC4 KO cells proliferated poorly than the wild-type counterpart. Accordingly, a significant decrease in the viability of KLHDC4 KO cells compared to control cells was observed by cell counting kit-8 (CCK-8) assay (). Similarly, knockout of KLHDC4 dramatically prevented colony formation by these cells in soft agar compared to control wild-type cells, indicating that anchorage-dependent growth was suppressed by KLHDC4 loss (). We next evaluated the in vivo effects of KLHDC4 deficiency on tumor growth by subcutaneous injection of parental or KLHDC4 KO CNE2 cells into nude mice and tumor growth were monitored. In contrast to the parental CNE2 cells, which formed large tumors within 4 weeks (8 of 8 mice), mice injected with KLHDC4 KO cells displayed either no tumor formation or greatly retarded tumor growth (3 of 8 mice; ). Consistently, the average weight of the xenograft tumors were also significantly reduced in the absence of KLHDC4 ().

These results suggest that KLHDC4 is required for NPC cell growth and proliferation both in vitro and in vivo. Loss of KLHDC4 reduces NPC cell migration and invasion Aberrant cell migration and invasion is frequently associated with cancer, and enhanced migration and invasion capacity is generally believed to be associated with tumor metastasis. Thus, we performed wound healing and Transwell assays to determine whether KLHDC4 expression is related to cell migration and invasion, respectively. At 20 hours after scratch injury, the KLHDC4 KO cells were able to cover only 53.3% of the scratch, whereas the control cells covered 88.0% ( P. Loss of KLHDC4 induces apoptosis in NPC cells Apoptosis has long been known to be associated with tumor growth and metastasis. Induction of apoptosis is also a therapeutic strategy for the treatment of NPC.

To define the underlying molecular mechanism by which KLHDC4 inhibits NPC progression, we first examined morphological and biochemical changes in cells after loss of KLHDC4. The control CNE2 cells exhibited a normal cobblestone epithelial morphology and apoptotic cells were hardly observed.

In contrast, around 10% of the KLHDC4 KO cells displayed the typical characteristics of apoptosis, such as cell shrinkage, membrane blebbing, and nuclear condensation (). Compared to the control cells, annexin V/PI staining showed a significant increase in apoptotic cells (annexin V positive/PI negative and annexin V positive/PI positive) among the KLHDC4 KO population under both non-treated and cis-platin treated conditions (). In addition, the activity of caspase-3 and PARP, as demonstrated by the appearance of their cleaved forms, were markedly elevated in KLHDC4 KO cells, indicating that the cell death induced by KLHDC4 deficiency via a route of apoptosis (). In agreement with the aforementioned in vitro results, IHC of the KLHDC4 knockout xenograft biopsies showed a substantial increase in the number of cleaved caspase-3 and cleaved PARP-positive cells and decreased Ki-67 positive cells (), further supporting the role of KLHDC4 in promoting NPC progression through regulating apoptosis.

Discussion NPC is an uncommon cancer exhibiting complex interactions of multiple etiologic factors. An overall understanding of the molecular pathogenesis of this disease is still lacking, and the identification of effective prognostic and diagnostic biomarkers are urgently needed. In this study, we reported that KLHDC4, an uncharacterized kelch protein, is associated with nasopharyngeal carcinoma tumorigenesis and development.

We found that KLHDC4 is markedly upregulated in NPC clinical specimens and cell lines. Furthermore, gene expression profiles from the GEO repository also reveal upregulation of KLHDC4 mRNA in tumor sections of different origins (). These results clearly indicated that KLHDC4 expression is involved in cancer development. However, it remains to be defined how KLHDC4 expression is regulated during pathogenesis. Next, we focused on the potential relevance of KLHDC4 expression to clinicopathologic characteristics and its application in prognostic evaluation of NPC patients. Our data showed significant correlations of KLHDC4 expression levels with tumor staging.

High KLHDC4 protein expression also predicts a poorer overall survival and metastasis-free survival in NPC patients (). These results strongly suggest an oncogenic role of KLHDC4 in NPC development and highlight its potential use in NPC prognostic prediction. Consistent with our observations in clinical samples, we found that knockout of KLHDC4 gene in NPC cell line reduced cell growth, migration and invasion, as well as tumorigenesis in xenograft mice model (Figs and ). Together with the clinical data, these results indicate that KLHDC4 may play an oncogenic role in NPC.

CNE2 is a cell line commonly used in NPC studies due to its undifferentiated characteristic and easy maintenance []. However, studies of KLHDC4 gene in additional NPC cell lines are warranted to further confirm the role of KLHDC4 in NPC development.

The pathogenesis of NPC is complex and involves a number of signaling pathways related to tumor cell growth and metastasis []. Among them, the apoptosis signaling pathway is crucial for cells survival and tumorigenesis. Importantly, defective or inefficient apoptosis has become an hallmark of cancer cells []. Recently, several reports have demonstrated that dysregulation of gene expression in NPC were found to inhibit cancer cell growth and metastasis through induction apoptosis [, ]. Since we noticed that KLHDC4 KO cells displayed typical characteristic of apoptosis, we tried to define whether KLHDC4 participates in cellular apoptosis regulation. A higher proportion of annexin V-positive cells among the KLHDC4 KO population were found under both static and cis-platin induced conditions.

Furthermore, elevated expression of cleaved caspase-3 and cleaved PARP were seen in tumor cells and xenografts after depletion of KLHDC4 (). These data indicate that loss of KLHDC4 induces activation of cellular apoptosis in CNE2 cells and possibly hinders viability and tumor cell growth and metastasis. We also attempted to further elucidate the downstream signal that mediates KLHDC4’s anti-apoptotic effect. Several reports highlighted that kelch proteins participate in regulating cellular apoptosis. KEAP1, a protein with BTB-BACK domain and six tandem kelch repeats, serves as a substrate adaptor of the Cullin3 E3 ligase complex. Investigations demonstrated that KEAP1 plays a key role in regulating apoptosis via modulation of the cellular redox balance by targeting NRF2, a transcription factor that is often overexpressed in many human cancers, for proteasome-mediated degradation [–].

In addition, Sekine et al. [] found that KLHDC10, a protein with BC box and six tandem kelch repeats, was required for H 2O 2-induced sustained activation of ASK1 and cell death in Neuro2A cells. Another research by Mahrour et al. [] reported that two other kelch proteins, KLHDC2 and KLHDC3, were able to interact with Cullin2/5 proteins via their BC boxes. These information prompted us to assess whether KLHDC4 associates with cullin proteins. Immunoprecipitation assay revealed that KLHDC4 was not able to interact with Cullin2 or Cullin3 ().Besides, purification of KLHDC4 protein complex in human cells did not found any proteins that substantially associates with KLHDC4, none of any cullin protein family members, or other kelch proteins, have been identified in mass spectrometry data (data not shown). Structure analysis also implied that KLHDC4 lacks consensus modules mediated its interaction with cullin proteins ().All the evidences suggest that KLHDC4 may exert its functions other than CRL-mediated protein degradation pathway, or not even through canonical protein-protein interaction.

Quantitative proteomics and DNA microarray are needed in the future to fully understand the molecular mechanism by which KLHDC4 participates in anti-apoptosis process. S1 Fig KLHDC4 does not interact with Cullin2 or Cullin3.

(A-B) 293T cells were transiently tranfected with plasmids encoding SFB-tagged KLHDC3, KLHDC4, KLHDC10, KEAP1, or empty vector together with plasmid encoding HA-tagged Cullin2 or Cullin3 as indicated. Cell lysates were precipitated with S-protein beads and immunoblotted with indicated antibodies. (C) Protein structures of KLHDC4, KLHDC3, KLHDC10 and KEAP1. Red rectangle: Kelch repeat; Blue hexagon: BC box; Yellow rhombus: BTB domain; Green rhombus: BACK domain. Funding Statement This work was supported by the National Nature Science Foundation of China (grant number: 81572781) (), the Specialized Research Fund for the Doctoral Program of Higher Education (No. 0099) (), the National Basic Research Program of China (973 Program: 2013CB910303) () and the National High Technology Research and Development Program of China (863 Program: 2012AA02A206 and 2012AA02A501) ().

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.