CRISPR/Cas13a-based supersensitive circulating tumor DNA assay for detecting EGFR mutations in plasma.

Despite recent technological advancements in cell tumor DNA (ctDNA) mutation detection, challenges persist in identifying low-frequency mutations due to inadequate sensitivity and coverage of current procedures. Herein, we introduce a super-sensitivity and specificity technique for detecting ctDNA mutations, named HiCASE. The method utilizes PCR-based CRISPR, coupled with the restriction enzyme. In this work, HiCASE focuses on testing a series of EGFR mutations to provide enhanced detection technology for non-small cell lung cancer (NSCLC), enabling a detection sensitivity of 0.01% with 40 ng cell free DNA standard. When applied to a panel of 140 plasma samples from 120 NSCLC patients, HiCASE exhibits 88.1% clinical sensitivity and 100% specificity with 40 µL of plasma, higher than ddPCR and Super-ARMS assay. In addition, HiCASE can also clearly distinguish T790M/C797S mutations in different positions at a 1% variant allele frequency, offering valuable guidance for drug utilization. Indeed, the established HiCASE assay shows potential for clinical applications.

Phosphoglycerate Kinase 1: An Effective Therapeutic Target in Cancer.

Phosphoglycerate kinase 1 (PGK1) serves as a pivotal enzyme in the cellular glycolysis pathway, facilitating adenosine-triphosphate (ATP) production in tumor cells and driving the Warburg effect. PGK1 generates ATP through the reversible phosphorylation reaction of 1,3-bisphosphoglycerate (1,3-BPG) to Mg-adenosine-5'-diphosphate (Mg-ADP). In addition to its role in regulating cellular metabolism, PGK1 plays a pivotal role in autophagy induction, regulation of the tricarboxylic acid cycle (TCA), and various mechanisms including tumor cell drug resistance, and so on. Given its multifaceted functions within cells, the involvement of PGK1 in many types of cancer, including breast cancer, astrocytoma, metastatic colon cancer, and pancreatic ductal adenocarcinoma, is intricate. Notably, PGK1 can function as an intracellular protein kinase to coordinate tumor growth, migration, and invasion via posttranslational modifications (PTMs). Furthermore, elevated expression levels of PGK1 have been observed in cancer tissues, indicating its association with unfavorable treatment outcomes and prognosis. This review provides a comprehensive summary of PGK1's expression pattern, structural features, functional properties, involvement in PTMs, and interaction with tumors. Additionally highlighted are the prospects for developing and applying related inhibitors that confirm the indispensable value of PGK1 in tumor progression.

Targeted disruption of the BCR-ABL fusion gene by Cas9/dual-sgRNA inhibits proliferation and induces apoptosis in chronic myeloid leukemia cells.

The BCR-ABL fusion gene, formed by the fusion of the breakpoint cluster region protein ( BCR) and the Abl Oncogene 1, Receptor Tyrosine Kinase ( ABL) genes, encodes the BCR-ABL oncoprotein, which plays a crucial role in leukemogenesis. Current therapies have limited efficacy in patients with chronic myeloid leukemia (CML) because of drug resistance or disease relapse. Identification of novel strategies to treat CML is essential. This study aims to explore the efficiency of novel CRISPR-associated protein 9 (Cas9)/dual-single guide RNA (sgRNA)-mediated disruption of the BCR-ABL fusion gene by targeting BCR and cABL introns. A co-expression vector for Cas9 green fluorescent protein (GFP)/dual-BA-sgRNA targeting BCR and cABL introns is constructed to produce lentivirus to affect BCR-ABL expression in CML cells. The effects of dual-sgRNA virus-mediated disruption of BCR-ABL are analyzed via the use of a genomic sequence and at the protein expression level. Cell proliferation, cell clonogenic ability, and cell apoptosis are assessed after dual sgRNA virus infection, and phosphorylated BCR-ABL and its downstream signaling molecules are detected. These effects are further confirmed in a CML mouse model via tail vein injection of Cas9-GFP/dual-BA-sgRNA virus-infected cells and in primary cells isolated from patients with CML. Cas9-GFP/dual-BA-sgRNA efficiently disrupts BCR-ABL at the genomic sequence and gene expression levels in leukemia cells, leading to blockade of the BCR-ABL tyrosine kinase signaling pathway and disruption of its downstream molecules, followed by cell proliferation inhibition and cell apoptosis induction. This method prolongs the lifespan of CML model mice. Furthermore, the effect is confirmed in primary cells derived from patients with CML.

Fiber Optic SPR POCT: A Novel Nucleic Acid Detection Biosensor for Environmental Viruses.

In the post-COVID-19 pandemic era, the long-term surveillance of pathogens is still important. The rapid detection of pathogens facilitates the accurate and convenient real-time monitoring of microbial contamination and improves the management of diseases. Here, a novel surface plasmon resonance (SPR)-based point of care testing (POCT) approach of microorganism nucleic acids with the guidance of CRISPR enzyme is described, including the application of optical fiber-based detection of trace SARS-CoV2 virus in sewage water on SPR and validation of the plasmonic biosensor for the detection of single-nucleotide mutations in natural water samples.

TRIM3 inhibits colorectal cancer cell migration and lipid droplet formation by promoting FABP4 degradation.

This study is to investigate the regulation of TRIM3/FABP4 on colorectal cancer (CRC) cell migration and lipid metabolism. After transfection of HCT116, LoVo, or SW480 cells, the expression of FABP4, TRIM3, N-cadherin, Vimentin, E-cadherin, and lipid droplet (LD) formation-related genes was measured by qRT-PCR or western blot assays. Wound healing and Transwell assays were applied to detect CRC cell migration and invasion abilities. The levels of triglyceride (TG) and total cholesterol (TC) were measured and the formation of LDs was observed. Additionally, the relationship between FABP4 and TRIM3 was confirmed by Co-IP and ubiquitination assays. Furthermore, a liver metastasis model of CRC was established to explore the effect of FABP4 on CRC tumor metastasis in vivo. FABP4 was upregulated in CRC cells. Downregulation of FABP4 or upregulation of TRIM3 resulted in repressed cell migration and invasion, decreased TG and TC levels, and reduced numbers of LDs. In nude mice, knockdown of FABP4 reduced metastatic nodules in the liver. Mechanistically, TRIM3 combined FABP4 and decreased its protein expression by ubiquitination. Overexpressed FABP4 reversed the influence of TRIM3 upregulation on CRC cell migration and LD formation. In conclusion, underexpressed TRIM3 suppressed FABP4 ubiquitination and accelerated CRC cell migration and LD formation.

Liver proteomic analysis reveals the key proteins involved in host immune response to sepsis.

Background: Sepsis is a serious infection-induced response in the host, which can result in life-threatening organ dysfunction. It is of great importance to unravel the relationship between sepsis and host immune response and its mechanisms of action. Liver is one of the most vulnerable organs in sepsis, however, the specific pathogenesis of septic liver injury has not been well understood at the protein level. Methods: A total of 12 healthy Sprague-Dawley (SD) male rats aged from 6 to 8 weeks were adaptively housed in individual cages in the specific pathogen free animal room. These lab rats were grouped into two groups: treatment (N = 9) and control (N = 3) groups; only three mice from the treatment group survived and were used for subsequent experiments. A TMT-based proteomic analysis for liver tissue was performed in the septic rat model. Results: A total of 37,012 unique peptides were identified, and then 6,166 proteins were determined, among which 5,701 were quantifiable. Compared to the healthy control group, the septic rat group exhibited 162 upregulated and 103 downregulated differentially expressed proteins (DEPs). The upregulated and downregulated DEPs were the most significantly enriched into the complement and coagulation cascades and metabolic pathways. Protein-protein interaction (PPI) analysis further revealed that the upregulated and downregulated DEPs each clustered in a PPI network. Several highly connected upregulated and downregulated DEPs were also enriched into the complement and coagulation cascades pathways and metabolic pathways, respectively. The parallel reaction monitoring (PRM) results of the selected DEPs were consistent with the results of the TMT analysis, supporting the proteomic data. Conclusion: Our findings highlight the roles of complement and coagulation cascades and metabolic pathways that may play vital roles in the host immune response. The DEPs may serve as clinically potential treatment targets for septic liver injury.

Fusobacterium nucleatum-derived small extracellular vesicles facilitate tumor growth and metastasis via TLR4 in breast cancer.

BACKGROUND: The contributive role of the microbiome in tumor progression has been reported in multiple studies, such as the Fusobacterium nucleatum (F. nucleatum) in breast cancer (BC). This study aimed to explore the role of F. nucleatum-derived small extracellular vesicles (Fn-EVs) in BC and preliminarily uncover the mechanism. METHODS: Ten normal and 20 cancerous breast tissues were harvested to investigate the gDNA expression of F. nucleatum and its relation with the clinical characteristics of BC patients. After isolating Fn-EVs by ultracentrifugation from F. nucleatum (ATCC 25,586), both MDA-MB-231 and MCF-7 cells were treated with PBS, Fn, or Fn-EVs, followed by being subjected to CCK-8, Edu staining, wound healing, and Transwell assays to detect their cell viability, proliferation, migration, and invasion. TLR4 expression in BC cells with diverse treatments was assessed by western blot. In vivo experiments were performed to verify its role in tumor growth and liver metastasis. RESULTS: The F. nucleatum gDNA levels of breast tissues in BC patients were significantly higher than those in normal subjects, and positively associated with tumor size and metastasis. Fn-EVs administration significantly enhanced the cell viability, proliferation, migration, and invasion of BC cells, while knocking down TLR4 in BC cells could block these effects. Furthermore, in vivo study verified the contributive role of Fn-EVs in tumor growth and metastasis of BC, which might rely on its regulation of TLR4. CONCLUSIONS: Collectively, our results suggest that F. nucleatum plays an important role in BC tumor growth and metastasis by regulating TLR4 through Fn-EVs. Thus, a better understanding of this process may aid in the development of novel therapeutic agents.

Kruppel-like factor 5 enhances proliferation, lipid droplet formation and oxaliplatin resistance in colorectal cancer by promoting fatty acid binding protein 6 transcription.

Oxaliplatin (OXA) is a standard agent for colorectal cancer (CRC) adjuvant chemotherapy. However, acquired and intrinsic OXA resistance is a primary challenge for CRC treatment. This study investigates the function of the Kruppel-like factor 5/fatty acid binding proteins 6 (KLF5/FABP6) axis in CRC proliferation, lipid droplet formation and OXA resistance. OXA-resistant CRC cell lines were constructed, and FABP6 and KLF5 expression was assessed in parental and OXA-resistant CRC cells. Subsequent to gain- and loss-of-function experiments, CRC cell proliferation was assessed by cell counting kit-8 (CCK-8) and clone formation assays, the intracellular lipid synthesis by oil red O staining and the protein expression of lipid metabolism genes by western blot. OXA resistance of CRC cells was assessed by CCK-8 assay. The binding of KLF5 to FABP6 was analyzed by the dual-luciferase reporter and ChIP assays. A tumorigenicity assay in nude mice was adopted to examine the impact of KLF5 on CRC tumor growth and OXA resistance in vivo . FABP6 and KLF5 expression was high in CRC cell lines. Downregulation of FABP6 or KLF5 restrained CRC cell proliferation and lipid droplet formation in vitro . FABP6 and KLF5 expression was elevated in OXA-resistant CRC cells. Downregulation of FABP6 or KLF5 repressed the OXA resistance of OXA-resistant CRC cells. Mechanistically, KLF5 facilitated the transcription of FABP6. FABP6 overexpression counteracted the suppressive effects of KLF5 downregulation on CRC cell growth, lipid droplet formation and OXA resistance. KLF5 downregulation restrained CRC tumor growth and OXA resistance in vivo . In conclusion, KLF5 knockdown reduced FABP6 transcription to protect against proliferation, lipid droplet formation and OXA resistance in CRC.

Expression of AKRs superfamily and prognostic in human gastric cancer.

The human aldo-keto reductase (AKRs) superfamily is involved in the development of various tumors. However, the different expression patterns of AKRs and their prognostic value in gastric cancer (GC) have not been clarified. In this study, we analyzed the gene expression and gene methylation level of AKRs in GC patients and the survival data and immune infiltration based on AKRs expression, using data from different databases. We found that the expression levels of AKR1B10, AKR1C1, AKR1C2, and AKR7A3 in GC tissues were lower and the expression level of AKR6A5 was higher in GC tissues than in normal tissue. These differentially expressed genes (AKR1B10, AKR1C1, AKR1C2, AKR7A3, and AKR6A5) were significantly correlated with the infiltration level. The expression of SPI1 and AKR6A5 in GC was positively correlated. Survival analysis showed that GC levels of AKR6A5 reduced or increased mRNA levels of AKR7A3, and AKR1B10 was expected to have higher overall survival (OS), first progression (FP) survival, and postprogression survival (PPS) rates and a better prognosis. Moreover, the expression of AKR1B1 was found to be correlated with the staging of GC. The methylation of AKR6A5 (KCNAB2) at cg05307871 and cg01907457 was significantly associated with the classification of GC. Meta-analysis and ROC curve analysis show that the expression level of AKR1B1 and the methylation of cg16156182 (KCNAB1), cg11194299 (KCNAB2), cg16132520 (AKR1B1), and cg13801416 (AKR1B1) had a high hazard ratio and a good prognostic value. These data suggest that the expression and methylation of AKR1B1 and AKR6A5 are significantly related to the prognosis.

A Cascade Targeted and Mitochondrion-Dysfunctional Nanomedicine Capable of Overcoming Drug Resistance in Hepatocellular Carcinoma.

Chemoresistance is a formidable issue in clinical anticancer therapy and is pertinent to the lowered efficacies of chemotherapeutics and the activated tumor self-repairing proceedings. Herein, bifunctional amphiphiles containing galactose ligands and high-density disulfide are synthesized for encapsulating mitochondrion-targeting tetravalent platinum prodrugs to construct a cascade targeted and mitochondrion-dysfunctional nanomedicine (Gal-NP@TPt). Subsequent investigations verify that Gal-NP@TPt with sequential targeting functions toward tumors and mitochondria improved the spatiotemporal level of platinum. In addition, glutathione depletion by Gal-NP@TPt appear to substantially inhibit the proceedings of platinum detoxification, inducing the susceptibility to the mitochondrial platinum. Moreover, the strategic transportation of platinum to mitochondria lacking DNA repair machinery by Gal-NP@TPt lowers the possibility of platinum deactivation. Eventually, Gal-NP@TPt demonstrates appreciable antitumor effects for the systemic treatment of patient-derived tumor xenografts of hepatocellular carcinoma. Note that these strategies in overcoming drug resistance have also been confirmed to be valid based on genome-wide analysis via RNA-sequencing. Therefore, an intriguing multifunctional nanomedicine capable of resolving formidable chemoresistance is achieved, which should be greatly emphasized in practical applications for the treatment of intractable tumors.

Regulation of progesterone receptor expression in endometriosis, endometrial cancer, and breast cancer by estrogen, polymorphisms, transcription factors, epigenetic alterations, and ubiquitin-proteasome system.

The uterus and breasts are hormone-responsive tissues. Progesterone and estradiol regulate gonadotropin secretion, prepare the endometrium for implantation, maintain pregnancy, and regulate the differentiation of breast tissue. Dysregulation of these hormones causes endometriosis, endometrial cancer, and breast cancer, damaging the physical and mental health of women. Emerging evidence has shown that progesterone resistance or elevated progesterone activity is the primary hormonal substrate of these diseases. Since progesterone acts through its specific nuclear receptor, the abnormal expression of the progesterone receptor (PR) dysregulates progesterone function. This review discusses the regulatory mechanisms of PR expression in patients with endometriosis, and endometrial or breast cancer, including estrogen, polymorphisms, transcription factors, epigenetics, and the ubiquitin-proteasome system. (1) Estrogen promotes the expression of PRA (a PR isoform) mRNA and protein through the interaction of estrogen receptors (ERs) and Sp1 with half-ERE/Sp1 binding sites. ERs also affect the binding of Sp1 and Sp1 sites to promote the expression of PRB (another PR isoform)(2) PR polymorphisms, mainly PROGINS and + 331 G/A polymorphism, regulate PR expression by affecting DNA methylation and transcription factor binding. (3) The influence of epigenetic alterations on PR expression occurs through DNA methylation, histone modification, and microRNA. (4) As one of the main protein degradation pathways in vivo, the ubiquitin-proteasome system (UPS) regulates PR expression by participating in protein degradation. These mechanisms may provide new molecular targets for diagnosing and treating endometriosis, endometrial, and breast cancer.

Circulating Tumor DNA-A Novel Biomarker of Tumor Progression and Its Favorable Detection Techniques.

Cancer is the second leading cause of death in the world and seriously affects the quality of life of patients. The diagnostic techniques for tumors mainly include tumor biomarker detection, instrumental examination, and tissue biopsy. In recent years, liquid technology represented by circulating tumor DNA (ctDNA) has gradually replaced traditional technology with its advantages of being non-invasive and accurate, its high specificity, and its high sensitivity. ctDNA may carry throughout the circulatory system through tumor cell necrosis, apoptosis, circulating exosome secretion, etc., carrying the characteristic changes in tumors, such as mutation, methylation, microsatellite instability, gene rearrangement, etc. In this paper, ctDNA mutation and methylation, as the objects to describe the preparation process before ctDNA analysis, and the detection methods of two gene-level changes, including a series of enrichment detection techniques derived from PCR, sequencing-based detection techniques, and comprehensive detection techniques, are combined with new materials. In addition, the role of ctDNA in various stages of cancer development is summarized, such as early screening, diagnosis, molecular typing, prognosis prediction, recurrence monitoring, and drug guidance. In summary, ctDNA is an ideal biomarker involved in the whole process of tumor development.

Yin Yang 1 promotes aggressive cell growth in high-grade breast cancer by directly transactivating kinectin 1.

Invasive cancer growth and metastasis account for the poor prognosis of high-grade breast cancer. Recently, we reported that kinectin 1 (KTN1), a member of the kinesin-binding protein family, promotes cell invasion of triple-negative breast cancer and high-grade breast cancer cells by augmenting the NF-κB signaling pathway. However, the upstream mechanism regulating KTN1 is unknown. Therefore, this functional study was performed to decipher the regulatory cohort of KTN1 in high-grade breast cancer. Bioinformatic analysis indicated that transcription factor Yin Yang 1 (YY1) was a potential transactivator of KTN1. High YY1 expression correlated positively with pathological progression and poor prognosis of high-grade breast cancer. Additionally, YY1 promoted cell invasive growth both in vitro and in vivo, in a KTN1-dependent manner. Mechanistically, YY1 could transactivate the KTN1 gene promoter. Alternatively, YY1 could directly interact with a co-factor, DEAD-box helicase 3 X-linked (DDX3X), which significantly co-activated YY1-mediated transcriptional expression of KTN1. Moreover, DDX3X augmented YY1-KTN1 signaling-promoted invasive cell growth of breast cancer. Importantly, overexpression of YY1 enhanced tumor aggressive growth in a mouse breast cancer model. Our findings established a novel DDX3X-assisted YY1-KTN1 regulatory axis in breast cancer progression, which could lead to the development novel therapeutic targets for breast cancer.

Carbon Dots in Biomedicine: A Review.

Despite the rapid development of science and technology, the effective treatment of cancer still threatens human life and health. However, the success of cancer treatment is closely related to early diagnosis, identification, and effective treatment. In recent years, with the strengthening of the development and research of nanomaterials for cancer diagnosis and treatment, researchers have found that carbon dots (CDs) have the advantages of wide absorption, excellent biocompatibility, diverse imaging characteristics, and photostability and are widely used in various fields, such as sensing, imaging, and drug/gene transportation. Recently, researchers also discovered that CDs could be used as an effective photosensitizer to generate active oxygen or convert light energy into heat under the stimulation of the external lasers, making them have the effects of photothermal and photodynamic therapy for cancer. In this review, we first outline the single-modal and multimodal imaging analysis of CDs in cancer cells. After introducing diversified imaging functions, we focused on the design and the latest research progress of CDs in phototherapy and introduced in detail the strategies of CDs in phototherapy treatment and the challenges faced by clinical applications. We hope that this overview can provide important insights for researchers and accelerate the pace of research on CDs in imaging-guided phototherapy treatment.

Stabilization of UCA1 by N6-methyladenosine RNA methylation modification promotes colorectal cancer progression.

BACKGROUND: UCA1 is frequently upregulated in a variety of cancers, including CRC, and it can play an oncogenic role by various mechanisms. However, how UCA1 is regulated in cancer is largely unknown. In this study, we aimed to determine whether RNA methylation at N6-methyladenosine (m6A) can impact UCA1 expression in colorectal cancer (CRC). METHODS: qRT-PCR was performed to detect the level of UCA1 and IGF2BP2 in CRC samples. CRISPR/Cas9 was employed to knockout (KO) UCA1, METTL3 and WTAP in DLD-1 and HCT-116 cells, while rescue experiments were carried out to re-express METTL3 and WTAP in KO cells. Immunoprecipitation using m6A antibody was performed to determine the m6A modification of UCA1. In vivo pulldown assays using S1m tagging combined with site-direct mutagenesis was carried out to confirm the recognition of m6A-modified UCA1 by IGF2BP2. Cell viability was measured by MTT and colony formation assays. The expression of UCA1 and IGF2BP2 in TCGA CRC database was obtained from GEPIA ( http://gepia.cancer-pku.cn ). RESULTS: Our results revealed that IGF2BP2 serves as a reader for m6A modified UCA1 and that adenosine at 1038 of UCA1 is critical to the recognition by IGF2BP2. Importantly, we showed that m6A writers, METTL3 and WTAP positively regulate UCA1 expression. Mechanically, IGF2BP2 increases the stability of m6A-modified UCA1. Clinically, IGF2BP2 is upregulated in CRC tissues compared with normal tissues. CONCLUSION: These results suggest that m6A modification is an important factor contributing to upregulation of UCA1 in CRC tissues.

AKR1B10 promotes breast cancer cell proliferation and migration via the PI3K/AKT/NF-κB signaling pathway.

BACKGROUND: Aberrant expression of Aldo-Keto reductase family 1 member B10 (AKR1B10) was associated with tumor size and metastasis of breast cancer in our published preliminary studies. However, little is known about the detailed function and underlying molecular mechanism of AKR1B10 in the pathological process of breast cancer. METHODS: The relationship between elevated AKR1B10 expression and the overall survival and disease-free survival of breast cancer patients was analyzed by Kaplan-Meier Plotter database. Breast cancer cell lines overexpressing AKR1B10 (MCF-7/AKR1B10) and breast cancer cell lines with knockdown of AKR1B10 (BT-20/shAKR1B10) were constructed to analyze the impact of AKR1B10 expression on cell proliferation and migration of breast cancer. The expression levels of AKR1B10 were detected and compared in the breast cancer cell lines and tissues by RT-qPCR, western blot and immunohistochemistry. The proliferation of breast cancer cells was monitored by CCK8 cell proliferation assay, and the migration and invasion of breast cancer cells was observed by cell scratch test and transwell assay. The proliferation- and EMT-related proteins including cyclinD1, c-myc, Survivin, Twist, SNAI1, SLUG, ZEB1, E-cadherin, PI3K, p-PI3K, AKT, p-AKT, IKBα, p-IKBα, NF-κB p65, p-NF-κB p65 were detected by western blot in breast cancer cells. MCF-7/AKR1B10 cells were treated with LY294002, a PI3K inhibitor, to consider the impact of AKR1B10 overexpression on the PI3K/AKT/NF-κB signal cascade and the presence of NF-κB p65 in nuclear. In vivo tumor xenograft experiments were used to observe the role of AKR1B10 in breast cancer growth in mice. RESULTS: AKR1B10 expression was significantly greater in breast cancer tissue compared to paired non-cancerous tissue. The expression of AKR1B10 positively correlated with lymph node metastasis, tumor size, Ki67 expression, and p53 expression, but inversely correlated with overall and disease-free survival rates. Gene Ontology analysis showed that AKR1B10 activity contributes to cell proliferation. Overexpression of AKR1B10 facilitated the proliferation of MCF-7 cells, and induced the migration and invasion of MCF-7 cells in vitro in association with induction of epithelial-mesenchymal transition (EMT). Conversely, knockdown of AKR1B10 inhibited these effects in BT-20 cells. Mechanistically, AKR1B10 activated PI3K, AKT, and NF-κB p65, and induced nuclear translocation of NF-κB p65, and expression of proliferation-related proteins including c-myc, cyclinD1, Survivin, and EMT-related proteins including ZEB1, SLUG, Twist, but downregulated E-cadherin expression in MCF-7 cells. AKR1B10 silencing reduced the phosphorylation of PI3K, AKT, and NF-κB p65, the nuclear translocation of NF-κB p65, and the expression of proliferation- and migration-related proteins in BT-20 cells. LY294002, a PI3K inhibitor, attenuated the phosphorylation of PI3K, AKT, and NF-κB p65, and the nuclear translocation of NF-κB p65. In vivo tumor xenograft experiments confirmed that AKR1B10 promoted breast cancer growth in mice. CONCLUSIONS: AKR1B10 promotes the proliferation, migration and invasion of breast cancer cells via the PI3K/AKT/NF-κB signaling pathway and represents a novel prognostic indicator as well as a potential therapeutic target in breast cancer.

[Preparation and identification of rabbit polyclonal antibody against BCR-ABL b3a2 fusion protein].

Objective To prepare and identify rabbit anti-breakpoint cluster region-Abelson leukemia virus oncogene (BCR-ABL) b3a2 subtype polyclonal antibody. Methods A peptide containing the fusion sequence of the b3a2 subtype BCR-ABL fusion protein was designed and synthesized with the purity higher than 90%. The fusion polypeptide was coupled to Keyhole Limpet hemocyanin (KLH) and used to immune New Zealand rabbits. Antiserum was purified after multiple immunizations, in addition to using the b3a2 subtype fusion polypeptide for affinity purification. Peptides harboring only BCR or c-ABL amino acid sequences were also synthesized and used to purify the antibody in the secondary purification. The antibody that only bound to part of the epitope was absorbed and removed. ELISA and Western blotting were performed to determine the antibody titer and specificity. Results The rabbit serum background was low before immunization. The titer of the polyclonal antibody reached 1:32 000 after immunization, which met the experimental requirements. Western blotting showed that the antibody could specifically recognize the b3a2 subtype fusion protein of BCR-ABL. Conclusion The experiment has prepared the specific rabbit polyclonal antibody against BCR-ABL b3a2 subtype.

Vorinostat targets UBE2C to reverse epithelial-mesenchymal transition and control cervical cancer growth through the ubiquitination pathway.

Vorinostat is a histone deacetylase inhibitor (HDACi) that was demonstrated in our previous study to inhibit the proliferation, migration, and invasion of cervical cancer cells by regulating the PI3K/Akt signaling pathway. However, the molecular mechanism of vorinostat in cervical cancer treatment remains to be further elucidated. A nude mouse xenograft model was established to analyze the antitumor effect of vorinostat in vivo. The combination of iTRAQ-based proteomics and parallel reaction monitoring (PRM) technology has proven to be an efficient and reliable method to identify potential targets for cancer chemotherapy. In this study, 254 differentially expressed proteins in vorinostat-treated cervical cancer cells, among which 180 were upregulated and 74 were downregulated, were identified by using an iTRAQ-based proteomic strategy. Subsequent bioinformatic and PRM analysis of these differentially expressed proteins indicated that UBE2C is a promising target of vorinostat in the inhibition of cervical cancer cell proliferation. We confirmed that the expression of endogenous UBE2C in cervical cancer cell lines was significantly higher than that in normal cervical epithelial cell lines. Additionally, we found that vorinostat downregulated the expression of UBE2C, SQSTM1/p62, N-cadherin, vimentin and upregulated E-cadherin in SiHa and HeLa cells. Our results also showed that vorinostat can downregulate the expression of SQSTM1/p62, N-cadherin, and vimentin during the treatment of cervical cancer cells by regulating UBE2C, while upregulating the expression of E-cadherin. In conclusion, vorinostat reverses epithelial-mesenchymal transition by targeting UBE2C and controls the proliferation of cervical cancer cells through the ubiquitination pathway. UBE2C can be used as a promising target for the development of vorinostat treatment strategies.