Nuclear Localization Signal Peptide Research Articles

Efficient deletion of LoxP-flanked selectable marker genes from the genome of transgenic pigs by an engineered Cre recombinase.

Genetically modified (GM) pigs hold great promises for pig genetic improvement, human health and life science. When GM pigs are produced, selectable marker genes (SMGs) are usually introduced into their genomes for host cell or animal recognition. However, the SMGs that remain in GM pigs might have multiple side effects. To avoid the possible side effects caused by the SMGs, they should be removed from the genome of GM pigs before their commercialization. The Cre recombinase is commonly used to delete the LoxP sites-flanked SMGs from the genome of GM animals. Although SMG-free GM pigs have been generated by Cre-mediated recombination, more efficient and cost-effective approaches are essential for the commercialization of SMG-free GM pigs. In this article we describe the production of a recombinant Cre protein containing a cell-penetrating and a nuclear localization signal peptide in one construct. This engineered Cre enzyme can efficiently excise the LoxP-flanked SMGs in cultured fibroblasts isolated from a transgenic pig, which then can be used as nuclear donor cells to generate live SMG-free GM pigs harboring a desired transgene by somatic cell nuclear transfer. This study describes an efficient and far-less costly method for production of SMG-free GM pigs.

Telomerase inhibition, telomere attrition and proliferation arrest of cancer cells induced by phosphorothioate ASO-NLS conjugates targeting hTERC and siRNAs targeting hTERT

Telomerase activity has been regarded as a critical step in cellular immortalization and carcinogenesis and because of this, regulation of telomerase represents an attractive target for anti-tumor specific therapeutics. Recently, one avenue of cancer research focuses on antisense strategy to target the oncogenes or cancer driver genes, in a sequence specific fashion to down-regulate the expression of the target gene. The protein catalytic subunit, human telomerase reverse transcriptase (hTERT) and the template RNA component (hTERC) are essential for telomerase function, thus theoretically, inhibition of telomerase activity can be achieved by interfering with either the gene expression of hTERT or the hTERC of the telomerase enzymatic complex. The present study showed that phosphorothioate antisense oligonucleotide (sASO)-nuclear localization signal (NLS) peptide conjugates targeting hTERC could inhibit telomerase activity very efficiently at 5 μM concentration but less efficiently at 1 μM concentration. On the other hand, siRNA targeting hTERT mRNA could strongly suppress hTERT expression at 200 nM concentration. It was also revealed that siRNA targeting hTERT could induce telomere attrition and then irreversible arrest of proliferation of cancer cells.

Developing high-efficiency base editors by combining optimized synergistic core components with new types of nuclear localization signal peptide

The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) system has been widely used for genome editing. In this system, the cytosine base editor (CBE) and adenine base editor (ABE) allow generating precise and irreversible base mutations in a programmable manner and have been used in many different types of cells and organisms. However, their applications are limited by low editing efficiency at certain genomic target sites or at specific target cytosine (C) or adenine (A) residues. Using a strategy of combining optimized synergistic core components, we developed a new multiplex super-assembled ABE (sABE) in rice that showed higher base-editing efficiency than previously developed ABEs. We also designed a new type of nuclear localization signal (NLS) comprising a FLAG epitope tag with four copies of a codon-optimized NLS (F4NLSr2) to generate another ABE named F4NLS-sABE. This new NLS increased editing efficiency or edited additional A at several target sites. A new multiplex super-assembled CBE (sCBE) and F4NLSr2 involved F4NLS-sCBE were also created using the same strategy. F4NLS-sCBE was proven to be much more efficient than sCBE in rice. These optimized base editors will serve as powerful genome-editing tools for basic research or molecular breeding in rice and will provide a reference for the development of superior editing tools for other plants or animals.

Discovering nuclear targeting signal sequence through protein language learning and multivariate analysis

Nuclear localization signals (NLSs) are peptides that target proteins to the nucleus by binding to carrier proteins in the cytoplasm that transport their cargo across the nuclear membrane. Accurate identification of NLSs can help elucidate the functions of nuclear protein complexes. The currently known NLS predictors are usually specific to certain species or largely dependent on prior knowledge of NLS basic residues. Thus, a more general predictor is highly desired to reduce the potentially high false positives or false negatives in discovering new NLSs. Here, we report a new method, INSP (Identification Nucleus Signal Peptide), to effectively identify NLS mainly based on statistical knowledge and machine learning algorithms. In our NLS machine learning model, we considered the query protein sequence as text and extracted the sequence context features using a natural language model. These word-vector features encode discriminative knowledge of NLS motif frequency and are thus useful for model recognition. The output of the machine learning model will be fused with statistical knowledge of the query sequence to build a final multivariate regression model for NLS peptide identification. The experimental results demonstrate a promising performance of the new INSP approach. INSP is freely available at: www.csbio.sjtu.edu.cn/bioinf/INSP/for academic use.

Competitive inhibition by NAG-1/GDF-15 NLS peptide enhances its anti-cancer activity

Non-steroidal anti-inflammatory drug activated gene-1 (NAG-1), also known as growth differentiation factor 15 (GDF15), is a TGF-β (transforming growth factor beta) superfamily protein with a distinctive secretion pathway. NAG-1 is associated with multiple diseases including cancer, wherein it plays a role in both pro- and anti-cancer activities. We previously reported that NAG-1 is translocated to different subcellular compartments and its activity depends on its localization. In this paper, we report that the transfection of a novel peptide corresponding to the nuclear localization signal (NLS) of NAG-1 blocks its translocation to the nucleus. Further, accumulation of NAG-1 in the cytoplasm decreased mitochondrial membrane potential, thus implying apoptosis induction as a consequence. Overall, our results indicate that the novel peptide derived from NAG-1 NLS sequence is a promising tool for enhancing the anti-tumorigenic activity of NAG-1.

Cytoplasm Localized ARID1B Promotes Oncogenesis in Pancreatic Cancer by Activating RAF-ERK Signaling

The ARID1B/BAF250b subunit of the human SWI/SNF chromatin remodeling complex is a canonical nuclear tumor suppressor. Immunohistochemistry on a pancreatic cancer tissue microarray revealed significant ARID1B cytoplasmic localization that correlated with advanced tumor stage and lymph node positivity. Identification of the nuclear localization signal (NLS) using in silico prediction and subcellular localization studies facilitated evaluation of a possible cytoplasmic function for ARID1B. A cytoplasm-restricted ARID1B-NLS mutant was significantly compromised to regulate transcription activation and tumor suppression functions, as expected. Surprisingly however, cytoplasm-localized ARID1B could bind c-RAF and PPP1CA causing stimulation of RAS-RAF-ERK signaling and β-catenin transcription activity in pancreatic cancer cells. More importantly, cytoplasmic ARID1B resulted in an induction of cell growth and migration in pancreatic cancer cell lines that was dependent on ERK signaling and caused increased tumorigenesis in nude mice. NLS peptides representing mutations identified from pancreatic cancer samples exhibiting ARID1B cytoplasmic localization or curated from cancer somatic mutation database were significantly compromised to effect nuclear localization of a reporter protein. ARID1B cytoplasmic localization correlated significantly with active forms of ERK and β-catenin in primary pancreatic tumor samples. ARID1B may therefore promote oncogenesis through non-canonical cytoplasm-based gain of function mechanisms in addition to dysregulation in the nucleus.

Nuclear localization signal peptide enhances transfection efficiency and decreases cytotoxicity of poly(agmatine/N,N'-cystamine-bis-acrylamide)/pDNA complexes.

At present, nonviral gene vectors develop rapidly, especially cationic polymers. A series of bioreducible poly(amide amine) (PAA) polymers containing guanidino groups have been synthesized by our research team. These novel polymer vectors demonstrated significantly higher transfection efficiency and lower cytotoxicity than polyethylenimine (PEI)-25kDa. However, compared with viral gene vectors, relatively low transfection efficiency, and high cytotoxicity are still critical problems confronting these polymers. In this study, poly(agmatine/N,N'-cystamine-bis-acrylamide) p(AGM-CBA) was selected as a model polymer, nuclear localization signal (NLS) peptide PV7 (PKKKRKV) with good biocompatibility and nuclear localization effect was introduced to investigate its impact on transfection efficiency and cytotoxicity. NLS peptide-mediated in vitro transfection was performed in NIH 3T3 cells by directly incorporating NLS peptide with the complexes of p(AGM-CBA)/pDNA. Meanwhile, the transfection efficiency and cytotoxicity of these complexes were evaluated. The results showed that the transfection efficiency could be increased by 5.7 times under the appropriate proportion, and the cytotoxicity brought by the polymer vector could be significantly reduced.

Chimeric peptide engineered exosomes for dual-stage light guided plasma membrane and nucleus targeted photodynamic therapy

Targeted delivery of the drug to its therapeutically active site with low immunogenicity and system toxicity is critical for optimal tumor therapy. In this paper, exosomes as naturally-derived nano-sized membrane vesicles are engineered by chimeric peptide for plasma membrane and nucleus targeted photosensitizer delivery and synergistic photodynamic therapy (PDT). Importantly, a dual-stage light strategy is adopted for precise PDT by selectively and sequentially destroying the plasma membrane and nucleus of tumor cells. Briefly, plasma membrane-targeted PDT of chimeric peptide engineered exosomes (ChiP-Exo) could directly disrupt the membrane integrity and cause cell death to some extent. More interestingly, the photochemical internalization (PCI) and lysosomal escape triggered by the first-stage light significantly improve the cytosolic delivery of ChiP-Exo, which could enhance its nuclear delivery due to the presence of nuclear localization signals (NLS) peptide. Upon the second-stage light irradiation, the intranuclear ChiP-Exo would activate reactive oxygen species (ROS) in situ to disrupt nuclei for robust and synergistic PDT. Based on exosomes, this dual-stage light guided subcellular dual-targeted PDT strategy exhibits a greatly enhanced therapeutic effect on the inhibition of tumor growth with minimized system toxicity, which also provides a new insight for the development of individualized biomedicine for precise tumor therapy.

Gene Delivery by PAMAM Dendrimer Conjugated with the Nuclear Localization Signal Peptide Derived from Influenza B Virus Nucleoprotein

Polyamidoamine (PAMAM) dendrimer is one of the cationic polymers widely used in nonviral vector-based gene therapy. As PAMAM comprises biodegradable peptide bonds, it shows biocompatibility and relatively low cytotoxicity. In comparison with polyethylenimine (PEI, 25 kDa) that shows high transfection efficiency, the native PAMAM dendrimer has limitations as a gene carrier. To improve the gene expression efficiency, we introduced KRTR peptides derived from the influenza B virus nucleoprotein, known as a nuclear localization signal (NLS), to PAMAM generation 3. We synthesized PAMAM-RTRK and PAMAM-HRTRK and evaluated the effects of the histidine residue on cytotoxicity. The transfection efficiency of PAMAM-RTRK and PAMAM-HRTRK was similar to that of PEI in HepG2 cells, while their gene expression capacities were much higher than capacity of PEI in A549 cells. PAMAM-HRTRK showed relatively lower cytotoxicity than PAMAM-RTRK in the two cell lines, but the transfection capacity was similar for the two polymers. These results imply that the combination of histidine and KRTR peptide may potentially result in a novel PAMAM-based vector that could be used for prolonged gene therapy through the reduction in cytotoxicity and enhancement of gene expression.

Induction of a protective response in ducks vaccinated with a DNA vaccine encoding engineered duck circovirus Capsid protein

Duck circovirus (DuCV) is an immunosuppressive pathogen that causes a huge economic loss in the avian industry. Efficient vaccination has become a necessary strategy for preventing DuCV infection in the breeding industry. Three DNA vaccines encoding the Capsid (Cap) protein of DuCV were developed in this study, which were based on the eukaryotic vector pcDNA3.1 containing (i) the full length of Cap gene, pcDNA3.1-Cap, (ii) the Cap gene with a deletion of its nuclear localization signal (NLS) peptide encoding sequence, pcDNA3.1-CapΔNLS, and (iii) the Cap gene without NLS but harboring a fragment encoding the secretory signal peptide of tissue plasminogen activator (tPA), pcDNA3.1-tPA-CapΔNLS. Production of Cap protein-derived antigens from these three DNA vaccines was confirmed in vitro. The deletion of the NLS coding sequence of the Cap gene changed the subcellular location of the Capsid protein from the nucleus to the cytoplasm. Secretion of the Cap protein was observed in pcDNA3.1-tPA-CapΔNLS-transfected cells. The immunogenicity of these three DNA vaccines was assessed in vivo by measuring Cap-specific antibody and related cytokine levels. The results demonstrated that all these vaccines could induce a significant, specific immune response to protect ducks from DuCV challenge. Notably, higher titers of Cap-specific antibody were produced in ducks vaccinated with pcDNA3.1-tPA-CapΔNLS, which provided the highest protective efficacy at a rate of 90% in the challenge experiment. Taken together, DNA vaccines expressing the DuCV Cap protein show promising immunogenicity, which can be enhanced by replacing the NLS of the Cap protein with a secretory signal peptide of tPA.

Variations in nuclear localization strategies among pol X family enzymes.

Despite the essential roles of pol X family enzymes in DNA repair, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional nuclear localization signal (NLS) in DNA polymerase β, indicating the importance of active nuclear targeting, even for enzymes likely to leak into and out of the nucleus. The current studies further explore the active nuclear transport of these enzymes by identifying and structurally characterizing the functional NLS sequences in the three remaining human pol X enzymes: terminal deoxynucleotidyl transferase (TdT), DNA polymerase mu (pol μ) and DNA polymerase lambda (pol λ). NLS identifications are based on Importin α (Impα) binding affinity determined by fluorescence polarization of fluorescein-labeled NLS peptides, X-ray crystallographic analysis of the Impα∆IBB•NLS complexes and fluorescence-based subcellular localization studies. All three polymerases use NLS sequences located near their N-terminus; TdT and pol μ utilize monopartite NLS sequences, while pol λ utilizes a bipartite sequence, unique among the pol X family members. The pol μ NLS has relatively weak measured affinity for Impα, due in part to its proximity to the N-terminus that limits non-specific interactions of flanking residues preceding the NLS. However, this effect is partially mitigated by an N-terminal sequence unsupportive of Met1 removal by methionine aminopeptidase, leading to a 3-fold increase in affinity when the N-terminal methionine is present. Nuclear targeting is unique to each pol X family enzyme with variations dependent on the structure and unique functional role of each polymerase.

Dual-Function, Cationic, Peptide-Coated Nanodiamond Systems: Facilitating Nuclear-Targeting Delivery for Enhanced Gene Therapy Applications

Nuclear-targeting therapy is considered to be a promising strategy of disease treatment. So far, developing biocompatible and nucleus-permeable delivery systems remains a great challenge. Here, we report a nuclear-targeted delivery platform based on 30 nm nanodiamonds (NDs) which were coated with dual-function, cationic peptides consisting of the human immounodeficiency virus TAT protein and a nuclear localization signal (NLS) peptide in aqueous media. As compared to uncoated NDs, cationic peptide-functionalized NDs were confirmed as a small, safe, and efficient carrier which not only facilitates the enhanced cellular uptake and delivery of loaded cargos to the nucleus in a number of cell lines but also shows their advantages of low cytotoxicity and high affinity to antisense oligonucleotides. This peptide-based modification strategy does not contribute greatly to the size of the ND which is important in its use in constructing nuclear targeting vehicles. Compared with traditional gene silencing in cytopl...

A Universal GSH-Responsive Nanoplatform for the Delivery of DNA, mRNA, and Cas9/sgRNA Ribonucleoprotein.

The long-sought promise of gene therapy for the treatment of human diseases remains unfulfilled, largely hindered by the lack of an efficient and safe delivery vehicle. In this study, we have developed a universal glutathione-responsive nanoplatform for the efficient delivery of negatively charged genetic biomacromolecules. The cationic block copolymer, poly(aspartic acid-(2-aminoethyl disulfide)-(4-imidazolecarboxylic acid))-poly(ethylene glycol), bearing imidazole residues and disulfide bonds, can form polyplexes with negatively charged DNA, mRNA, and Cas9/sgRNA ribonucleoprotein (RNP) through electrostatic interactions, which enable efficient cellular uptake, endosomal escape, and cytosol unpacking of the payloads. To facilitate the nuclear transport of DNA and RNP, the nuclear localization signal peptide was integrated into the DNA or RNP polyplexes. All three polyplex systems were fully characterized and optimized in vitro. Their relatively high transfection efficiency and low cytotoxicity, as well as convenient surface functionalization merit further investigation.

Developing a flippase-mediated maker recycling protocol for the oleaginous yeast Rhodosporidium toruloides.

To establish a recombinase flippase (FLP) and flippase recognition target (FRT) system-mediated protocol for post-integration excision of exogenous DNA fragments in the oleaginous yeast Rhodosporidium toruloides. Binary vectors were constructed to harbor FLP expressing cassette together with the hygromycin-resistance marker. Results showed that R. toruloides transformants produced FLP, but failed to mediate removal of the bleomycin-resistance marker within two FRT sites. When FLP was fused with a native nuclear localization signal (NLS) peptide, the system was found functional. Moreover, R. toruloides recombinant strains expressing the NLS-fused FLP under the control of PADH2, an promoter of alcohol dehydrogenase 2 gene (RHTO_03062), were obtained to realize homologous recombination upon growing in glucose-deficient medium. We have devised a homologous recombination method for R. toruloides based on the FLP/FRT system, which may facilitate further metabolic engineering and designing advanced cell factories for value-added chemicals.

Enzymatically synthesized poly(amino-co-ester) polyplexes for systemic delivery of pcDNA-miRNA-214 to suppress colorectal cancer liver metastasis.

Liver metastases from colorectal cancer (CRC) are the major cause of cancer-related deaths in CRC patients. In our previous study, microRNA-214 (miR-214) was identified in CRC patients as a novel regulator of CRC liver metastasis, which could serve as a therapeutic target to inhibit CRC proliferation and metastasis. In this study, we aim to develop a new CRC treatment strategy based on miR-214 gene therapy using biodegradable non-viral gene vectors. We developed multifunctional quaternary polyplexes that consist of cationic poly(ω-pentadecalactone-co-N-methyldiethyleneamine-co-sebacate) (PPMS) for DNA condensation to form a nano-sized polyplex core, hyaluronic acids (HA) grafted with a poly(ethylene glycol) (PEG) (HA-g-mPEG) shell for polyplex stabilization and targeted delivery, and nuclear localization signal (NLS) peptides for enhanced intracellular transport of pDNA to the nucleus. The results showed that the DNA/NLS/PPMS/HA-g-mPEG quaternary polyplexes could enhance DNA condensation, increase cellular uptake efficiency and decrease cytotoxicity. Most importantly, the quaternary polyplexes showed favorable transfection efficiency both in vitro and in vivo. The colony formation and migration ability were significantly inhibited in HCT116 cells transfected with pcDNA-miR-214 quaternary polyplexes. The up-regulation of miR-214 in HCT116 cells by pre-transfection of polyplexes-miR-214 could remarkably inhibit tumor growth and liver metastases in a xenograft mouse model. Furthermore, systemic administration of miR-214 using this multifunctional vector resulted in dramatic inhibition of liver metastasis without obvious toxicity in CRC xenografted mice. Collectively, systemic delivery of pcDNA-miR-214 by this multifunctional vector could be a powerful and highly specific therapeutic approach in the treatment of CRC liver metastasis.

Cancer Therapeutic Proficiency of Dual-Targeted Mesoporous Silica Nanocomposite Endorses Combination Drug Delivery

The cargo-loaded mesoporous silica nanoparticles (MSNs) with convenient surface modification can facilitate the development of the innovative nanodrug system. Herein, the present investigation described the electrostatically self-assembled MSNs as a nanosized drug carrier to realize potent synergistic chemotherapy based on the specificity in targeting cytoplasm and nucleus of tumor cells. In this context, the primarily constructed MSNs were subjected with anticancer drug topotecan (TPT) into its large pores. Then, the selective TAT peptide (a nuclear localization signal peptide) was anchored onto TPT-loaded MSNs (TPT-MSN). Subsequently, the positive surface of TPT-MSN-TAT was capped with negatively charged components, poly(acrylic acid) (PAA)-cRGD peptide and citraconic anhydride (CAH)-metformin (MT), and acted as a smart gatekeeper. Comparatively, PAA-cRGD attached onto MSNs serving as the targeted molecules could upsurge by invasion into cancer cells. Interestingly, the acidic pH of the lysosomal compartment in tumor cells triggers the conjugated CAH from the polymer decorated mesoporous silica (PMS) nanocomposite and could efficiently release MT into the cytoplasm. Consequently, the remaining TPT-MSN-TAT efficiently targets the nucleus and delivers the TPT to improve synergistic chemotherapeutic effects. The precisely released drugs were individually enhanced in the in vitro and in vivo cell killing efficiencies. Thus, the study provides a potential drug delivery podium through combined drugs to realize cancer cell targeting approach.

The AtMC4 regulates the stem cell homeostasis in Arabidopsis by catalyzing the cleavage of AtLa1 protein in response to environmental hazards

The AtLa1 protein is an RNA binding factor that initiates the translation of WUSCHEL (WUS) mRNA in Arabidopsis. The AtLa1 protein can regulate the stem cell homeostasis via the nuclear-to-cytoplasmic translocation in response to environmental hazards. However, the translocation mechanism of AtLa1 protein remains to be elucidated. In the present study, we have explored the role of AtMC4 protein in the subcellular translocation of AtLa1 protein. Our results showed that the AtLa1 protein is a substrate of AtMC4 protein. The AtMC4 protein can interact with AtLa1 protein and catalyze the cleavage of the C-terminal nuclear localization signal peptide of AtLa1 protein. The AtMC4 protein is mainly distributed in the cytoplasm. In the presence of environmental stresses, the cytoplasmic-to-nuclear translocation of AtMC4 protein is enhanced with the result that more AtLa1 protein can be cleaved and transported from nucleus to cytoplasm, where AtLa1 protein further initiates the translation of WUS mRNA. By contrast, knockdown of AtMC4 expression inhibits the nuclear-to-cytoplasmic translocation of AtLa1 protein and the WUS protein translation. Based on these results, we conclude that the AtMC4 protein regulates stem cell homeostasis by catalyzing the cleavage of AtLa1 protein in response to environmental hazards.

Structural characterisation of TNRC6A nuclear localisation signal in complex with importin-alpha.

The GW182/TNRC6 family of proteins are central scaffolds that link microRNA-associated Argonaute proteins to the cytoplasmic decay machinery for targeted mRNA degradation processes. Although nuclear roles for the GW182/TNRC6 proteins are unknown, recent reports have demonstrated nucleocytoplasmic shuttling activity that utilises the importin-α and importin-β transport receptors for nuclear translocation. Here we describe the structure of mouse importin-α in complex with the TNRC6A nuclear localisation signal peptide. We further show that the interactions observed between TNRC6A and importin-α are conserved between mouse and human complexes. Our results highlight the ability of monopartite cNLS sequences to maximise contacts at the importin-α major binding site, as well as regions outside the main binding cavities.

Inhibition of EGFR nuclear shuttling decreases irradiation resistance in HeLa cells.

Cervical cancer is a leading cause of mortality in women worldwide. The resistance to irradiation at the advanced stage is the main reason for the poor prognosis and high mortality. This work aims to elucidate the molecular mechanism underlying the radio-resistance. In this study, we determined the pEGFR-T654 and pDNA-PK-T2609 expression level changes in irradiated HeLa cells treated with T654 peptide, a nuclear localization signal (NLS) inhibitor, to inhibit EGFR nuclear transport. Cell viability, cell cycle and migratory capacity were analyzed. Xenograft animal model was used to evaluate the effect of EGFR nuclear transport inhibition on the tumor growth in vivo. The enhanced translocation of nuclear EGFR in the irradiated HeLa cells correlated with the increasing level of pEGFR-T654 and pDNA-PK-T2609. Inhibition of EGFR nuclear translocation by NLS peptide inhibitor attenuated DNA damage repair in the irradiated HeLa cells, decreased cell viability and promoted cell death through arrest at G0 phase. NLS peptide inhibitor impaired the migratory capacity of irradiated HeLa cells, and negatively affected tumorigenesis in xenograft mice. This work puts forward a potential molecular mechanism of the irradiation resistance in cervical cancer cells, providing a promising direction towards an efficient therapy of cervical cancer.

Inhibition of EGFR nuclear translocation attenuate radioresistance through decreased p-DNA-PK expression in cervical cancer cells.

e17011 Background: The commonly used treatment for cervical cancer is radiotherapy. However, the resistance to irradiation and metastasis at the advanced stage is a common reason for the poor prognosis and high mortality. This study was designed to elucidate the role of epidermal growth factor receptor (EGFR) nuclear translocation in radioresistance, and its correlation with DNA damage repair pathway in the cervical cancer cells. Methods: The dynamic expression of EGFR, DNA-dependent protein kinase (DNA-PK), PDK-1, PKN1 and their phosphorylation level in irradiated cervical cancer cell line CaSki at 0 10 20 40 minutes was determined by western blotting. Besides, nuclear localization signal (NLS) peptide inhibitor and control peptides was synthesized and treated cells before irradiation to elucidate the correlation between EGFR nuclear translocation and DNA damage repair after irradiation. Results: Expression of EGFR, protein kinase N1 (PKN1), and DNA-PK in nucleus was increased after irradiation in CaSki cells. Irradiation also enhanced the phosphorylation level of EGFR at Thr654, PKN1 at T774 and DNA-PK at T2609. Inhibition of EGFR nuclear translocation by NLS peptide decreased the expression level of EGFR and DNA-PK in the nucleus, and attenuated their phosphorylation process. Conclusions: EGFR nuclear translocation riggered by irradiation promoted DNA damage repair in irradiated cervical cancer cells. This work facilitated us to understand the possible molecular mechanism of the resistance to irradiation in the treatment of cervical cancer, providing a potentially potent clinical method to cancer therapy.