The X-ray crystal structure of human A15C neuroglobin reveals both native/de novo disulfide bonds and unexpected ligand-binding sites.

Human neuroglobin (Ngb) contains a heme group and three Cys residues (Cys46, Cys55, and Cys120) in the polypeptide chain. By introducing an additional Cys at position 15, the X-ray structure of A15C Ngb mutant was solved at a high resolution of 1.35 Å, which reveals the formation of both the native (C46C55) and the engineered (C15C120) disulfide bonds, likely playing a functional and structural role, respectively, according to the geometry analysis. Unexpectedly, 1,4-dioxane from the crystallization reagents was bound not only to the protein surface, but also to the heme distal pocket, providing insights into protein-ligand interactions for the globin and guiding the design of functional heme enzymes.

Photonic generation of frequency-tunable biphase and quadriphase coded pulse signals without background interference enabled by vector modulation and balanced detection.

An approach to generating frequency-tunable biphase and quadriphase coded pulse signals without background interference based on a polarization division multiplexing dual-parallel Mach-Zehnder modulator (PDM-DPMZM) is presented and demonstrated. Two ternary baseband code sequences are separately encoded into a pair of orthogonal optical carriers by exploiting a polyphase encoder on the basis of the principle of vector modulation, which in turn can be mapped to the phase shifts of the generated phase coded waveforms after the balanced detection. The frequency tunability can also be achieved by controlling the bias voltage of the associated modulator, so that the carrier frequency can be tuned to either fundamental or doubled frequency. Additionally, by designing different phase codes, the generated pulse signals can be conveniently switched between the quadriphase and biphase coding waveforms. The major advantage of the proposed approach is that four phase shifts can be obtained by simply adjusting the polarity of the ternary code sequences, overcoming the power-dependent limitation of the previous work. A proof-of-principle experiment is conducted to assess the feasibility of the proposed approach built on the Barker code and Frank code phase coded pulse signals generation. Experimental results show the phase coded pulse signals at 12 and 24 GHz carrier frequency are well behaved in terms of peak-to-sidelobe ratio (PSR), range-Doppler coupling and Doppler tolerance.

Selective cadmium regulation mediated by a cooperative binding mechanism in CadR.

Detoxification of the highly toxic cadmium element is essential for the survival of living organisms. Pseudomonas putida CadR, a MerR family transcriptional regulator, has been reported to exhibit an ultraspecific response to the cadmium ion. Our crystallographic and spectroscopic studies reveal that the extra cadmium selectivity of CadR is mediated by the unexpected cooperation of thiolate-rich site I and histidine-rich site II. Cadmium binding in site I mediates the reorientation of protein domains and facilitates the assembly of site II. Subsequently, site II bridge-links 2 DNA binding domains through ligands His140/His145 in the C-terminal histidine-rich tail. With dynamic transit between 2 conformational states, this bridge could stabilize the regulator into an optimal conformation that is critical for enhancing the transcriptional activity of the cadmium detoxification system. Our results provide dynamic insight into how nature utilizes the unique cooperative binding mechanism in multisite proteins to recognize cadmium ions specifically.

Conversion of Human Neuroglobin into a Multifunctional Peroxidase by Rational Design.

Protein design has received much attention in the last decades. With an additional disulfide bond to enhance the protein stability, human A15C neuroglobin (Ngb) is an ideal protein scaffold for heme enzyme design. In this study, we rationally converted A15C Ngb into a multifunctional peroxidase by replacing the heme axial His64 with an Asp residue, where Asp64 and the native Lys67 at the heme distal site were proposed to act as an acid-base catalytic couple for H2O2 activation. Kinetic studies showed that the catalytic efficiency of A15C/H64D Ngb was much higher (∼50-80-fold) than that of native dehaloperoxidase, which even exceeds (∼3-fold) that of the most efficient native horseradish peroxidase. Moreover, the dye-decolorizing peroxidase activity was also comparable to that of some native enzymes. Electron paramagnetic resonance, molecular docking, and isothermal titration calorimetry studies provided valuable information for the substrate-protein interactions. Therefore, this study presents the rational design of an efficient multifunctional peroxidase based on Ngb with potential applications such as in bioremediation for environmental sustainability.

Structural basis of Zn(II) induced metal detoxification and antibiotic resistance by histidine kinase CzcS in Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is a major opportunistic human pathogen, causing serious nosocomial infections among immunocompromised patients by multi-determinant virulence and high antibiotic resistance. The CzcR-CzcS signal transduction system in P. aeruginosa is primarily involved in metal detoxification and antibiotic resistance through co-regulating cross-resistance between Zn(II) and carbapenem antibiotics. Although the intracellular regulatory pathway is well-established, the mechanism by which extracellular sensor domain of histidine kinase (HK) CzcS responds to Zn(II) stimulus to trigger downstream signal transduction remains unclear. Here we determined the crystal structure of the CzcS sensor domain (CzcS SD) in complex with Zn(II) at 1.7 Å resolution. This is the first three-dimensional structural view of Zn(II)-sensor domain of the two-component system (TCS). The CzcS SD is of α/ß-fold in nature, and it senses the Zn(II) stimulus at micromole level in a tetrahedral geometry through its symmetry-related residues (His55 and Asp60) on the dimer interface. Though the CzcS SD resembles the PhoQ-DcuS-CitA (PDC) superfamily member, it interacts with the effector in a novel domain with the N-terminal α-helices rather than the conserved ß-sheets pocket. The dimerization of the N-terminal H1 and H1' α-helices is of primary importance for the activity of HK CzcS. This study provides preliminary insight into the molecular mechanism of Zn(II) sensing and signaling transduction by the HK CzcS, which will be beneficial to understand how the pathogen P. aeruginosa resists to high levels of heavy metals and antimicrobial agents.

Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage.

The AlkB repair enzymes, including Escherichia coli AlkB and two human homologues, ALKBH2 and ALKBH3, are iron(II)- and 2-oxoglutarate-dependent dioxygenases that efficiently repair N(1)-methyladenine and N(3)-methylcytosine methylated DNA damages. The development of small molecule inhibitors of these enzymes has seen less success. Here we have characterized a previously discovered natural product rhein and tested its ability to inhibit AlkB repair enzymes in vitro and to sensitize cells to methyl methane sulfonate that mainly produces N(1)-methyladenine and N(3)-methylcytosine lesions. Our investigation of the mechanism of rhein inhibition reveals that rhein binds to AlkB repair enzymes in vitro and promotes thermal stability in vivo In addition, we have determined a new structural complex of rhein bound to AlkB, which shows that rhein binds to a different part of the active site in AlkB than it binds to in fat mass and obesity-associated protein (FTO). With the support of these observations, we put forth the hypothesis that AlkB repair enzymes would be effective pharmacological targets for cancer treatment.

Structural Insights into the Redox-Sensing Mechanism of MarR-Type Regulator AbfR.

As a master redox-sensing MarR-family transcriptional regulator, AbfR participates in oxidative stress responses and virulence regulations in Staphylococcus epidermidis. Here, we present structural insights into the DNA-binding mechanism of AbfR in different oxidation states by determining the X-ray crystal structures of a reduced-AbfR/DNA complex, an overoxidized (Cys13-SO2H and Cys13-SO3H) AbfR/DNA, and 2-disulfide cross-linked AbfR dimer. Together with biochemical analyses, our results suggest that the redox regulation of AbfR-sensing displays two novel features: (i) the reversible disulfide modification, but not the irreversible overoxidation, significantly abolishes the DNA-binding ability of the AbfR repressor; (ii) either 1-disulfide cross-linked or 2-disulfide cross-linked AbfR dimer is biologically significant. The overoxidized species of AbfR, resembling the reduced AbfR in conformation and retaining the DNA-binding ability, does not exist in biologically significant concentrations, however. The 1-disulfide cross-linked modification endows AbfR with significantly weakened capability for DNA-binding. The 2-disulfide cross-linked AbfR adopts a very "open" conformation that is incompatible with DNA-binding. Overall, the concise oxidation chemistry of the redox-active cysteine allows AbfR to sense and respond to oxidative stress correctly and efficiently.

Novobiocin binding to NalD induces the expression of the MexAB-OprM pump in Pseudomonas aeruginosa.

NalD was reported to be the secondary repressor of the MexAB-OprM multidrug efflux pump, the major system contributing to intrinsic multidrug resistance in Pseudomonas aeruginosa. Here, we show that novobiocin binds directly to NalD, which leads NalD to dissociate from the DNA promoter, and thus de-represses the expression of the MexAB-OprM pump. In addition, we have solved the crystal structure of NalD at a resolution of 2.90 Å. The structural alignment of NalD to its homologue TtgR reveals that the residues N129 and H167 in NalD are involved in its novobiocin-binding ability. We have confirmed the function of these two amino acids by EMSA and plate assay. The results presented here highlight the importance and diversity of regulatory mechanism in bacterial antibiotic resistance, and provide further insight for novel antimicrobial development.

A Whole Blood Assay for AR-V7 and ARv567es in Patients with Prostate Cancer.

PURPOSE: Most prostate cancer mortality can be attributed to metastatic castration resistant prostate cancer, an advanced stage that remains incurable despite recent advances. The AR (androgen receptor) signaling axis remains active in castration resistant prostate cancer. Recent studies suggest that expression of the AR-V (AR splice variant) AR-V7 may underlie resistance to abiraterone and enzalutamide. However, controversy exists over the optimal assay. Our objective was to develop a fast and sensitive assay for AR-Vs in patients. MATERIALS AND METHODS: Two approaches were assessed in this study. The first approach was based on depletion of leukocytes and the second one used RNA purified directly from whole blood preserved in PAXgene® tubes. Transcript expression was analyzed by quantitative reverse transcription-polymerase chain reaction. RESULTS: Through a side-by-side comparison we found that the whole blood approach was suitable to detect AR-Vs. The specificity of the assay was corroborated in a cancer-free cohort. Using the PAXgene assay samples from a cohort of 46 patients with castration resistant prostate cancer were analyzed. Overall, AR-V7 and ARv567es were detected in 67.53% and 29.87% of samples, respectively. Statistical analysis revealed a strong association of AR-V positivity with a history of second line hormonal therapies. CONCLUSIONS: To our knowledge this is the first study to demonstrate that PAXgene preserved whole blood can be used to obtain clinically relevant information regarding the expression of 2 AR-Vs. These data on a castration resistant prostate cancer cohort support a role for AR-Vs in resistance to therapies targeting the AR ligand-binding domain.

Overexpression of Eg5 correlates with high grade astrocytic neoplasm.

To investigate the relationship between Eg5 and histopathological grade of astrocytoma, Eg5 expression was evaluated by immunohistochemical examination on 88 specimens including 25 cases of glioblastoma (WHO grade IV), 22 cases of anaplastic astrocytoma (WHO grade III), 20 cases of diffuse astrocytoma (WHO grade II), and 21 cases of pilocytic astrocytoma (WHO grade I). The histopathological characteristics and Eg5 expression level of each tumor were assessed and statistically analyzed. Astrocytic tumors exhibited significant correlation of expression of Eg5 with higher WHO histopathological grades (p < 0.001). Eg5 is expressed in 51-98% (mean 76.88%) of neoplastic cells in glioblastoma, 34-57% (mean 43.59%) of neoplastic cells in anaplastic astrocytoma, 6-36% (mean 18.60%) of neoplastic cells in diffuse astrocytoma, and 2-28% (mean 13.48%) of neoplastic cells in pilocytic astrocytoma. In conclusion, overexpression of Eg5 associates with high-grade astrocytic neoplasm, and it may represent an independent diagnostic and prognostic factor in grading astrocytic tumors and predicting prognosis of astrocytic tumor patients.

Structural Basis for the Selective Pb(II) Recognition of Metalloregulatory Protein PbrR691.

The transcription regulator PbrR691, one of the MerR family proteins, shows extremely high sensitivity and selectivity toward Pb(II) in Ralstonia metallidurans CH34. Here, we present the crystal structure of PbrR691 in complex with Pb(II) at 2.0 Å resolution. The Pb(II) coordinates with three conserved cysteines and adopts a unique trigonal-pyramidal (hemidirected) geometry. To our knowledge, the PbrR691-Pb(II) structure provides the first three-dimensional visualization of a functional hemidirected lead(II) thiolate coordinate geometry in a protein.

Methylselenocysteine preventing castration-resistant progression of prostate cancer.

BACKGROUND: Castration-resistant progression of prostate cancer after androgen deprivation therapy remains a critical challenge in the clinical management of prostate cancer. Resurgent androgen receptor activity is an established driver of castration-resistant progression, and upregulation of androgen receptor expression has been implicated to contribute to the resurgent androgen receptor activity. We reported previously that methylselenocysteine can decrease the expression and activity of androgen receptor. Here we investigated the ability of methylselenocysteine to inhibit castration-resistant progression of prostate cancer. METHODS: The regrowth of LNCaP prostate cancer xenografts after castration was monitored. The levels of prostate-specific antigen in mouse serum were measured by ELISA. Tumor cell proliferation and apoptosis were analyzed via Ki-67 immunohistochemistry and TUNEL assay, respectively. Intratumoral angiogenesis was assessed by immunohistochemistry staining of vascular endothelial growth factor and CD31. RESULTS: We showed that methylselenocysteine delayed castration-resistant regrowth of LNCaP xenograft tumors after androgen deprivation. This was accompanied by decreased serum levels of prostate-specific antigen, inhibition of prostate cancer cell proliferation and tumor angiogenesis, as well as downregulation of androgen receptor and induction of apoptosis in the relapsed tumors. CONCLUSIONS: The present study represents the first to show the preclinical efficacy of methylselenocysteine in delaying castration-resistant progression of prostate cancer. The findings provide a rationale for evaluating the clinical application of combining methylselenocysteine with androgen deprivation therapy for the treatment of advanced prostate cancer.

20(S)-protopanaxadiol-aglycone downregulation of the full-length and splice variants of androgen receptor.

As a public health problem, prostate cancer engenders huge economic and life-quality burden. Developing effective chemopreventive regimens to alleviate the burden remains a major challenge. Androgen signaling is vital to the development and progression of prostate cancer. Targeting androgen signaling via blocking the production of the potent ligand dihydrotestosterone has been shown to decrease prostate cancer incidence. However, the potential of increasing the incidence of high-grade prostate cancers has been a concern. Mechanisms of disease progression after the intervention may include increased expression of androgen receptor (AR) in prostate tissue and expression of the constitutively active AR splice variants (AR-Vs) lacking the ligand-binding domain. Thus, novel agents targeting the receptor, preferentially both the full-length and AR-Vs, are urgently needed. In the present study, we show that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) effectively downregulates the expression and activity of both the full-length AR and AR-Vs. The effects of PPD on AR and AR-Vs are manifested by an immediate drop in proteins followed by a reduction in transcripts, attributed to PPD induction of proteasome-mediated degradation and inhibition of the transcription of the AR gene. We further show that although PPD inhibits the growth as well as AR expression and activity in LNCaP xenograft tumors, the morphology and AR expression in normal prostates are not affected. This study is the first to show that PPD suppresses androgen signaling through downregulating both the full-length AR and AR-Vs, and provides strong rationale for further developing PPD as a promising agent for the prevention and/or treatment of prostate cancer.

A Modified Purse-String Stapling Technique for Intracorporeal Circular Stapled Esophagojejunostomy During Laparoscopic Total Gastrectomy: Comparison with Extracorporeal Reconstruction Technique.

Background: Intracorporeal esophagojejunostomy (EJ) in the context of laparoscopic total gastrectomy remains a complex and technically demanding procedure. We have previously introduced a novel method of intracorporeal circular stapled EJ utilizing a conventional purse-string suture instrument. Since May 2018, we have refined this technique, and the aim of this study was to assess its safety and efficacy. Methods: Between May 2018 and June 2022, we enrolled 92 patients who underwent laparoscopic total gastrectomy with the modified intracorporeal reconstruction method. In addition, between March 2014 and June 2022, we enrolled 121 patients who underwent the procedure with the extracorporeal reconstruction method. We retrospectively collected and compared the clinical data of these 2 patient cohorts. Results: Intracorporeal reconstruction group experienced lower postoperative pain scores (2.7 ± 1.3 versus 4.5 ± 1.4, P = .032), reduced administration of analgesics (3.1 ± 2.2 versus 5.0 ± 3.5, P = .041), and shorter postoperative hospital stays (4.9 ± 2.3 versus 6.3 ± 3.5, P = .045) compared with the extracorporeal reconstruction group. In addition, anastomotic time and postoperative pain score were not increased in the overweight patients in the intracorporeal reconstruction group. Anastomotic leakage occurred in 2 (2.2%) patients in the intracorporeal reconstruction group and 4 (3.3%) patients in the extracorporeal reconstruction group. Anastomotic stricture occurred in 1 (1.1% and 0.8%) patient in each group. There was no significant difference in the overall postoperative complication rate between the 2 groups. Conclusions: The modified intracorporeal purse-string stapling technique for EJ during laparoscopic total gastrectomy is a safe and viable option, exhibiting less invasiveness and comparable outcomes to the extracorporeal reconstruction method, especially suitable for obese patients.

The oxidative nuclease activity of human cytochrome c with mutations in Ω-loop C/D.

Natural and artificial nucleases have extensive applications in biotechnology and biomedicine. The exploration of protein with potential DNA cleavage activity also inspires the design of artificial nuclease and helps to understand the physiological process of DNA damage. In this study, we engineered four human cytochrome c (Cyt c) mutants (N52S, N52A, I81N, and I81D Cyt c), which showed enhanced DNA cleavage activity and degradation in comparison with WT Cyt c, especially under acidic conditions. The mechanism assays revealed that the superoxide (O2•-) plays an important role in the nuclease reaction. The kinetic assays showed that the peroxidase activity of the I81D Cyt c mutant enhanced up to 9-fold at pH 5. This study suggests that the mutations of Ile81 and Asn52 in Ω-loop C/D are critical for the nuclease activity of Cyt c, which may have physiological significance in DNA damage and potential applications in biomedicine.

Naturally Occurring I81N Mutation in Human Cytochrome c Regulates Both Inherent Peroxidase Activity and Interactions with Neuroglobin.

Human cytochrome c (hCyt c) is a crucial heme protein and plays an indispensable role in energy conversion and intrinsic apoptosis pathways. The sequence and structure of Cyt c were evolutionarily conserved and only a few naturally occurring mutants were detected in humans. Among those variable sites, position 81 was proposed to act as a peroxidase switch in the initiation stages of apoptosis. In this study, we show that Ile81 not only suppresses the intrinsic peroxidase activity but also is essential for Cyt c to interact with neuroglobin (Ngb), a potential protein partner. The kinetic assays showed that the peroxidase activity of the naturally occurring variant I81N was enhanced up to threefold under pH 5. The local stability of the Ω-loop D (residues 70-85) in the I81N variant was decreased. Moreover, the Alphafold2 program predicted that Ile81 forms stable contact with human Ngb. Meanwhile, the Ile81 to Asn81 missense mutation abolishes the interaction interface, resulting in a ∼40-fold decrease in binding affinity. These observations provide an insight into the structure-function relationship of the conserved Ile81 in vertebrate Cyt c.

A Novel RNA-Binding Protein Signature to Predict Clinical Outcomes and Guide Clinical Therapy in Gastric Cancer.

Objective: This study aimed to develop an RNA-binding protein (RBP)-based signature for risk stratification and guiding clinical therapy in gastric cancer. Methods: Based on survival-related RBPs, an RBP-based signature was established by LASSO regression analysis in TCGA dataset. Kaplan-Meier curves were drawn between high- and low-risk groups. The predictive efficacy of this signature was assessed via ROCs at 1-, 3-, and 5-year survival. Its generalizability was verified in an external dataset. Following adjustment with other clinicopathological characteristics, the independency of survival prediction was evaluated via multivariate Cox regression and subgroup analyses. GSEA was utilized in identifying activated pathways in two groups. Stromal score, immune score, tumor purity, and infiltration levels of 22 immune cells were determined in each sample via the ESTIMATE and CIBERSORT algorithms. The sensitivity to chemotherapy drugs was assessed through the GDSC database. Results: Data showed that patients with high risk exhibited unfavorable clinical outcomes than those with low risk. This signature possessed good performance in predicting 1-, 3-, and 5-year survival and can be independently predictive of patients' survival. Calcium, ECM receptor interaction, and focal adhesion were highly enriched in high-risk samples. High-risk samples presented increased stromal and immune scores and reduced tumor purity. Moreover, this signature presented close relationships with immune infiltrations. Low-risk specimens were more sensitive to sorafenib, gefitinib, vinorelbine, and gemcitabine than high-risk specimens. Conclusion: This RBP-based signature may be a promising tool for predicting clinical outcomes and guiding clinical therapy in gastric cancer.

Survivin gene silencing sensitizes prostate cancer cells to selenium growth inhibition.

BACKGROUND: Prostate cancer is a leading cause of cancer-related death in men worldwide. Survivin is a member of the inhibitor of apoptosis (IAP) protein family that is expressed in the majority of human tumors including prostate cancer, but is barely detectable in terminally differentiated normal cells. Downregulation of survivin could sensitize prostate cancer cells to chemotherapeutic agents in vitro and in vivo. Selenium is an essential trace element. Several studies have shown that selenium compounds inhibit the growth of prostate cancer cells. The objective of this study is to investigate whether survivin gene silencing in conjunction with selenium treatment could enhance the therapeutic efficacy for prostate cancer and to elucidate the underlying mechanisms. METHODS: Expression of survivin was analyzed in a collection of normal and malignant prostatic tissues by immunohistochemical staining. In vitro studies were conducted in PC-3M, C4-2B, and 22Rv1 prostate cancer cells. The effect of selenium on survivin expression was analyzed by Western blotting and semi-quantitative RT-PCR. Survivin gene knockdown was carried out by transfecting cells with a short hairpin RNA (shRNA) designed against survivin. Cell proliferation was quantitated by the 3-(4,5-Dimethylthiazol-2-yl)- 2,5-Diphenyltetrazolium Bromide (MTT) assay and apoptosis by propidium iodide staining followed by flow cytometry analysis. Finally, in vivo tumor growth assay was performed by establishing PC-3M xenograft in nude mice and monitoring tumor growth following transfection and treatment. RESULTS: We found that survivin was undetectable in normal prostatic tissues but was highly expressed in prostate cancers. Survivin knockdown or selenium treatment inhibited the growth of prostate cancer cells, but the selenium effect was modest. In contrast to what have been observed in other cell lines, selenium treatment had little or no effect on survivin expression in several androgen-independent prostate cancer cell lines. Survivin knockdown sensitized these cells to selenium growth inhibition and apoptosis induction. In nude mice bearing PC-3M xenografts, survivin knockdown synergizes with selenium in inhibiting tumor growth. CONCLUSIONS: Selenium could inhibit the growth of hormone-refractory prostate cancer cells both in vitro and in vivo, but the effects were modest. The growth inhibition was not mediated by downregulating survivin expression. Survivin silencing greatly enhanced the growth inhibitory effects of selenium.

A Five-Gene Signature Based on Stromal/Immune Scores in the Tumor Microenvironment and Its Clinical Implications for Liver Cancer.

Increasing evidence highlights the clinical significance of stromal cells and immune cells in the liver cancer microenvironment. However, reliable prognostic models have not been well established. This study aimed to develop a gene signature for liver cancer based on stromal and immune scores. Using the estimation of stromal and immune cells in malignant tumor tissues using expression data (ESTIMATE) algorithm, stromal and immune scores were estimated based on the transcriptome profile of The Cancer Genome Atlas (TCGA) liver cancer cohort. Stromal-/immune-related differentially expressed genes were identified, followed by functional enrichment analysis. The Cox regression model was used to select prognostic genes and construct a gene signature. Its predictive potential was evaluated by receiver operating characteristic (ROC). The correlation between the risk score and immune cell infiltration was analyzed using Tumor Immune Estimation Resource (TIMER). Three hundred sixty-four upregulated and 10 downregulated stromal-/immune-related genes were identified, were mainly enriched in immune-related processes and pathways. Through univariate and multivariate cox survival analysis, a five-gene risk score was constructed, composed of FABP3, HTRA3, OLFML2B, PDZD4 and SLAMF6. Patients with high score indicated a poorer prognosis than those with low risk score. The areas under the ROC curves of overall survival (OS), progression-free interval, 3-, 5-year, OS status were 0.68, 0.57, 0.72, 0.74 and 0.728, indicating its well performance on predicting patients' prognoses. Furthermore, the risk score and the five genes were significantly correlated with immune cell infiltration in the tumor microenvironment. In this study, we proposed a prognostic five-gene signature based on stromal/immune scores in the liver cancer microenvironment.

Long Non-Coding RNA BLACAT1 Promotes the Tumorigenesis of Gastric Cancer by Sponging microRNA-149-5p and Targeting KIF2A.

OBJECTIVE: Gastric cancer (GC) is a gastrointestinal tumor. This study is aimed to explore the regulatory mechanism of long non-coding RNA BLACAT1 (BLACAT1)/microRNA-149-5p (miR-149-5p)/KIF2A cascade on GC. METHODS: The expression of BLACAT1, miR-149-5p and KIF2A in GC was detected by qRT-PCR. The proliferation, migration and invasion of GC cells in vitro were analyzed by MTT, wound-healing and transwell assay, respectively. The xenograft tumor model was constructed in nude mice to confirm the inhibition effect of BLACAT1 knockdown on GC in vivo. Then, dual-luciferase reporter assay was used to detect the interactions among BLACAT1, miR-149-5p and KIF2A. Western blot assay was performed to determine the protein expression of KIF2A. RESULTS: The expression of BLACAT1 and KIF2A was up-regulated in GC, but miR-149-5p expression was down-regulated. Silencing of BLACAT1 retarded the proliferation, migration and invasion of GC cells in vitro and the growth of tumor xenograft in vivo. Moreover, BLACAT1 acted as the molecular sponge of miR-149-5p to up-regulate KIF2A expression. At last, feedback experiments suggested that BLACAT1 accelerated the proliferation, migration and invasion of GC cells by regulating miR-149-5p/KIF2A axis. CONCLUSION: BLACAT1 facilitated the tumorigenesis of GC through regulating miR-149-5p/KIF2A axis, which indicated BLACAT1/miR-149-5p/KIF2A cascade may be a new therapeutic target.