Small-Molecule Inhibitors of TIPE3 Protein Identified through Deep Learning Suppress Cancer Cell Growth In Vitro.

Tumor necrosis factor-α-induced protein 8-like 3 (TNFAIP8L3 or TIPE3) functions as a transfer protein for lipid second messengers. TIPE3 is highly upregulated in several human cancers and has been established to significantly promote tumor cell proliferation, migration, and invasion and inhibit the apoptosis of cancer cells. Thus, inhibiting the function of TIPE3 is expected to be an effective strategy against cancer. The advancement of artificial intelligence (AI)-driven drug development has recently invigorated research in anti-cancer drug development. In this work, we incorporated DFCNN, Autodock Vina docking, DeepBindBC, MD, and metadynamics to efficiently identify inhibitors of TIPE3 from a ZINC compound dataset. Six potential candidates were selected for further experimental study to validate their anti-tumor activity. Among these, three small-molecule compounds (K784-8160, E745-0011, and 7238-1516) showed significant anti-tumor activity in vitro, leading to reduced tumor cell viability, proliferation, and migration and enhanced apoptotic tumor cell death. Notably, E745-0011 and 7238-1516 exhibited selective cytotoxicity toward tumor cells with high TIPE3 expression while having little or no effect on normal human cells or tumor cells with low TIPE3 expression. A molecular docking analysis further supported their interactions with TIPE3, highlighting hydrophobic interactions and their shared interaction residues and offering insights for designing more effective inhibitors. Taken together, this work demonstrates the feasibility of incorporating deep learning and MD simulations in virtual drug screening and provides inhibitors with significant potential for anti-cancer drug development against TIPE3-.

Novel Dihydropteridinone Derivatives As Potent Inhibitors of the Understudied Human Kinases Vaccinia-Related Kinase 1 and Casein Kinase 1δ/ε.

Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.

Design, synthesis and biological evaluation of 4-(indolin-1-yl)-6-substituted-pyrido[3,2-d]pyrimidine derivatives as Mnk1/2 inhibitors.

The Mnk-eIF4E axis plays a crucial role in tumor development, and inhibiting Mnk kinases is a promising approach for cancer therapy. Starting with fragment WS23, a series of 4-(indolin-1-yl)-6-substituted-pyrido[3,2-d]pyrimidine derivatives were designed and synthesized. Among these derivatives, compound 15b showed the highest potency with IC50 values of 0.8 and 1.5 nM against Mnk1 and Mnk2, respectively. Additionally, it demonstrated good selectivity among 30 selected kinases. 15b significantly suppressed MOLM-13 and K562 cell lines growth and caused cell cycle arrest. Furthermore, the Western blot assay revealed that 15b effectively downregulated the downstream proteins p-eIF4E, Mcl-1, and c-myc. Additionally, 15b exhibited remarkable stability in rat plasma and rat and human microsomes. In vivo anti-tumor activity study suggested that treatment with 15b suppressed tumor growth in LL/2 syngeneic models. These findings highlight the potential of 15b as a novel and potent Mnks inhibitor, which deserves further investigation.

Ribosome subunit attrition and activation of the p53-MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition.

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the 'WIN' site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

13-Oxyingenol-dodecanoate inhibits the growth of non-small cell lung cancer cells by targeting ULK1.

Lung cancer is the leading cause of cancer deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 80-85% of all lung cancers. Euphorbia kansui yielded 13-oxyingenol-dodecanoate (13OD), an ingenane-type diterpenoid, which had a strong cytotoxic effect on NSCLC cells. The underlying mechanism and potential target, however, remained unknown. The study found that 13OD effectively inhibited the cell proliferation and colony formation of NSCLC cells (A549 and H460 cells), with less toxicity in normal human lung epithelial BEAS-2B cells. Moreover, 13OD can cause mitochondrial dysfunction, and apoptosis in NSCLC cells. Mechanistically, the transcriptomics results showed that differential genes were mainly enriched in the mTOR and AMPK signaling pathways, which are closely related to cellular autophagy, the related indicators were subsequently validated. Additionally, bafilomycin A1 (Baf A1), an autophagy inhibitor, reversed the mitochondrial damage caused by 13OD. Furthermore, the Omics and Text-based Target Enrichment and Ranking (OTTER) method predicted ULK1 as a potential target of 13OD against NSCLC cells. This hypothesis was further confirmed using molecular docking, the cellular thermal shift assay (CETSA), and Western blot analysis. Remarkably, ULK1 siRNA inhibited 13OD's toxic activity in NSCLC cells. In line with these findings, 13OD was potent and non-toxic in the tumor xenograft model. Our findings suggested a possible mechanism for 13OD's role as a tumor suppressor and laid the groundwork for identifying targets for ingenane-type diterpenoids.

The Role of Intramolecular Reactions and Chemical Degradation in the Apparent Biotransformation Pathways of a Series of SYK Inhibitors.

In vitro metabolism studies of the spleen tyrosine kinase inhibitors AZ-A and AZ-B identified four unusual metabolites. M1 (mass-to-charge ratio 411) was formed by both molecules and was common to several analogs (AZ-C to AZ-H) sharing the same core structure, appearing to derive from the complete loss of a pendent 3,4-diaminotetrahydropyran ring and pyrazole ring cleavage resulting in a nonobvious metabolite. M2-M4 were formed by AZ-A and a subset of the other compounds only and apparently resulted from a sequential loss of H2 from parent. Initial attempts to isolate M3 for identification were unsuccessful due to sample degradation, and it was subsequently found that M2 and M3 underwent sequential chemical degradation steps to M4. M4 was successfully isolated and shown by mass spectrometry and NMR spectroscopy to be a tricyclic species incorporating the pyrazole and the 3,4-diaminotetrahydropyran groups. We propose that this arises from an intramolecular reaction between the primary amine on the tetrahydropyran and a putative epoxide intermediate on the adjacent pyrazole ring, evidence for which was generated in a ß-mercaptoethanol-trapping experiment. The loss of the tetrahydropyran moiety observed in M1 was found to be enhanced in an analog that was unable to undergo the intramolecular reaction step, leading us to propose two possible reaction pathways originating from the reactive intermediate. Ultimately, we conclude that the apparently complex and unusual metabolism of this series of compounds likely resulted from a single metabolic activation step forming an epoxide intermediate, which subsequently underwent intramolecular rearrangement and/or chemical degradation to form the final observed products. SIGNIFICANCE STATEMENT: The current work provides an unusual biotransformation example showing the potential for intramolecular reactions and chemical degradation to give the appearance of complex metabolism arising from a single primary route of metabolism.

N1-Benzoylated 5-(4-pyridinyl)indazole-based kinase inhibitors: Attaining haspin and Clk4 selectivity via modulation of the benzoyl substituents.

Haspin and Clk4 are both understudied protein kinases (PKs), offering potential targets for the development of new anticancer agents. Thus, the identification of new inhibitors targeting these PKs is of high interest. However, the inhibitors targeting haspin or Clk4 developed to date show a poor selectivity profile over other closely related PKs, increasing the risk of side effects. Herein, we present two newly developed N1-benzyolated 5-(4-pyridinyl)indazole-based inhibitors (18 and 19), derived from a newly identified indazole hit. These inhibitors exhibit an exceptional inhibitory profile toward haspin and/or Clk4. Compound 18 (2-acetyl benzoyl) showed a preference to inhibit Clk4 and haspin over a panel of closely related kinases, with sixfold selectivity for Clk4 (IC50 = 0.088 and 0.542 µM, respectively). Compound 19 (4-acetyl benzoyl) showed high selectivity against haspin over the common off-target kinases (Dyrks and Clks) with an IC50 of 0.155 µM for haspin. Molecular docking studies explained the remarkable selectivity of 18 and 19, elucidating how the new scaffold can be modified to toggle between inhibition of haspin or Clk4, despite the high homology of the ATP-binding sites. Their distinguished profile allows these compounds to be marked as interesting chemical probes to assess the selective inhibition of haspin and/or Clk4.

Mechanism of Resistance to the WDR5 Inhibitor in MLL-Rearranged Leukemia.

Drug resistance is a major problem often limiting the long-term effectiveness of targeted cancer therapeutics. Resistance can be acquired through mutations or amplification of the primary drug targets or activation of bypass signaling pathways. Considering the multifaceted function of WDR5 in human malignancies, WDR5 has emerged as an attractive drug target for the discovery of small-molecule inhibitors. In this study, we investigated if cancer cells might develop resistance to a highly potent WDR5 inhibitor. We established a drug-adapted cancer cell line and discovered that WDR5P173L mutation occurs in the resistant cells, which confers resistance by preventing target engagement of the inhibitor. This work elucidated the WDR5 inhibitor's potential resistance mechanism in a preclinical study as a reference for future study in the clinical stage.

Structure-based discovery of potent WD repeat domain 5 inhibitors that demonstrate efficacy and safety in preclinical animal models.

WD repeat domain 5 (WDR5) is a core scaffolding component of many multiprotein complexes that perform a variety of critical chromatin-centric processes in the nucleus. WDR5 is a component of the mixed lineage leukemia MLL/SET complex and localizes MYC to chromatin at tumor-critical target genes. As a part of these complexes, WDR5 plays a role in sustaining oncogenesis in a variety of human cancers that are often associated with poor prognoses. Thus, WDR5 has been recognized as an attractive therapeutic target for treating both solid and hematological tumors. Previously, small-molecule inhibitors of the WDR5-interaction (WIN) site and WDR5 degraders have demonstrated robust in vitro cellular efficacy in cancer cell lines and established the therapeutic potential of WDR5. However, these agents have not demonstrated significant in vivo efficacy at pharmacologically relevant doses by oral administration in animal disease models. We have discovered WDR5 WIN-site inhibitors that feature bicyclic heteroaryl P7 units through structure-based design and address the limitations of our previous series of small-molecule inhibitors. Importantly, our lead compounds exhibit enhanced on-target potency, excellent oral pharmacokinetic (PK) profiles, and potent dose-dependent in vivo efficacy in a mouse MV4:11 subcutaneous xenograft model by oral dosing. Furthermore, these in vivo probes show excellent tolerability under a repeated high-dose regimen in rodents to demonstrate the safety of the WDR5 WIN-site inhibition mechanism. Collectively, our results provide strong support for WDR5 WIN-site inhibitors to be utilized as potential anticancer therapeutics.

LACC1 contributes to inflammation and cognitive disorder after stroke via the AMPK/NLRP3 pathway.

The current study aimed to investigate the effects of LACC1 on cognitive disorder due to stroke, as well as its underlying mechanism. LACC1 promoted inflammation and aggravated cognitive impairment in a mouse model of stroke. In an in vitro model of stroke, inhibition of LACC1 reduced inflammation and ROS­induced oxidative stress by activating AMP­activated protein kinase (AMPK) expression and suppressing NLPR3 expression. Furthermore, our studies revealed that inhibition of AMPK activity reduced the effects of si­LACC1 on cognitive disorder in mice after stroke via the AMPK/NLPR3 pathway. AMPK activation also reduced the effects of LACC1 on inflammation and ROS­induced oxidative stress via the NLPR3 pathway in the in vitro model that we evaluated. Our study suggests that LACC1­aggravated inflammation causes cognitive impairment after stroke via the AMPK/NLRP3 pathway, which may provide a new therapeutic target for stroke and other neurological diseases and their associated complications. In sum, we identified an important role and regulatory mechanism for LACC1 in maintaining stroke­induced cognitive disorder via the AMPK/NLRP3 pathway.

Delta-like ligand 3-targeted radioimmunotherapy for neuroendocrine prostate cancer.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer with limited meaningful treatment options. NEPC lesions uniquely express delta-like ligand 3 (DLL3) on their cell surface. Taking advantage of DLL3 overexpression, we developed and evaluated lutetium-177 (177Lu)-labeled DLL3-targeting antibody SC16 (177Lu-DTPA-SC16) as a treatment for NEPC. SC16 was functionalized with DTPA-CHX-A" chelator and radiolabeled with 177Lu to produce 177Lu-DTPA-SC16. Specificity and selectivity of 177Lu-DTPA-SC16 were evaluated in vitro and in vivo using NCI-H660 (NEPC, DLL3-positive) and DU145 (adenocarcinoma, DLL3-negative) cells and xenografts. Dose-dependent treatment efficacy and specificity of 177Lu-DTPA-SC16 radionuclide therapy were evaluated in H660 and DU145 xenograft-bearing mice. Safety of the agent was assessed by monitoring hematologic parameters. 177Lu-DTPA-SC16 showed high tumor uptake and specificity in H660 xenografts, with minimal uptake in DU145 xenografts. At all three tested doses of 177Lu-DTPA-SC16 (4.63, 9.25, and 27.75 MBq/mouse), complete responses were observed in H660-bearing mice; 9.25 and 27.75 MBq/mouse doses were curative. Even the lowest tested dose proved curative in five (63%) of eight mice, and recurring tumors could be successfully re-treated at the same dose to achieve complete responses. In DU145 xenografts, 177Lu-DTPA-SC16 therapy did not inhibit tumor growth. Platelets and hematocrit transiently dropped, reaching nadir at 2 to 3 wk. This was out of range only in the highest-dose cohort and quickly recovered to normal range by week 4. Weight loss was observed only in the highest-dose cohort. Therefore, our data demonstrate that 177Lu-DTPA-SC16 is a potent and safe radioimmunotherapeutic agent for testing in humans with NEPC.

Discovery of a dual WDR5 and Ikaros PROTAC degrader as an anti-cancer therapeutic.

WD repeat domain 5 (WDR5), an integral component of the MLL/KMT2A lysine methyltransferase complex, is critically involved in oncogenesis and represents an attractive onco-target. Inhibitors targeting protein-protein interactions (PPIs) between WDR5 and its binding partners, however, do not inhibit all of WDR5-mediated oncogenic functions and exert rather limited antitumor effects. Here, we report a cereblon (CRBN)-recruiting proteolysis targeting chimera (PROTAC) of WDR5, MS40, which selectively degrades WDR5 and the well-established neo-substrates of immunomodulatory drugs (IMiDs):CRBN, the Ikaros zinc finger (IKZF) transcription factors IKZF1 and IKZF3. MS40-induced WDR5 degradation caused disassociation of the MLL/KMT2A complex off chromatin, resulting in decreased H3K4me2. Transcriptomic profiling revealed that targets of both WDR5 and IMiDs:CRBN were significantly repressed by treatment of MS40. In MLL-rearranged leukemias, which exhibit IKZF1 high expression and dependency, co-suppression of WDR5 and Ikaros by MS40 is superior in suppressing oncogenesis to the WDR5 PPI inhibitor, to MS40's non-PROTAC analog controls (MS40N1 and MS40N2, which do not bind CRBN and WDR5, respectively), and to a matched VHL-based WDR5 PROTAC (MS169, which degrades WDR5 but not Ikaros). MS40 suppressed the growth of primary leukemia patient cells in vitro and patient-derived xenografts in vivo. Thus, dual degradation of WDR5 and Ikaros is a promising anti-cancer strategy.

SINBAD, structural, experimental and clinical characterization of STAT inhibitors and their potential applications.

The abnormal activation of signal transducer and activator of transcription (STAT) protein family is recognized as cause or driving force behind multiple diseases progression. Therefore, searching for potential treatment strategy is pursued by multiple scientific groups. We consider that providing comprehensive, integrated and unified dataset for STAT inhibitory compounds may serve as important tool for other researchers. We developed SINBAD (STAT INhbitor Biology And Drug-ability) in response to our experience with inhibitory compound research, knowing that gathering detailed information is crucial for effective experiment design and also for finding potential solutions in case of obtaining inconclusive results. SINBAD is a curated database of STAT inhibitors which have been published and described in scientific articles providing prove of their inhibitory properties. It is a tool allowing easy analysis of experimental conditions and provides detailed information about known STAT inhibitory compounds.

Inhibition of VRK1 suppresses proliferation and migration of vascular smooth muscle cells and intima hyperplasia after injury via mTORC1/ß-catenin axis.

Characterized by abnormal proliferation and migration of vascular smooth muscle cells (VSMCs), neointima hyperplasia is a hallmark of vascular restenosis after percutaneous vascular interventions. Vaccinia-related kinase 1 (VRK1) is a stress adaptionassociated ser/thr protein kinase that can induce the proliferation of various types of cells. However, the role of VRK1 in the proliferation and migration of VSMCs and neointima hyperplasia after vascular injury remains unknown. We observed increased expression of VRK1 in VSMCs subjected to platelet-derived growth factor (PDGF)-BB by western blotting. Silencing VRK1 by shVrk1 reduced the number of Ki-67-positive VSMCs and attenuated the migration of VSMCs. Mechanistically, we found that relative expression levels of ß-catenin and effectors of mTOR complex 1 (mTORC1) such as phospho (p)-mammalian target of rapamycin (mTOR), p-S6, and p-4EBP1 were decreased after silencing VRK1. Restoration of ß-catenin expression by SKL2001 and re-activation of mTORC1 by Tuberous sclerosis 1 siRNA (siTsc1) both abolished shVrk1-mediated inhibitory effect on VSMC proliferation and migration. siTsc1 also rescued the reduced expression of ß-catenin caused by VRK1 inhibition. Furthermore, mTORC1 re-activation failed to recover the attenuated proliferation and migration of VSMC resulting from shVrk1 after silencing ß-catenin. We also found that the vascular expression of VRK1 was increased after injury. VRK1 inactivation in vivo inhibited vascular injury-induced neointima hyperplasia in a ß-catenin-dependent manner. These results demonstrate that inhibition of VRK1 can suppress the proliferation and migration of VSMC and neointima hyperplasia after vascular injury via mTORC1/ß-catenin pathway. [BMB Reports 2022; 55(5): 244-249].

Inhibiting SCAP/SREBP exacerbates liver injury and carcinogenesis in murine nonalcoholic steatohepatitis.

Enhanced de novo lipogenesis mediated by sterol regulatory element-binding proteins (SREBPs) is thought to be involved in nonalcoholic steatohepatitis (NASH) pathogenesis. In this study, we assessed the impact of SREBP inhibition on NASH and liver cancer development in murine models. Unexpectedly, SREBP inhibition via deletion of the SREBP cleavage-activating protein (SCAP) in the liver exacerbated liver injury, fibrosis, and carcinogenesis despite markedly reduced hepatic steatosis. These phenotypes were ameliorated by restoring SREBP function. Transcriptome and lipidome analyses revealed that SCAP/SREBP pathway inhibition altered the fatty acid (FA) composition of phosphatidylcholines due to both impaired FA synthesis and disorganized FA incorporation into phosphatidylcholine via lysophosphatidylcholine acyltransferase 3 (LPCAT3) downregulation, which led to endoplasmic reticulum (ER) stress and hepatocyte injury. Supplementation with phosphatidylcholines significantly improved liver injury and ER stress induced by SCAP deletion. The activity of the SCAP/SREBP/LPCAT3 axis was found to be inversely associated with liver fibrosis severity in human NASH. SREBP inhibition also cooperated with impaired autophagy to trigger liver injury. Thus, excessively strong and broad lipogenesis inhibition was counterproductive for NASH therapy; this will have important clinical implications in NASH treatment.

WIN site inhibition disrupts a subset of WDR5 function.

WDR5 nucleates the assembly of histone-modifying complexes and acts outside this context in a range of chromatin-centric processes. WDR5 is also a prominent target for pharmacological inhibition in cancer. Small-molecule degraders of WDR5 have been described, but most drug discovery efforts center on blocking the WIN site of WDR5, an arginine binding cavity that engages MLL/SET enzymes that deposit histone H3 lysine 4 methylation (H3K4me). Therapeutic application of WIN site inhibitors is complicated by the disparate functions of WDR5, but is generally guided by two assumptions-that WIN site inhibitors disable all functions of WDR5, and that changes in H3K4me drive the transcriptional response of cancer cells to WIN site blockade. Here, we test these assumptions by comparing the impact of WIN site inhibition versus WDR5 degradation on H3K4me and transcriptional processes. We show that WIN site inhibition disables only a specific subset of WDR5 activity, and that H3K4me changes induced by WDR5 depletion do not explain accompanying transcriptional responses. These data recast WIN site inhibitors as selective loss-of-function agents, contradict H3K4me as a relevant mechanism of action for WDR5 inhibitors, and indicate distinct clinical applications of WIN site inhibitors and WDR5 degraders.

Targeting the Non-Catalytic Functions: a New Paradigm for Kinase Drug Discovery?

Protein kinases have been highly fruitful targets for cancer drug discovery in the past two decades, while most of these drugs bind to the "adenosine triphosphate (ATP)-site" and inhibit kinase catalytic activity. Recently, accumulated evidence suggests that kinases possess functions beyond catalysis through their scaffolds, and the scaffolding functions could play critical roles in multiple cellular signaling and cell fate controls. Small molecules modulating the noncatalytic functions of kinases are rarely reported but emerge as new promising therapeutic strategies for various diseases. Herein, we summarize the characterized noncatalytic functions of kinases, and highlight the recent progress on developing small-molecule modulators of the noncatalytic functions of kinases. Mechanisms and characteristics of different kinds of modulators are also discussed. It is also speculated that targeting the noncatalytic functions would represent a new direction for kinase-based drug discovery.

Loss of TIPE3 reduced the proliferation, survival and migration of lung cancer cells through inactivation of Akt/mTOR, NF-κB, and STAT-3 signaling cascades.

Lung cancer is the foremost cause of cancer related mortality among men and one of the most fatal cancers among women. Notably, the 5-year survival rate of lung cancer is very low; 5% in developing countries. This low survival rate can be attributed to factors like late stage diagnosis, rapid postoperative recurrences in the patients undergoing treatment and development of chemoresistance against different agents used for treating lung cancer. Therefore, in this study we evaluated the potential of a recently identified protein namely TIPE3 which is known as a transfer protein of lipid second messengers as a lung cancer biomarker. TIPE3 was found to be significantly upregulated in lung cancer tissues indicating its role in the positive regulation of lung cancer. Supporting this finding, knockout of TIPE3 was also found to reduce the proliferation, survival and migration of lung cancer cells and arrested the G2 phase of cell cycle through inactivation of Akt/mTOR, NF-κB, STAT-3 signaling. It is well evinced that tobacco is the major risk factor of lung cancer which affects both males and females. Therefore, this study also evaluated the involvement of TIPE3 in tobacco mediated lung carcinogenesis. Notably, this study shows for the first time that TIPE3 positively regulates tobacco induced proliferation, survival and migration of lung cancer through modulation of Akt/mTOR signaling. Thus, TIPE3 plays critical role in the pathogenesis of lung cancer and hence it can be specifically targeted to develop novel therapeutic strategies.

miR-335-5p Inhibits Progression of Uterine Leiomyoma by Targeting ARGLU1.

Studies have demonstrated that miR-335-5p exhibits an essential role in the progress of multiple tumors, including thyroid cancer, pancreatic cancer, and non-small-cell lung cancer. However, the possible expression, the detailed role, and the underlying mechanisms of miR-335-5p in uterine leiomyoma (UL) still remained unclear. Therefore, the present study was designed to investigate the mechanism and function of miR-335-5p in UL. In our study, microRNA-335-5p (miR-335-5p) is significantly downregulated in UL tissues and UL cell lines, especially in HCC1688 and SK-UT-1 cells. Functionally, overexpression of miR-335-5p notably inhibits the viability of UL cell lines by CCK-8 assay. Besides, upregulation of miR-335-5p inhibits proliferation of UL cell lines by colony formation assay and decreases the protein levels of PCNA and Ki-67 detected by western blot assay. In addition, overexpression of miR-335-5p induces UL cell cycle arrest at G1 phase. Upregulation of miR-335-5p decreases the levels of Cyclin A1, Cyclin B1, and Cyclin D2 and upregulates the expression of p27 protein. Additionally, upregulation of miR-335-5p promotes the apoptosis of UL cell lines, increases the protein levels of Bax, Cleaved caspase-3, and Cleaved caspase-9, and decreases the protein expression of Bcl-2. Moreover, Arginine and Glutamate-Rich protein 1 (ARGLU1) is predicted as a target of miR-335-5p by ENCORI and miRDB and confirmed by dual-luciferase reporter assay. ARGLU1 is negatively associated with miR-335-5p. Furthermore, overexpression of ARGLU1 partly restores the effects of miR-335-5p mimic on the viability, proliferation, cell cycle, and apoptosis of UL cell lines. To conclude, miR-335-5p may play a repressive role in UL by targeting ARGLU1 and serve as a potential therapeutic target for the treatment of UL.