LIM Domain Kinase 1 Research Articles

<i>Alpinia zerumbet</i>: A Review of the Chemistry, Quantity, and Pharmacological Properties of Selected Kavalactones

Alpinia zerumbet or shell ginger is a ginger plant with diverse chemical constituents and medicinal and non-medicinal uses. Dihydro-5,6-dehydrokawain (DDK) and Dehydrokawain (DK) are two kavalactones (also known as kava pyrones or styrylpyrones) from A. zerumbet. Both DDK and DK have a carbonyl group at C2, a methoxy group at C4, and a double bond at C5 and C6. DK has a double bond at C7 and C8 that is absent in DDK. Quantity of DDK in A. zerumbet can be ranked as rhizome > leaf > flower > stem > seed. The pericarp and seed placenta of the fruit has higher quantity of DDK than the leaf. In most plant parts, the contents of DDK are higher than those of DK. Hispidin (HP) is synthesized from DK by hydrolysis. These three kavalactones from A. zerumbet have the most promising pharmacological properties that include insecticidal, fungicidal, antioxidant, inhibition of enzymes, inhibition of Advanced Glycation End-products (AGEs), inhibition of p21-activated kinase 1 (PAK1), inhibition of LIM domain kinase 1 (LIMK1), promotion of hair growth, anti-cancer, inhibition of melanogenesis, anti-inflammatory, anti-obesity, HIV-1 integrase inhibition, neuraminidase inhibition, osteogenic, anti-platelet aggregation, cytoprotective, anti-ulcerative, and singlet oxygen quenching activities. Some fields for further research are suggested. Sources of information in this review were from Google, Google Scholar, Science Direct, PubMed, J-Stage, China National Knowledge Infrastructure (CNKI), and PubChem.

Novel Histopathological Biomarkers in Prostate Cancer: Implications and Perspectives.

Prostate cancer (PCa) is the second most frequently diagnosed cancer in men. Despite the significant progress in cancer diagnosis and treatment over the last few years, the approach to disease detection and therapy still does not include histopathological biomarkers. The dissemination of PCa is strictly related to the creation of a premetastatic niche, which can be detected by altered levels of specific biomarkers. To date, the risk factors for biochemical recurrence include lymph node status, prostate-specific antigen (PSA), PSA density (PSAD), body mass index (BMI), pathological Gleason score, seminal vesicle invasion, extraprostatic extension, and intraductal carcinoma. In the future, biomarkers might represent another prognostic factor, as discussed in many studies. In this review, we focus on histopathological biomarkers (particularly CD169 macrophages, neuropilin-1, cofilin-1, interleukin-17, signal transducer and activator of transcription protein 3 (STAT3), LIM domain kinase 1 (LIMK1), CD15, AMACR, prostate-specific membrane antigen (PSMA), Appl1, Sortilin, Syndecan-1, and p63) and their potential application in decision making regarding the prognosis and treatment of PCa patients. We refer to studies that found a correlation between the levels of biomarkers and tumor characteristics as well as clinical outcomes. We also hypothesize about the potential use of histopathological markers as a target for novel immunotherapeutic drugs or targeted radionuclide therapy, which may be used as adjuvant therapy in the future.

Actin remodeling mediates ROS production and JNK activation to drive apoptosis-induced proliferation.

Stress-induced cell death, mainly apoptosis, and its subsequent tissue repair is interlinked although our knowledge of this connection is still very limited. An intriguing finding is apoptosis-induced proliferation (AiP), an evolutionary conserved mechanism employed by apoptotic cells to trigger compensatory proliferation of their neighboring cells. Studies using Drosophila as a model organism have revealed that apoptotic caspases and c-Jun N-terminal kinase (JNK) signaling play critical roles to activate AiP. For example, the initiator caspase Dronc, the caspase-9 ortholog in Drosophila, promotes activation of JNK leading to release of mitogenic signals and AiP. Recent studies further revealed that Dronc relocates to the cell cortex via Myo1D, an unconventional myosin, and stimulates production of reactive oxygen species (ROS) to trigger AiP. During this process, ROS can attract hemocytes, the Drosophila macrophages, which further amplify JNK signaling cell non-autonomously. However, the intrinsic components connecting Dronc, ROS and JNK within the stressed signal-producing cells remain elusive. Here, we identified LIM domain kinase 1 (LIMK1), a kinase promoting cellular F-actin polymerization, as a novel regulator of AiP. F-actin accumulates in a Dronc-dependent manner in response to apoptotic stress. Suppression of F-actin polymerization in stressed cells by knocking down LIMK1 or expressing Cofilin, an inhibitor of F-actin elongation, blocks ROS production and JNK activation, hence AiP. Furthermore, Dronc and LIMK1 genetically interact. Co-expression of Dronc and LIMK1 drives F-actin accumulation, ROS production and JNK activation. Interestingly, these synergistic effects between Dronc and LIMK1 depend on Myo1D. Therefore, F-actin remodeling plays an important role mediating caspase-driven ROS production and JNK activation in the process of AiP.

LIMK1: A promising prognostic and immune infiltration indicator in colorectal cancer.

Studies have shown that LIM domain kinase 1 (LIMK1) is upregulated in a variety of tumors and may be a potential detection target. The present study analyzed the expression difference of LIMK1 and its relationship with tumor clinicopathological characteristics and tumor microenvironment in colorectal cancer (CRC). The transcriptomic data of LIMK1 with CRC were downloaded from The Cancer Genome Atlas (TCGA) database and GEO databases for analyzing the expression of LIMK1 mRNA and the correlation with the prognosis of patients. The protein expression of LIMK1 was obtained from the Human Protein Atlas. The receiver operating characteristic (ROC) curve and Kaplan-Meier was used to evaluate the expression characteristics and prognostic differences of LIMK1 in CRC. STRING was used to analyze co-expression genes of LIMK1. The tumor immune estimation resource was applied to the correlation between LIMK1 expression and immune infiltrates. The present study verified LIMK1 expression at the level of clinical samples collected from the Tianjin Medical University General Hospital and cell lines using reverse transcription-quantitative PCR. The mRNA and protein expression of LIMK1 were both upregulated in tumor tissues compared with adjacent tissues in CRC. The expression levels of LIMK1 were positively associated with clinical-pathological features of CRC including lymphatic invasion (P=4.00×10−2) and high pathologic stages (P=4.20×10−2). The AUC value of LIMK1 in CRC was 0.937 (95% CI: 0.918-0.957) through ROC analysis. Under the best cut-off value (4.009), the sensitivity and specificity were 98 and 81.9%. LIMK1 expression was mainly related to CD4+ T cells, macrophages and dendritic cells in the immune microenvironment of CRC. In conclusion, the high expression of LIMK1 in CRC was closely related to the clinical features and prognosis of patients. Therefore, LIMK1 was a promising prognostic indicator and a potential target for immunotherapy in CRC.

LncRNA MIR9-3HG enhances LIMK1 mRNA and protein levels to contribute to the carcinogenesis of lung squamous cell carcinoma via sponging miR-138–5p and recruiting TAF15

The aberrantly expressed long non-coding RNAs (lncRNAs) are closely correlated with the malignant progression of cancer cells. In our study, we identified lncRNA MIR9–3 host gene (MIR9–3HG) as the research target and explored its roles in lung squamous cell carcinoma (LUSC). RT-qPCR was conducted to reveal that MIR9–3HG was observably overexpressed in LUSC cells. Functional assays encompassing colony formation, 5-ethynyl-2′-deoxyuridine (EdU) staining, transwell and flow cytometry assays and western blot detecting related proteins demonstrated that MIR9–3HG depletion hampered cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) while accelerating cell apoptosis in LUSC. Subcellular fractionation assay were performed to demonstrate that MIR9–3HG was prominently distributed in the cytoplasm of LUSC cells. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), immunofluorescence (IF), fluorescent in situ hybridization (FISH) and RNA pull down assays were implemented to confirm that MIR9–3HG modulates LIM domain kinase 1 (LIMK1) mRNA and protein levels by sequestering microRNA-138–5p (miR-138–5p) and recruiting TATA-box binding protein associated factor 15 (TAF15) protein. Taken together, our research determined that MIR9–3HG up-regulated LIMK1 mRNA and protein levels to promote LUSC carcinogenesis, which offers a novel insight into mechanisms of LUSC.

Electroacupuncture promotes the repair of neurological function in ischemic stroke rats by inhi-biting cofilin rod formation

To observe the effect of electroacupuncture (EA) on modified neurological severity score (mNSS), cerebral infarction volume, expression of Lim domain kinase-1 (LIMK1) and slingshot homolog-1 (SSH1) proteins, Cofilin rod formation and neural cell apoptosis in rats with ischemic stroke (IS), so as to explore its mechanisms underlying improvement of IS. Male SD rats were randomly divided into normal, model and EA groups, with 13 rats in each group. The IS model was established by occlusion of the middle cerebral artery (MCAO) according to Zea Longa's method. EA was applied to "Quchi" (LI11) and "Zusanli" (ST36) for 30 min, once a day for 7 consecutive days. The behavioral changes of mNSS were observed before and after modeling. The volume of cerebral infarction was measured by using a small animal magnetic resonance imaging. The protein expressions of LIMK1 and SSH1 in the cerebral ischemic tissues were detected by Western blot. The density of Cofilin rod and neural cell apoptosis in cerebral ischemic area were determined by immunofluorescence staining and TUNEL staining, separately. After modeling, the mNSS score, cerebral infarction volume ratio, expression level of SSH1, density of Cofilin rod and the number of apoptotic cells were significantly increased (P<0.01), while the expression level of LIMK1 protein was obviously decreased in the model group relevant to the normal group (P<0.01). After 7 days' treatment, all the increased and decreased levels of the indexes mentioned above were reversed in the EA group relevant to the model group (P<0.01, P<0.05). EA of LI11 and ST36 can improve neurological function and reduce infarction range in MCAO rats, which may be related to its action in regulating the expression of LIMK1 and SSH1, inhibiting the formation of Cofilin rod and reducing apoptosis of neural cells.

Avian Hepatitis E Virus ORF2 Protein Interacts with Rap1b to Induce Cytoskeleton Rearrangement That Facilitates Virus Internalization.

ABSTRACTAvian hepatitis E virus (HEV) causes liver diseases and multiple extrahepatic disorders in chickens. However, the mechanisms involved in avian HEV entry remain elusive. Herein, we identified the RAS-related protein 1b (Rap1b) as a potential HEV-ORF2 protein interacting candidate. Experimental infection of chickens and cells with an avian HEV isolate from China (CaHEV) led to upregulated expression and activation of Rap1b both in vivo and in vitro. By using CaHEV capsid as mimic of virion to treat cell in vitro, it appears that the interaction between the viral capsid and Rap1b promoted cell membrane recruitment of the downstream effector Rap1-interacting molecule (RIAM). In turn, RIAM further enhanced Talin-1 membrane recruitment and retention, which led to the activation of integrin α5/β1, as well as integrin-associated membrane protein kinases, including focal adhesion kinase (FAK). Meanwhile, FAK activation triggered activation of downstream signaling molecules, such as Ras-related C3 botulinum toxin substrate 1 RAC1 cell division cycle 42 (CDC42), p21-activated kinase 1 (PAK1), and LIM domain kinase 1 (LIMK1). Finally, F-actin rearrangement induced by Cofilin led to the formation of lamellipodia, filopodia, and stress fibers, contributes to plasma membrane remodeling, and might enhance CaHEV virion internalization. In conclusion, our data suggested that Rap1b activation was triggered during CaHEV infection and appeared to require interaction between CaHEV-ORF2 and Rap1b, thereby further inducing membrane recruitment of Talin-1. Membrane-bound Talin-1 then activates key Integrin-FAK-Cofilin cascades involved in modulation of actin kinetics, and finally leads to F-actin rearrangement and membrane remodeling to potentially facilitate internalization of CaHEV virions into permissive cells.IMPORTANCE Rap1b is a multifunctional protein that is responsible for cell adhesion, growth, and differentiation. The inactive form of Rap1b is phosphorylated and distributed in the cytoplasm, while active Rap1b is prenylated and loaded with GTP to the cell membrane. In this study, the activation of Rap1b was induced during the early stage of avian HEV infection under the regulation of PKA and SmgGDS. Continuously activated Rap1b recruited its effector RIAM to the membrane, thereby inducing the membrane recruitment of Talin-1 that led to the activation of membrane α5/β1 integrins. The triggering of the signaling pathway-associated Integrin α5/β1-FAK-CDC42&RAC1-PAK1-LIMK1-Cofilin culminated in F-actin polymerization and membrane remodeling that might promote avian HEV virion internalization. These findings suggested a novel mechanism that is potentially utilized by avian HEV to invade susceptible cells.

Structural Aspects of LIMK Regulation and Pharmacology.

Malfunction of the actin cytoskeleton is linked to numerous human diseases including neurological disorders and cancer. LIMK1 (LIM domain kinase 1) and its paralogue LIMK2 are two closely related kinases that control actin cytoskeleton dynamics. Consequently, they are potential therapeutic targets for the treatment of such diseases. In the present review, we describe the LIMK conformational space and its dependence on ligand binding. Furthermore, we explain the unique catalytic mechanism of the kinase, shedding light on substrate recognition and how LIMK activity is regulated. The structural features are evaluated for implications on the drug discovery process. Finally, potential future directions for targeting LIMKs pharmacologically, also beyond just inhibiting the kinase domain, are discussed.

LIM domain kinase 1

Background Despite recent advancement, prostate cancer (PC) remains to represent a leading cause of cancer mortality and morbidity in men. Diagnosis of PC faces many challenges, especially on core biopsies. Multiple signaling pathways have been involved in PC progression. However, castration resistance eventually develops, especially in metastatic PC. Identifying a marker that helps in distinguishing invasive tumor from benign and precursor lesions, as well as predicts its metastatic and prognostic potentials, is needed. LIM-domain kinase 1 (LIMK1) is a newly identified marker that affects the cytoskeleton of cancer cells. Its role in PC is still not well understood. This study aimed at evaluating LIMK1 expression in benign prostatic hyperplasia (30 cases), high-grade prostatic intraepithelial neoplasia (27 cases), and PC prostatic adenocarcinoma (60 cases) and its association with the prognostic clinicopathological parameters in PC. Results No strong expression was detected in benign prostatic hyperplasia expression compared with 25.9% and 48.3% in high-grade prostatic intraepithelial neoplasia and prostatic adenocarcinoma (Pca) groups. The expression was statistically higher in the Pca group than the other groups. There was significant association with poor prognostic parameters in Pca groups, including higher prostate-specific antigen levels, tumor percentage, Gleason scores, grade groups, T stage, positive lymph-node metastases, extracapsular extension, seminal vesicle invasion, distant metastasis, and prognostic-stage group. Conclusion LIMK1 is considered a promising diagnostic and prognostic marker in Pca.

Upregulation of LIMK1 Is Correlated With Poor Prognosis and Immune Infiltrates in Lung Adenocarcinoma.

BackgroundProtein-coding gene LIM Domain Kinase 1 (LIMK1) is upregulated in various tumors and reported to promote tumor invasion and metastasis. However, the prognostic values of LIMK1 and correlation with immune infiltrates in lung adenocarcinoma are still not understood. Therefore, we evaluated the prognostic role of LIMK1 and its correlation with immune infiltrates in lung adenocarcinoma.MethodsTranscriptional expression profiles of LIMK1 between lung adenocarcinoma tissues and normal tissues were downloaded from the Cancer Genome Atlas (TCGA). The LIMK1 protein expression was assessed by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and the Human Protein Atlas. Receiver operating characteristic (ROC) curve was used to differentiate lung adenocarcinoma from adjacent normal tissues. Kaplan-Meier method was conducted to assess the effect of LIMK1 on survival. Protein-protein interaction (PPI) networks were constructed by the STRING. Functional enrichment analyses were performed using the “ClusterProfiler” package. The relationship between LIMK1 mRNA expression and immune infiltrates was determined by tumor immune estimation resource (TIMER) and tumor-immune system interaction database (TISIDB).ResultsThe expression of LIMK1 in lung adenocarcinoma tissues was significantly upregulated than those in adjacent normal tissues. Increased LIMK1 mRNA expression was associated with lymph node metastases and high TNM stage. The ROC curve analysis showed that with a cutoff level of 4.908, the accuracy, sensitivity, and specificity for LIMK1 differentiate lung adenocarcinoma from adjacent controls were 69.5, 93.2, and 71.9%, respectively. Kaplan-Meier survival analysis showed lung adenocarcinoma patients with high- LIMK1 had a worse prognosis than those with low- LIMK1 (43.1 vs. 55.1 months, P = 0.028). Correlation analysis indicated LIMK1 mRNA expression was correlated with tumor purity and immune infiltrates.ConclusionUpregulated LIMK1 is significantly correlated with poor survival and immune infiltrates in lung adenocarcinoma. Our study suggests that LIMK1 can be used as a biomarker of poor prognosis and potential immune therapy target in lung adenocarcinoma.

LIMK1 promotes peritoneal metastasis of gastric cancer and is a therapeutic target

Peritoneal metastasis is a common form of metastasis among advanced gastric cancer patients. In this study, we reported the identification of LIM domain kinase 1 (LIMK1) as a promoter of gastric cancer peritoneal metastasis, and its potential to be a therapeutic target of dabrafenib (DAB). Using transcriptomic sequencing of paired gastric cancer peritoneal metastasis, primary tumors, and normal gastric tissues, we first unveiled that LIMK1 is selectively up-regulated in metastatic tumors. Increased LIMK1 in gastric cancer peritoneal metastasis was validated by immunohistochemistry analysis of an independent patient cohort. In vitro functional studies demonstrated that LIMK1 knockout or knockdown significantly inhibited cell migration and invasion of gastric cancer cells. LIMK1 knockout also abrogated peritoneal and liver metastases of gastric cancer cells in nude mice in vivo. Dabrafenib, a small molecule targeting LIMK1, was found to decrease cell migration and invasion of gastric cancer cells in vitro and abolish peritoneal and liver metastasis formation in vivo. Mechanistically, either LIMK1 knockout or Dabrafenib inhibited LIMK1 expression and phosphorylation of its downstream target cofilin. Taken together, our results demonstrated that LIMK1 functions as a metastasis promoter in gastric cancer by inhibiting LIMK1-p-cofilin and that Dabrafenib has the potential to serve as a novel treatment for gastric cancer peritoneal metastasis.

FOXD3‑AS1/miR‑128‑3p/LIMK1 axis regulates cervical cancer progression

Long non-coding RNA forkhead box D3 antisense RNA 1 (FOXD3-AS1) functions as an oncogenic regulator in several types of cancer, including breast cancer, glioma and cervical cancer. However, the effects and mechanisms underlying FOXD3-AS1 in cervical cancer (CC) are not completely understood. The present study aimed to investigate the biological functions and potential molecular mechanisms underlying FOXD3-AS1 in CC progression. Reverse transcription-quantitative PCR was performed to detect FOXD3-AS1, microRNA (miR)-128-3p and LIM domain kinase 1 (LIMK1) expression levels in CC tissues and cells. Immunohistochemical staining and western blotting were conducted to assess LIMK1 protein expression levels in CC tissues and cells, respectively. Cell Counting Kit-8 and BrdU assays were used to determine the role of FOXD3-AS1 in regulating cell proliferation. CC cell migration and invasion were assessed by performing Transwell assays. Dual-luciferase reporter assays were conducted to verify the binding between miR-128-3p and FOXD3-AS1. FOXD3-AS1 expression was significantly increased in CC tissues and cell lines compared with adjacent healthy tissues and normal cervical epithelial cells, respectively. High FOXD3-AS1 expression was significantly associated with poor differentiation of tumor tissues, increased tumor size and positive lymph node metastasis. FOXD3-AS1 overexpression significantly increased CC cell proliferation, migration and invasion compared with the negative control (NC) group, whereas FOXD3-AS1 knockdown resulted in the opposite effects compared with the small interfering RNA-NC group. Moreover, the results demonstrated that FOXD3-AS1 targeted and negatively regulated miR-128-3p, which indirectly upregulated LIMK1 expression. Therefore, the present study demonstrated that FOXD3-AS1 upregulated LIMK1 expression via competitively sponging miR-128-3p in CC cells, promoting CC progression.

Arhgef6 (alpha-PIX) cytoskeletal regulator signals to GTPases and Cofilin to couple T cell migration speed and persistence.

Immunity is governed by successful T cell migration, optimized to enable a T cell to fully scan its environment without wasted movement by balancing speed and turning. Here we report that the Arhgef6 RhoGEF (aka alpha-PIX; αPIX; Cool-2), an activator of small GTPases, is required to restrain cell migration speed and cell turning during spontaneous migration on 2D surfaces. In Arhgef6-/- T cells, expression of Arhgef7 (beta-PIX; βPIX; Cool-1), a homolog of Arhgef6, was increased and correlated with defective activation and localization of Rac1 and CDC42 GTPases, respectively. Downstream of Arhgef6, PAK2 (p21-activated kinase 2) and LIMK1 phosphorylation was reduced, leading to increased activation of Cofilin, the actin-severing factor. Consistent with defects in these signaling pathways, Arhgef6-/- T cells displayed abnormal bilobed lamellipodia and migrated faster, turned more, and arrested less than wild-type (WT) T cells. Using pharmacologic inhibition of LIMK1 (LIM domain kinase 1) to induce Cofilin activation in WT T cells, we observed increased migration speed but not increased cell turning. In contrast, inhibition of Cdc42 increased cell turning but not speed. These results suggested that the increased speed of the Arhgef6-/- T cells is due to hyperactive Cofilin while the increased turning may be due to abnormal GTPase activation and recruitment. Together, these findings reveal that Arhgef6 acts as a repressor of T cell speed and turning by limiting actin polymerization and lamellipodia formation.

Human kidney stone matrix proteins alleviate hyperoxaluria induced renal stress by targeting cell-crystal interactions

Increased levels of urinary oxalate also known as hyperoxaluria, increase the likelihood of kidney stone formation through enhanced calcium oxalate (CaOx) crystallization. The management of lithiatic renal pathology requires investigations at the initial macromolecular stages. Hence, the current study was designed to unravel the protein make-up of human kidney stones and its impact on renal cells' altered proteome, induced as the consequence of CaOx injury. CaOx kidney stones were collected from patients; stones were pooled for entire cohort, followed by protein extraction. Immunocytochemistry, RT-PCR and flow-cytometric analysis revealed the promising antilithiatic activity of kidney stone matrix proteins. The iTRAQ analysis of renal cells showed up-regulation of 12 proteins and down-regulation of 41 proteins due to CaOx insult, however, this differential expression was normalized in the presence of kidney stone matrix proteins. Protein network analysis revealed involvement of up-regulated proteins in apoptosis, calcium-binding, inflammatory and stress response pathways. Moreover, seven novel antilithiatic proteins were identified from human kidney stones' matrix: Tenascin-X-isoform2, CCDC-144A, LIM domain kinase-1, Serine/Arginine receptor matrix protein-2, mitochondrial peptide methionine sulfoxide reductase, volume-regulated anion channel subunit-LRRC8A and BMPR2. In silico analysis concluded that these proteins exert antilithiatic potential through crystal binding, thereby inhibiting the crystal-cell interaction, a pre-requisite to initiate inflammatory response. Thus, the outcomes of this study provide insights into the molecular events of CaOx induced renal toxicity and subsequent progression into nephrolithiasis.

Exosomal lncRNA H19 promotes the progression of hepatocellular carcinoma treated with Propofol via miR‐520a‐3p/LIMK1 axis

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer‐related deaths globally. Herein, we explored the underlying mechanism by which Propofol inhibited the development of HCC.Methods3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay was carried out to detect the viability and proliferation. Quantitative real‐time polymerase chain reaction (qRT‐PCR) and Western blot were performed to detect the expression of long noncoding RNA (lncRNA) H19, microRNA‐520a‐3p (miR‐520a‐3p), LIM domain kinase 1 (LIMK1), metastasis‐associated markers (Snail, Twist, Vimentin and E‐cadherin) and exosome markers (CD9 and CD81). Transmission electron microscopy (TEM) was used to observe the morphology and structure of exosomes. The apoptosis and metastasis were measured by flow cytometry and transwell assays. StarBase software was utilized to predict the targets of H19 and miR‐520a‐3p. Dual‐luciferase reporter assay was performed to confirm the interaction between miR‐520a‐3p and H19 or LIMK1. Nude mice bearing tumors were used to validate the role of exosomal H19.RESULTSThe high expression of exosomal H19 accelerated the proliferation and motility while hampering the apoptosis of HCC cells. MiR‐520a‐3p could bind with H19. Exosomal H19 exacerbated HCC through sponging miR‐520a‐3p. The 3’ untranslated region (3’UTR) of LIMK1 could bind to miR‐520a‐3p. MiR‐520a‐3p mimic transfection reversed the inhibitory effect of high expression of exosomal LIMK1 on the apoptosis of HCC cells and the promoting effects on the proliferation and metastasis of HCC cells. The mRNA and protein levels of LIMK1 were regulated by H19/miR‐520a‐3p signaling. The high level of exosomal H19 promoted the growth of HCC tumors in vivo.ConclusionCirculating H19 promoted the proliferation, migration and invasion and inhibited the apoptosis of HCC cells treated with Propofol through upregulating LIMK1 via sponging miR‐520a‐3p.

Recognition of physiological phosphorylation sites by p21-activated kinase 4

Many serine/threonine protein kinases discriminate between serine and threonine substrates as a filter to control signaling output. Among these, the p21-activated kinase (PAK) group strongly favors phosphorylation of Ser over Thr residues. PAK4, a group II PAK, almost exclusively phosphorylates its substrates on serine residues. The only well documented exception is LIM domain kinase 1 (LIMK1), which is phosphorylated on an activation loop threonine (Thr508) to promote its catalytic activity. To understand the molecular and kinetic basis for PAK4 substrate selectivity we compared its mode of recognition of LIMK1 (Thr508) with that of a known serine substrate, β-catenin (Ser675). We determined X-ray crystal structures of PAK4 in complex with synthetic peptides corresponding to its phosphorylation sites in LIMK1 and β-catenin to 1.9 Å and 2.2 Å resolution, respectively. We found that the PAK4 DFG + 1 residue, a key determinant of phosphoacceptor preference, adopts a sub-optimal orientation when bound to LIMK1 compared to β-catenin. In peptide kinase activity assays, we find that phosphoacceptor identity impacts catalytic efficiency but does not affect the Km value for both phosphorylation sites. Although catalytic efficiency of wild-type LIMK1 and β-catenin are equivalent, T508S mutation of LIMK1 creates a highly efficient substrate. These results suggest suboptimal phosphorylation of LIMK1 as a mechanism for controlling the dynamics of substrate phosphorylation by PAK4.

Up-regulation of LIMK1 expression in prostate cancer is correlated with poor pathological features, lymph node metastases and biochemical recurrence.

This study aimed to explore the association between LIM domain kinase 1 (LIMK1) expression in prostate cancer (PCa) tissues with advanced pathological features, lymph node metastases and biochemical recurrence. A total of 279 PCa specimens from patients who underwent radical prostatectomy and 50 benign prostatic hyperplasia (BPH) specimens were collected to construct tissue microarray, which were subjected to immunohistochemical staining for LIMK1 expression subsequently. Logistic and Cox regression analysis were used to evaluate the relationship between LIMK1 expression and clinicopathological features of patients with PCa. Immunohistochemical staining assay demonstrated that LIMK1 expression was significantly higher in PCa than BPH specimens (77.1% vs 26.0%; P < .001). LIMK1 expression was significantly higher in positive lymph node specimens than corresponding PCa specimens (P = .002; P < .001). Up‐regulation of LIMK1 was associated with prostate volume, prostate‐specific antigen, prostate‐specific antigen density, Gleason score, T stage, lymph node metastases, extracapsular extension and seminal vesicle invasion, and positive surgical margin. Multivariate logistic regression analysis demonstrated that LIMK1 was an independent risk factor for PCa lymph node metastasis (P < .05). Multivariate Cox regression analysis revealed that the up‐regulation of LIMK1 was an independent risk factor for biochemical recurrence. Kaplan‐Meier analysis indicated that up‐regulation LIMK1 was associated with shortened biochemical‐free survival (BFS) after radical prostatectomy (P < .001). In conclusion, LIMK1 was significantly up‐regulated in PCa and positive lymph node specimens and correlated with lymph node metastasis and shortened BFS of PCa. The underlying molecular mechanism of LIMK1 in PCa should be further evaluated.

Circular RNA hsa_circ_0012673 facilitates lung cancer cell proliferation and invasion via miR-320a/LIMK18521 axis.

Lung cancer is one of the most malignant tumors with high morbidity and mortality in the world. The incidence and mortality of lung cancer were increased per year in many countries over the past 50 years. The increasing studies had shown that circular RNA (circRNA) was involved in the progression of lung cancer. Therefore, it was significant to seek the molecular mechanism of circ_0012673 in lung cancer. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to estimate the expression levels of circ_0012673, miR-320a and LIM domain kinase 1 (LIMK1) in lung cancer tissues and cells. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT), flow cytometry and transwell assays were recruited to evaluate proliferation, apoptosis and mobility of lung cancer cells, respectively. The relative protein expression levels of Vimentin, N-cadherin, E-cadherin and LIMK1 were determined with Western blot assay. The relationships among circ_0012673, miR-320a and LIMK1 were analyzed by starBase database, dual-luciferase reporter assay, and Pearson's correlation. Circ_0012673 was overexpressed in lung cancer tissues and cell lines. Loss-of-functional experiment confirmed that knockdown of circ_0012673 constrained proliferation, motility and Epithelial-Mesenchymal Transition (EMT), but induced apoptosis by targeting miR-320a. Furthermore, LIMK1 was a target of miR-320a in lung cancer cells. Elevated LIMK1 could abolish the overexpression of miR-320a induced effects on lung cancer cells. Mechanistically, circ_0012673 contributed to lung cancer progression through mediating miR-320a /LIMK1 pathway. Circ_0012673 was a tumor-promoter in lung cancer via acting as competing endogenous RNA to regulate LIMK1 expression by binding miR-320a.

Diallyl disulfide induces downregulation and inactivation of cofilin1 differentiation via the Rac1/ROCK1/LIMK1 pathway in leukemia cells.

Cofilin is associated with cell differentiation; however, to the best of our knowledge, no data have indicated an association between the cofilin 1 pathway and leukemia cell differentiation. The present study investigated the involvement of the cofilin 1 signaling pathway in diallyl disulfide (DADS)-induced differentiation and the inhibitory effects on the proliferation, migration, and invasion of human leukemia HL-60 cells. First, it was identified that 8 µM DADS suppressed cell proliferation, migration and invasion, and induced differentiation based on the reduced nitroblue tetrazolium ability and increased CD11b and CD33 expression. DADS significantly downregulated the expression of cofilin 1 and phosphorylated cofilin 1 in HL-60 leukemia cells. Second, it was verified that silencing cofilin 1 markedly promoted 8 µM DADS-induced differentiation and the inhibitory effect on cell proliferation and invasion. Overexpression of cofilin 1 obviously suppressed 8 µM DADS-induced differentiation and the inhibitory effect on cell proliferation and invasion. Third, the present study examined the mechanisms by which 8 µM DADS decreases cofilin 1 expression and activation. The results revealed that 8 µM DADS inhibited the mRNA and protein expression of Rac1, Rho-associated protein kinase 1 (ROCK1) and LIM domain kinase 1 (LIMK1) as well as the phosphorylation of LIMK1 in HL-60 cells, while 8 µM DADS enhanced the effects of the Rac1-ROCK1-LIMK1 pathway in cells overexpressing cofilin 1 compared with that in control HL-60 cells. These results suggest that the anticancer function of DADS on HL-60 leukemia cells is regulated by the Rac1-ROCK1-LIMK1-cofilin 1 pathway, indicating that DADS could be a promising anti-leukemia therapeutic compound.

Up-regulation of miR-106a targets LIMK1 and contributes to cognitive impairment induced by isoflurane anesthesia in mice.

Postoperative cognitive dysfunction (POCD) had a great relationship with anesthesia during surgery, and miRNAs have been found involved in anesthesia-induced cognitive impairment. To explore the role and potential mechanism of miR-106a in isoflurane anesthesia-induced cognitive impairment. Adult male mice were treated with isoflurane anesthesia; Morris water maze tests and fear conditioning tests were performed; and expression levels of miR-106a and LIMK1 were determined by quantitative real-time PCR (qRT-PCR) and western blot. Dual luciferase reporter assay was used to determine the binding of miR-106a and 3'UTR of LIMK1. To verify the role of miR-106a, antagomir of miR-106a were intrahippocampally injected. Finally, expression of BCL2 apoptosis regulator (Bcl-2), LIM domain kinase 1 (LIMK1), BCL2-associated X, apoptosis regulator (Bax) and cleaved caspase3 was determined by western blot. In isoflurane anesthesia-treated group (IS), the percentage of target quadrant dwell time was significantly lower and the escape latency was significantly higher than in the control group (sham), and the freezing behavior of IS was significantly less in contextual fear conditioning tests. Expression levels of miR-106a were increased and those of LIMK1 were decreased in response to IS. Dual luciferase reporter assay showed that miR-106a could bind with the 3'UTR of LIMK1. Decreased expression levels of miR-106a improved the cognitive impairment of the mice treated with isoflurane. Intrahippocampally injected antagomir of miR-106a also increased LIMK1 and Bcl-2 levels, decreased the BAX and cleaved caspase3 expression levels in the mice treated with isoflurane. Decrease of LIMK1 expression by miR-106a played an important role in isoflurane anesthesia-induced cognitive impairment.