Live Cell Monitoring of Separase Activity, a Key Enzymatic Reaction for Chromosome Segregation, with Chimeric FRET-Based Molecular Sensor upon Cell Cycle Progression.

Separase is a key cysteine protease in the separation of sister chromatids through the digestion of the cohesin ring that inhibits chromosome segregation as a trigger of the metaphase-anaphase transition in eukaryotes. Its activity is highly regulated by binding with securin and cyclinB-CDK1 complex. These bindings prevent the proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties in biochemical examinations due to the instability of separase in vitro and the fact that few spatiotemporal resolution approaches exist for monitoring live separase activity throughout mitotic processes. Here, we have developed FRET-based molecular sensors, including GFP variants, with separase-cleavable sequences as donors and covalently attached fluorescent dyes as acceptor molecules. These are applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated the performance of equivalent molecular sensors, either localized or not localized inside the nucleus under cell cycle control, using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems that can be used specifically or statistically, which could lead to the elucidation of separase properties in detail.

Securin acetylation prevents precocious separase activation and premature sister chromatid separation.

Lysine acetylation of non-histone proteins plays crucial roles in many cellular processes. In this study, we examine the role of lysine acetylation during sister chromatid separation in mitosis. We investigate the acetylation of securin at K21 by cell-cycle-dependent acetylome analysis and uncover its role in separase-triggered chromosome segregation during mitosis. Prior to the onset of anaphase, the acetylated securin via TIP60 prevents its degradation by the APC/CCDC20-mediated ubiquitin-proteasome system. This, in turn, restrains precocious activation of separase and premature separation of sister chromatids. Additionally, the acetylation-dependent stability of securin is also enhanced by its dephosphorylation. As anaphase approaches, HDAC1-mediated deacetylation of securin promotes its degradation, allowing released separase to cleave centromeric cohesin. Blocking securin deacetylation leads to longer anaphase duration and errors in chromosome segregation. Thus, this study illustrates the emerging role of securin acetylation dynamics in mitotic progression and genetic stability.

Mild replication stress causes premature centriole disengagement via a sub-critical Plk1 activity under the control of ATR-Chk1.

A tight synchrony between the DNA and centrosome cycle is essential for genomic integrity. Centriole disengagement, which licenses centrosomes for duplication, occurs normally during mitotic exit. We recently demonstrated that mild DNA replication stress typically seen in cancer cells causes premature centriole disengagement in untransformed mitotic human cells, leading to transient multipolar spindles that favour chromosome missegregation. How mild replication stress accelerates the centrosome cycle at the molecular level remained, however, unclear. Using ultrastructure expansion microscopy, we show that mild replication stress induces premature centriole disengagement already in G2 via the ATR-Chk1 axis of the DNA damage repair pathway. This results in a sub-critical Plk1 kinase activity that primes the pericentriolar matrix for Separase-dependent disassembly but is insufficient for rapid mitotic entry, causing premature centriole disengagement in G2. We postulate that the differential requirement of Plk1 activity for the DNA and centrosome cycles explains how mild replication stress disrupts the synchrony between both processes and contributes to genomic instability.

Correlation Between Serum ESPL1 and Hepatitis B Virus-related Hepatocellular Carcinoma Histological Grade: A Chinese Single-center Case-control Study.

BACKGROUND/AIM: Serum markers to determine the histological grade of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) are still limited. This study aimed to investigate if serum extra spindle pole bodies-like 1 (ESPL1) protein could reflect the histological grade of HBV-related HCC. MATERIALS AND METHODS: A total of 154 patients with HBV-related HCC were enrolled in the experimental group and 41 non-HBV-related patients in the control. Enzyme-linked immunosorbent assay was used to detect serum ESPL1 levels. The differences in serological ESPL1, alpha-fetoprotein (AFP), and des-gamma-carboxy prothrombin (DCP) were compared between the two groups. HCC tumor diameter was measured, and pathological examination was performed to compare the relationship between ESPL1, AFP, and DCP and tumor size and histological grade. RESULTS: Serum AFP and DCP levels showed no significant difference between experimental group and control group, and increased when the tumor diameter increased but were not related to HCC histological grade. Serological ESPL1 levels were higher in the experimental group than those in the control group, and positively correlated with the histological grade. In the experimental group, tumor size and histological grade were almost independent (Kappa=0.000); patients with medium size tumors had the highest serum ESPL1 levels and the highest proportion of poorly differentiated carcinomas, whereas 75.6% of patients with small size tumors had moderately differentiated carcinomas and only 20% well differentiated carcinomas. CONCLUSION: Serum ESPL1 can reflect the malignant degree of HBV-related HCC and is helpful in identifying small size HCC tumors.

The molecular mechanisms of human separase regulation.

Sister chromatid segregation is the final irreversible step of mitosis. It is initiated by a complex regulatory system that ultimately triggers the timely activation of a conserved cysteine protease named separase. Separase cleaves the cohesin protein ring that links the sister chromatids and thus facilitates their separation and segregation to the opposite poles of the dividing cell. Due to the irreversible nature of this process, separase activity is tightly controlled in all eukaryotic cells. In this mini-review, we summarize the latest structural and functional findings on the regulation of separase, with an emphasis on the regulation of the human enzyme by two inhibitors, the universal inhibitor securin and the vertebrate-specific inhibitor CDK1-cyclin B. We discuss the two fundamentally different inhibitory mechanisms by which these inhibitors block separase activity by occluding substrate binding. We also describe conserved mechanisms that facilitate substrate recognition and point out open research questions that will guide studies of this fascinating enzyme for years to come.

An autonomous mathematical model for the mammalian cell cycle.

A mathematical model for the mammalian cell cycle is developed as a system of 13 coupled nonlinear ordinary differential equations. The variables and interactions included in the model are based on detailed consideration of available experimental data. A novel feature of the model is inclusion of cycle tasks such as origin licensing and initiation, nuclear envelope breakdown and kinetochore attachment, and their interactions with controllers (molecular complexes involved in cycle control). Other key features are that the model is autonomous, except for a dependence on external growth factors; the variables are continuous in time, without instantaneous resets at phase boundaries; mechanisms to prevent rereplication are included; and cycle progression is independent of cell size. Eight variables represent cell cycle controllers: the Cyclin D1-Cdk4/6 complex, APCCdh1, SCFßTrCP, Cdc25A, MPF, NuMA, the securin-separase complex, and separase. Five variables represent task completion, with four for the status of origins and one for kinetochore attachment. The model predicts distinct behaviors corresponding to the main phases of the cell cycle, showing that the principal features of the mammalian cell cycle, including restriction point behavior, can be accounted for in a quantitative mechanistic way based on known interactions among cycle controllers and their coupling to tasks. The model is robust to parameter changes, in that cycling is maintained over at least a five-fold range of each parameter when varied individually. The model is suitable for exploring how extracellular factors affect cell cycle progression, including responses to metabolic conditions and to anti-cancer therapies.

Pan-Cancer analysis and experimental validation identify the oncogenic nature of ESPL1: Potential therapeutic target in colorectal cancer.

Introduction: Extra spindle pole bodies like 1 (ESPL1) are required to continue the cell cycle, and its primary role is to initiate the final segregation of sister chromatids. Although prior research has revealed a link between ESPL1 and the development of cancer, no systematic pan-cancer analysis has been conducted. Combining multi-omics data with bioinformatics, we have thoroughly described the function of ESPL1 in cancer. In addition, we examined the impact of ESPL1 on the proliferation of numerous cancer cell lines. In addition, the connection between ESPL1 and medication sensitivity was verified using organoids obtained from colorectal cancer patients. All these results confirm the oncogene nature of ESPL1. Methods: Herein, we downloaded raw data from numerous publicly available databases and then applied R software and online tools to explore the association of ESPL1 expression with prognosis, survival, tumor microenvironment, tumor heterogeneity, and mutational profiles. To validate the oncogene nature of ESPL1, we have performed a knockdown of the target gene in various cancer cell lines to verify the effect of ESPL1 on proliferation and migration. In addition, patients' derived organoids were used to verify drug sensitivity. Results: The study found that ESPL1 expression was markedly upregulated in tumorous tissues compared to normal tissues, and high expression of ESPL1 was significantly associated with poor prognosis in a range of cancers. Furthermore, the study revealed that tumors with high ESPL1 expression tended to be more heterogeneous based on various tumor heterogeneity indicators. Enrichment analysis showed that ESPL1 is involved in mediating multiple cancer-related pathways. Notably, the study found that interference with ESPL1 expression significantly inhibited the proliferation of tumor cells. Additionally, the higher the expression of ESPL1 in organoids, the greater the sensitivity to PHA-793887, PAC-1, and AZD7762. Discussion: Taken together, our study provides evidence that ESPL1 may implicate tumorigenesis and disease progression across multiple cancer types, highlighting its potential utility as both a prognostic indicator and therapeutic target.

Separase and Roads to Disengage Sister Chromatids during Anaphase.

Receiving complete and undamaged genetic information is vital for the survival of daughter cells after chromosome segregation. The most critical steps in this process are accurate DNA replication during S phase and a faithful chromosome segregation during anaphase. Any errors in DNA replication or chromosome segregation have dire consequences, since cells arising after division might have either changed or incomplete genetic information. Accurate chromosome segregation during anaphase requires a protein complex called cohesin, which holds together sister chromatids. This complex unifies sister chromatids from their synthesis during S phase, until separation in anaphase. Upon entry into mitosis, the spindle apparatus is assembled, which eventually engages kinetochores of all chromosomes. Additionally, when kinetochores of sister chromatids assume amphitelic attachment to the spindle microtubules, cells are finally ready for the separation of sister chromatids. This is achieved by the enzymatic cleavage of cohesin subunits Scc1 or Rec8 by an enzyme called Separase. After cohesin cleavage, sister chromatids remain attached to the spindle apparatus and their poleward movement on the spindle is initiated. The removal of cohesion between sister chromatids is an irreversible step and therefore it must be synchronized with assembly of the spindle apparatus, since precocious separation of sister chromatids might lead into aneuploidy and tumorigenesis. In this review, we focus on recent discoveries concerning the regulation of Separase activity during the cell cycle.

Hexavalent Chromium Targets Securin to Drive Numerical Chromosome Instability in Human Lung Cells.

Hexavalent chromium [Cr(VI)] is a known human lung carcinogen with widespread exposure in environmental and occupational settings. Despite well-known cancer risks, the molecular mechanisms of Cr(VI)-induced carcinogenesis are not well understood, but a major driver of Cr(VI) carcinogenesis is chromosome instability. Previously, we reported Cr(VI) induced numerical chromosome instability, premature centriole disengagement, centrosome amplification, premature centromere division, and spindle assembly checkpoint bypass. A key regulator of these events is securin, which acts by regulating the cleavage ability of separase. Thus, in this study we investigated securin disruption by Cr(VI) exposure. We exposed human lung cells to a particulate Cr(VI) compound, zinc chromate, for acute (24 h) and prolonged (120 h) time points. We found prolonged Cr(VI) exposure caused marked decrease in securin levels and function. After prolonged exposure at the highest concentration, securin protein levels were decreased to 15.3% of control cells, while securin mRNA quantification was 7.9% relative to control cells. Additionally, loss of securin function led to increased separase activity manifested as enhanced cleavage of separase substrates; separase, kendrin, and SCC1. These data show securin is targeted by prolonged Cr(VI) exposure in human lung cells. Thus, a new mechanistic model for Cr(VI)-induced carcinogenesis emerges with centrosome and centromere disruption as key components of numerical chromosome instability, a key driver in Cr(VI) carcinogenesis.

Homologous chromosomes are stably conjoined for Drosophila male meiosis I by SUM, a multimerized protein assembly with modules for DNA-binding and for separase-mediated dissociation co-opted from cohesin.

For meiosis I, homologous chromosomes must be paired into bivalents. Maintenance of homolog conjunction in bivalents until anaphase I depends on crossovers in canonical meiosis. However, instead of crossovers, an alternative system achieves homolog conjunction during the achiasmate male meiosis of Drosophila melanogaster. The proteins SNM, UNO and MNM are likely constituents of a physical linkage that conjoins homologs in D. melanogaster spermatocytes. Here, we report that SNM binds tightly to the C-terminal region of UNO. This interaction is homologous to that of the cohesin subunits stromalin/Scc3/STAG and α-kleisin, as revealed by sequence similarities, structure modeling and cross-link mass spectrometry. Importantly, purified SU_C, the heterodimeric complex of SNM and the C-terminal region of UNO, displayed DNA-binding in vitro. DNA-binding was severely impaired by mutational elimination of positively charged residues from the C-terminal helix of UNO. Phenotypic analyses in flies fully confirmed the physiological relevance of this basic helix for chromosome-binding and homolog conjunction during male meiosis. Beyond DNA, SU_C also bound MNM, one of many isoforms expressed from the complex mod(mdg4) locus. This binding of MNM to SU_C was mediated by the MNM-specific C-terminal region, while the purified N-terminal part common to all Mod(mdg4) isoforms multimerized into hexamers in vitro. Similarly, the UNO N-terminal domain formed tetramers in vitro. Thus, we suggest that multimerization confers to SUM, the assemblies composed of SNM, UNO and MNM, the capacity to conjoin homologous chromosomes stably by the resultant multivalent DNA-binding. Moreover, to permit homolog separation during anaphase I, SUM is dissociated by separase, since UNO, the α-kleisin-related protein, includes a separase cleavage site. In support of this proposal, we demonstrate that UNO cleavage by tobacco etch virus protease is sufficient to release homolog conjunction in vivo after mutational exchange of the separase cleavage site with that of the bio-orthogonal protease.

CENP-F-dependent DRP1 function regulates APC/C activity during oocyte meiosis I.

Chromosome segregation is initiated by cohesin degradation, which is driven by anaphase-promoting complex/cyclosome (APC/C). Chromosome cohesin is removed by activated separase, with the degradation of securin and cyclinB1. Dynamin-related protein 1 (DRP1), a component of the mitochondrial fission machinery, is related to cyclin dynamics in mitosis progression. Here, we show that DRP1 is recruited to the kinetochore by centromeric Centromere protein F (CENP-F) after nuclear envelope breakdown in mouse oocytes. Loss of DRP1 during prometaphase leads to premature cohesin degradation and chromosome segregation. Importantly, acute DRP1 depletion activates separase by initiating cyclinB1 and securin degradation during the metaphase-to-anaphase transition. Finally, we demonstrate that DRP1 is bound to APC2 to restrain the E3 ligase activity of APC/C. In conclusion, DRP1 is a CENP-F-dependent atypical spindle assembly checkpoint (SAC) protein that modulates metaphase-to-anaphase transition by controlling APC/C activity during meiosis I in oocytes.

Separase Control and Cohesin Cleavage in Oocytes: Should I Stay or Should I Go?

The key to gametogenesis is the proper execution of a specialized form of cell division named meiosis. Prior to the meiotic divisions, the recombination of maternal and paternal chromosomes creates new genetic combinations necessary for fitness and adaptation to an ever-changing environment. Two rounds of chromosome segregation -meiosis I and II- have to take place without intermediate S-phase and lead to the creation of haploid gametes harboring only half of the genetic material. Importantly, the segregation patterns of the two divisions are fundamentally different and require adaptation of the mitotic cell cycle machinery to the specificities of meiosis. Separase, the enzyme that cleaves Rec8, a subunit of the cohesin complex constituting the physical connection between sister chromatids, has to be activated twice: once in meiosis I and immediately afterwards, in meiosis II. Rec8 is cleaved on chromosome arms in meiosis I and in the centromere region in meiosis II. This step-wise cohesin removal is essential to generate gametes of the correct ploidy and thus, embryo viability. Hence, separase control and Rec8 cleavage must be perfectly controlled in time and space. Focusing on mammalian oocytes, this review lays out what we know and what we still ignore about this fascinating mechanism.

Autocleavage of separase suppresses its premature activation by promoting binding to cyclin B1.

Accurate chromosome segregation requires timely activation of separase, a protease that cleaves cohesin during the metaphase-to-anaphase transition. However, the mechanism that maintains the inactivity of separase prior to this event remains unclear. We provide evidence that separase autocleavage plays an essential role in this process. We show that the inhibition of separase autocleavage results in premature activity before the onset of anaphase, accompanied by the formation of chromosomal bridges and spindle rocking. This deregulation is attributed to the reduced binding of cyclin B1 to separase that occurs during the metaphase-to-anaphase transition. Furthermore, when separase is mutated to render the regulation by cyclin B1 irrelevant, which keeps separase in securin-binding form, the deregulation induced by autocleavage inhibition is rescued. Our results reveal a physiological role of separase autocleavage in regulating separase, which ensures faithful chromosome segregation.

Depletion or cleavage of cohesin during anaphase differentially affects chromatin structure and segregation.

Chromosome segregation requires both the separation of sister chromatids and the sustained condensation of chromatids during anaphase. In yeast cells, cohesin is not only required for sister chromatid cohesion but also plays a major role determining the structure of individual chromatids in metaphase. Separase cleavage is thought to remove all cohesin complexes from chromosomes to initiate anaphase. It is thus not clear how the length and organisation of segregating chromatids is maintained during anaphase in the absence of cohesin. Here, we show that degradation of cohesin at the anaphase onset causes aberrant chromatid segregation. Hi-C analysis on segregating chromatids demonstrates that cohesin depletion causes loss of intrachromatid organisation. Surprisingly, tobacco etch virus (TEV)-mediated cleavage of cohesin does not dramatically disrupt chromatid organisation in anaphase, explaining why bulk segregation is achieved. In addition, we identified a small pool of cohesin complexes bound to telophase chromosomes in wild-type cells and show that they play a role in the organisation of centromeric regions. Our data demonstrates that in yeast cells cohesin function is not over in metaphase, but extends to the anaphase period when chromatids are segregating.

Protocol to measure cleavage efficiency of the meiotic cohesin subunit Rec8 by separase in mouse oocytes using a biosensor.

Here, we describe a biosensor to assess meiotic cohesin subunit Rec8 cleavage in mouse oocytes. We detail oocyte collection and microinjection of the mRNA expressing the biosensor. The biosensor is targeted to chromosomes and consists of two fluorophores flanking a Rec8 fragment containing separase cleavage sites. Cleavage leads to dissociation of one fluorophore from chromosomes, and the efficiency can be estimated by live imaging. We detail the use of this biosensor in mouse oocytes with or without Aurora B/C inhibitor. For complete details on the use and execution of this protocol, please refer to Nikalayevich et al. (2022).

Characterization of Transcriptional Responses to Genomovirus Infection of the White Mold Fungus, Sclerotinia sclerotiorum.

Soybean leaf-associated gemygorvirus-1 (SlaGemV-1) is a CRESS-DNA virus classified in the family Genomoviridae, which causes hypovirulence and abolishes sclerotia formation in infected fungal pathogens under the family Sclerotiniaceae. To investigate the mechanisms involved in the induction of hypovirulence, RNA-Seq was compared between virus-free and SlaGemV-1-infected Sclerotinia sclerotiorum strain DK3. Overall, 4639 genes were differentially expressed, with 50.5% up regulated and 49.5% down regulated genes. GO enrichments suggest changes in integral membrane components and transmission electron microscopy images reveal virus-like particles localized near the inner cell membrane. Differential gene expression analysis focused on genes responsible for cell cycle and DNA replication and repair pathways, ubiquitin proteolysis, gene silencing, methylation, pathogenesis-related, sclerotial development, carbohydrate metabolism, and oxalic acid biosynthesis. Carbohydrate metabolism showed the most changes, with two glycoside hydrolase genes being the most down regulated by -2396.1- and -648.6-fold. Genes relating to pathogenesis showed consistent down regulation with the greatest being SsNep1, SsSSVP1, and Endo2 showing, -4555-, -14.7-, and -12.3-fold changes. The cell cycle and DNA replication/repair pathways were almost entirely up regulated including a putative cyclin and separase being up regulated 8.3- and 5.2-fold. The oxalate decarboxylase genes necessary for oxalic acid catabolism and oxalic acid precursor biosynthesis genes and its metabolism show down regulations of -17.2- and -12.1-fold changes. Sclerotial formation genes also appear differentially regulated including a melanin biosynthesis gene Pks1 and a sclerotia formation gene Sl2 with fold changes of 3.8 and -2.9.

Recurrent but Short-Lived Duplications of Centromeric Proteins in Holocentric Caenorhabditis Species.

Centromeric histones (CenH3s) are essential for chromosome inheritance during cell division in most eukaryotes. CenH3 genes have rapidly evolved and undergone repeated gene duplications and diversification in many plant and animal species. In Caenorhabditis species, two independent duplications of CenH3 (named hcp-3 for HoloCentric chromosome-binding Protein 3) were previously identified in C. elegans and C. remanei. Using phylogenomic analyses in 32 Caenorhabditis species, we find strict retention of the ancestral hcp-3 gene and 10 independent duplications. Most hcp-3L (hcp-3-like) paralogs are only found in 1-2 species, are expressed in both males and females/hermaphrodites, and encode histone fold domains with 69-100% identity to ancestral hcp-3. We identified novel N-terminal protein motifs, including putative kinetochore protein-interacting motifs and a potential separase cleavage site, which are well conserved across Caenorhabditis HCP-3 proteins. Other N-terminal motifs vary in their retention across paralogs or species, revealing potential subfunctionalization or functional loss following duplication. An N-terminal extension in the hcp-3L gene of C. afra revealed an unprecedented protein fusion, where hcp-3L fused to duplicated segments from hcp-4 (nematode CENP-C). By extending our analyses beyond CenH3, we found gene duplications of six inner and outer kinetochore genes in Caenorhabditis, which appear to have been retained independent of hcp-3 duplications. Our findings suggest that centromeric protein duplications occur frequently in Caenorhabditis nematodes, are selectively retained for short evolutionary periods, then degenerate or are lost entirely. We hypothesize that unique challenges associated with holocentricity in Caenorhabditis may lead to this rapid "revolving door" of kinetochore protein paralogs.

Identification of a novel substrate motif of yeast separase and deciphering the recognition specificity using AlphaFold2 and molecular dynamics simulation.

Separase is a giant cysteine protease and has multiple crucial functions. The most well-known substrate of separase is the kleisin subunit of cohesin, the cleavage of which triggers chromosome segregation during cell division (Uhlmann et al., 1999; Kamenz and Hauf, 2016) [1,2]. Recently, separase has also been found to cleave MCL-1 or BCL-XL proteins to trigger apoptosis (Hellmuth and Stemmann, 2020) [3]. Although substrate recognition through a short sequence right upstream of the cleavage site is well established, recent studies suggested that sequence elements outside this minimum cleavage site are required for optimal cleavage activity and specificity (Rosen et al., 2019; Uhlmann et al., 2000) [4,5]. However, the sequences and their underlying mechanism are largely unknown. To further explore the substrate determinants and recognition mechanism, we carried out sequence alignments and found a conserved motif downstream of the cleavage site in budding yeast. Using Alphafold2 and molecular dynamics simulations, we found this motif is recognized by separase in a conserved cleft near the binding groove of its inhibitor securin. Their binding is mutually exclusive and requires conformation changes of separase. These findings provide deeper insights into substrate recognition and activation of separase, and paved the way for discovering more substrates of separase.

MicroRNA-202 safeguards meiotic progression by preventing premature SEPARASE-mediated REC8 cleavage.

MicroRNAs (miRNAs) are believed to play important roles in mammalian spermatogenesis but the in vivo functions of single miRNAs in this highly complex developmental process remain unclear. Here, we report that miR-202, a member of the let-7 family, plays an important role in spermatogenesis by phenotypic evaluation of miR-202 knockout (KO) mice. Loss of miR-202 results in spermatocyte apoptosis and perturbation of the zygonema-to-pachynema transition. Multiple processes during meiosis prophase I including synapsis and crossover formation are disrupted, and inter-sister chromatid synapses are detected. Moreover, we demonstrate that Separase mRNA is a miR-202 direct target and provides evidence that miR-202 upregulates REC8 by repressing Separase expression. Therefore, we have identified miR-202 as a new regulating noncoding gene that acts on the established SEPARASE-REC8 axis in meiosis.

Let-7c-5p Restrains Cell Growth and Induces Apoptosis of Lung Adenocarcinoma Cells via Targeting ESPL1.

Lung adenocarcinoma (LUAD) is a predominant malignancy, and its high mortality prompts us to incessantly probe the relevant targeted treatment. This work intended to study the molecular mechanism of ESPL1 in LUAD. Bioinformatics analysis was performed for pan-cancer and prognosis analysis as well as target gene prediction. Expression of ESPL1 mRNA and let-7c-5p was determined via qRT-PCR, and western blot was employed to detect protein level of ESPL1. Dual-luciferase reporter gene method verified the interaction between ESPL1 and let-7c-5p. Thereafter, CCK-8, wound healing, Transwell, and flow cytometry assays were utilized to investigate proliferation, migration, and apoptosis of LUAD cells. The results revealed that ESPL1 was upregulated in LUAD, which was associated with poor prognosis. Overexpressed ESPL1 promoted LUAD cells to invade, proliferate, and migrate. Furthermore, ESPL1 was a target gene of let-7c-5p. Let-7c-5p was downregulated in LUAD cells, and played a suppressive role in LUAD malignant development, while reversed by ESPL1. Taken together, it was posited that let-7c-5p/ESPL1 may be underlying therapeutic targets of LUAD.