MiR-107-3p Knockdown Alleviates Endothelial Injury in Sepsis via Kallikrein-Related Peptidase 5.

INTRODUCTION: Endothelial injury is a major characteristic of sepsis and contributes to sepsis-induced multiple-organ dysfunction. In this study, we investigated the role of miR-107-3p in sepsis-induced endothelial injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to 20 µg/mL of lipopolysaccharide (LPS) for 6-48 h. The levels of miR-107-3p and kallikrein-related peptidase 5 (KLK5) were examined. HUVECs were treated with LPS for 12 h and subsequently transfected with miR-107-3p inhibitor, KLK5 siRNA, or cotransfected with KLK5 siRNA and miR-107-3p inhibitor/negative control inhibitor. Cell survival, apoptosis, invasion, cell permeability, inflammatory response, and the Toll-like receptor 4/nuclear factor κB signaling were evaluated. In addition, the relationship between miR-107-3p and KLK5 expression was predicted and verified. RESULTS: LPS significantly elevated miR-107-3p levels, which peaked at 12 h. Conversely, the KLK5 level was lower in the LPS group than in the control group and was lowest at 12 h. MiR-107-3p knockdown significantly attenuated reductions in cell survival and invasion, apoptosis promotion, hyperpermeability and inflammation induction, and activation of the NF-κB signaling caused by LPS. KLK5 knockdown had the opposite effect. Additionally, KLK5 was demonstrated as a target of miR-107-3p. MiR-107-3p knockdown partially reversed the effects of KLK5 depletion in LPS-activated HUVECs. CONCLUSIONS: Our findings indicate that miR-107-3p knockdown may protect against sepsis-induced endothelial cell injury by targeting KLK5. This study identified a novel therapeutic target for sepsis-induced endothelial injury.

Genome-wide association study identifies kallikrein 5 in type 2 inflammation-low asthma.

BACKGROUND: Clinical studies of type 2 (T2) cytokine-related neutralizing antibodies in asthma have identified a substantial subset of patients with low levels of T2 inflammation who do not benefit from T2 cytokine neutralizing antibody treatment. Non-T2 mechanisms are poorly understood in asthma but represent a redefined unmet medical need. OBJECTIVE: We sought to gain a better understanding of genetic contributions to T2-low asthma. METHODS: We utilized an unbiased genome-wide association study of patients with moderate to severe asthma stratified by T2 serum biomarker periostin. We also performed additional expression and biological analysis for the top genetic hits. RESULTS: We identified a novel protective single nucleotide polymorphism at chr19q13.41, which is selectively associated with T2-low asthma and establishes Kallikrein-related peptidase 5 (KLK5) as the causal gene mediating this association. Heterozygous carriers of the single nucleotide polymorphisms have reduced KLK5 expression. KLK5 is secreted by human bronchial epithelial cells and elevated in asthma bronchial alveolar lavage. T2 cytokines IL-4 and IL-13 downregulate KLK5 in human bronchial epithelial cells. KLK5, dependent on its catalytic function, induces epithelial chemokine/cytokine expression. Finally, overexpression of KLK5 in airway or lack of an endogenous KLK5 inhibitor, SPINK5, leads to spontaneous airway neutrophilic inflammation. CONCLUSION: Our data identify KLK5 to be the causal gene at a novel locus at chr19q13.41 associated with T2-low asthma.

Cocktails of KLK5 Protease Inhibitors and Anti-TNFα Therapeutics: an Effective Treatment for Netherton Syndrome.

Netherton syndrome (NS) is a rare, severe type of ichthyosis, often lethal in neonates, for which there is no therapy. Spink5-/- mice recapitulate major NS hallmarks and die homogeneously within 5 h from birth due to severe epidermal barrier defect leading to dehydration. Spink5-/-Klk5-/- mice survive neonatal lethality, indicating that KLK5 could be a drug target for NS. Nevertheless, after a week, these mice developed epidermal inflammation and signs of barrier defect leading to lethality. Here we tested whether anti-TNFα strategy in combination with anti-KLK5 could provide a long-term effective therapy for NS. Deletion of Tnfa in Spink5-/- suppressed the inflammatory phenotype but did not rescue neonatal lethality of Spink5-/- indicating that anti-TNFα therapy alone would not be sufficient to treat NS. Interestingly, in Spink5-/-Klk5-/-Tnfa-/- mice, NS features were rescued, and mice lived normally for 16-18 months. For the first time, evidence is provided that a combination of anti-TNFα and anti-KLK5 therapeutics represents an effective therapeutic strategy for NS. Notably, anti-TNFα factors are marketed and used widely, while LMW KLK5 inhibitors are being developed.

STAT3 maintains skin barrier integrity by modulating SPINK5 and KLK5 expression in keratinocytes.

Skin barrier dysfunction induces skin inflammation. Signal transducer and activator of transcription 3 (STAT3) is known to be involved in Th17-mediated immune responses and barrier integrity in the cornea and intestine; however, its role in the skin barrier remains largely unknown. In this study, we elucidated the potential role of STAT3 in the skin barrier and its effect on kallikrein-related peptidase 5 (KLK5) and serine protease inhibitor Kazal-type 5 (SPINK5) expression using a mouse model with keratinocyte-specific ablation of STAT3. Keratinocyte-specific loss of STAT3 induced a cutaneous inflammatory phenotype with pruritus and intense scratching behaviour in mice. Transcriptomic analysis revealed that the genes associated with impaired skin barrier function, including KLK5, were upregulated. The effect of STAT3 on KLK5 expression in keratinocytes was not only substantiated by the increase in KLK5 expression following treatment with STAT3 siRNA but also by its decreased expression following STAT3 overexpression. Overexpression and IL-17A-mediated stimulation of STAT3 increased the expression of SPINK5, which was blocked by STAT3 siRNA. These results suggest that the expression of SPINK5 and KLK5 in keratinocytes could be dependent on STAT3 and that STAT3 might play an essential role in the maintenance of skin barrier homeostasis.

Identification of MFGE8 and KLK5/7 as mediators of breast tumorigenesis and resistance to COX-2 inhibition.

BACKGROUND: Cyclooxygenase 2 (COX-2) promotes stemness in triple negative breast cancer (TNBC), highlighting COX-2 as a promising therapeutic target in these tumors. However, to date, clinical trials using COX-2 inhibitors in breast cancer only showed variable patient responses with no clear significant clinical benefits, suggesting underlying molecular mechanisms contributing to resistance to COX-2 inhibitors. METHODS: By combining in silico analysis of human breast cancer RNA-seq data with interrogation of public patient databases and their associated transcriptomic, genomic, and clinical profiles, we identified COX-2 associated genes whose expression correlate with aggressive TNBC features and resistance to COX-2 inhibitors. We then assessed their individual contributions to TNBC metastasis and resistance to COX-2 inhibitors, using CRISPR gene knockout approaches in both in vitro and in vivo preclinical models of TNBC. RESULTS: We identified multiple COX-2 associated genes (TPM4, RGS2, LAMC2, SERPINB5, KLK7, MFGE8, KLK5, ID4, RBP1, SLC2A1) that regulate tumor lung colonization in TNBC. Furthermore, we found that silencing MFGE8 and KLK5/7 gene expression in TNBC cells markedly restored sensitivity to COX-2 selective inhibitor both in vitro and in vivo. CONCLUSIONS: Together, our study supports the establishment and use of novel COX-2 inhibitor-based combination therapies as future strategies for TNBC treatment.

Search for Non-Protein Protease Inhibitors Constituted with an Indole and Acetylene Core.

A possible inhibitor of proteases, which contains an indole core and an aromatic polar acetylene, was designed and synthesized. This indole derivative has a molecular architecture kindred to biologically relevant species and was obtained through five synthetic steps with an overall yield of 37% from the 2,2'-(phenylazanediyl)di(ethan-1-ol). The indole derivative was evaluated through docking assays using the main protease (SARS-CoV-2-Mpro) as a molecular target, which plays a key role in the replication process of this virus. Additionally, the indole derivative was evaluated as an inhibitor of the enzyme kallikrein 5 (KLK5), which is a serine protease that can be considered as an anticancer drug target.

Topical doxycycline monohydrate hydrogel 1% targeting proteases/PAR2 pathway is a novel therapeutic for atopic dermatitis.

Atopic Dermatitis (AD) is characterized by skin barrier disruption and an aberrant immune response. Doxycycline is tetracycline antibiotics broadly used systemically to treat inflammatory dermatologic conditions. Several studies have shown doxycycline has anti-inflammatory and pro-healing properties, mainly by blocking tissue proteolytic activity. It is our hypothesis that daily application of a novel doxycycline topical formulation in AD subjects will reduce severity of the disease, by blocking cutaneous proteases activity and restoring skin barrier function and inflammation. To test this hypothesis, we performed a proof of concept, open-label clinical study. Subjects enrolled in the study (n = 15) applied NanoDOX® Hydrogel 1% daily for 4 weeks on a chosen eczematous area. Investigational drug was well tolerated, and no local or systemic adverse events due to investigational drug were reported. Notably, a significant clinical improvement was observed based on a modified Eczema Area & Severity Index (EASI) score of the treated area from start of treatment to 14 and 28 days post-treatment (P < .001). A significant improvement of pruritus was also observed (P = .02). This proof of concept clinical trial is first to explore the impact of a non-systemic doxycycline treatment on AD patients. Our results provide evidence to investigate novel AD treatment strategies targeting cutaneous proteases activity.

Exonic mutations associated with atopic dermatitis disrupt lympho-epithelial Kazal-type related inhibitor action and enhance its degradation.

BACKGROUND: Skin desquamation is facilitated by serine proteases KLK5 and KLK7, which are tightly regulated by lympho-epithelial Kazal-type related inhibitor (LEKTI). LEKTI itself is controlled through degraded by mesotrypsin. Here, we sought to determine whether LEKTI exonic mutations associated with atopic dermatitis (AD) affect the protease inhibitory activity of LEKTI or its susceptibility to mesotrypsin degradation. METHODS: The inhibitory activities of the LEKTI domain 4 (D4) and D6 WT and AD-associated mutants on the enzyme activities of KLK5 and KLK7 were compared using fluorogenic substrates. A keratinocyte cell culture system using HaCat cells was established to assess the role of D6 WT and D386N on triggering inflammation via the induction of thymic stromal lymphopoietin (TSLP). A degradation assay was used to assess the susceptibility of D4 and D6 mutants to mesotrypsin degradation. RESULTS: Enzymatic assays revealed that the D6 D386N mutation affected the inhibitory activity of LEKTI on KLK5 but not KLK7. Other exonic mutations on D6 (N368S, V395M, and E420K) and D4 (R267Q) did not alter LEKTI inhibition. The D386N mutation disrupted the role of D6 in suppressing TSLP induction by KLK5 in HaCat cells. Although WT D4 is more susceptible to mesotrypsin degradation than WT D6, the D4 R267Q mutant was more resistant to mesotrypsin degradation, whereas the D6 E420K mutant showed enhanced mesotrypsin-mediated degradation. CONCLUSION: Exonic mutations in D6, which previously have been associated with AD, may cause a disruption of inhibitory activity on KLK5 or enhance the degradation by mesotrypsin.

Quantitative assessment and clinical relevance of kallikrein-related peptidase 5 mRNA expression in advanced high-grade serous ovarian cancer.

BACKGROUND: In ovarian cancer, dysregulation of mRNA expression of several components of the family of the kallikrein-related peptidases (KLKs) is observed. In this study, we have analyzed the KLK5 mRNA expression pattern in tumor tissue of patients suffering from high-grade serous ovarian cancer stage FIGO III/IV. Moreover, we have correlated the KLK5 mRNA levels with clinical outcome. METHODS: We assessed the mRNA expression levels of KLK5 in tumor tissue of 138 patients using quantitative PCR (qPCR). The mRNA levels were correlated with KLK5 antigen tumor tissue levels measured by ELISA (available for 41 of the 138 patients), established clinical features as well as patients' outcome, using Chi-square-tests, Mann-Whitney U-tests and Spearman rank calculations as well as Cox regression models, Kaplan-Meier survival analysis and the log-rank test. RESULTS: A highly significant correlation between the mRNA expression levels and protein levels of KLK5 in tumor tissues was observed (rs = 0.683, p < 0.001). In univariate Cox regression analysis, elevated KLK5 mRNA expression was remarkably associated with reduced progression-free survival (PFS; p = 0.047), but not with overall survival (OS). Association of KLK5 mRNA expression with PFS was validated in silico using The Cancer Genome Atlas. For this, Affymetrix-based mRNA data (n = 377) were analyzed applying the Kaplan-Meier Plotter tool (p = 0.027). In multivariable Cox analysis, KLK5 mRNA values revealed a trend towards statistical significance for PFS (p = 0.095), whereas residual tumor mass (0 mm vs. > 0 mm), but not ascites fluid volume (≤500 ml vs. > 500 ml), remained an independent indicator for both OS and PFS (p < 0.001, p = 0.005, respectively). CONCLUSIONS: These results obtained with a homogenous patient group with all patients suffering from advanced high-grade serous ovarian cancer support previous results suggesting elevated KLK5 mRNA levels as an unfavorable marker in ovarian cancer.

Structure guided drug design to develop kallikrein 5 inhibitors to treat Netherton syndrome.

The connection between Netherton syndrome and overactivation of epidermal/dermal proteases particularly KLK5 has been well established. To treat sufferers of this severe condition we wished to develop a topical KLK5 inhibitor in order to normalise epidermal shedding and reduce the associated inflammation and itching. In this paper we describe structure-based optimisation of a series of brightly coloured weak KLK5 inhibitors into colourless, non-irritant molecules with good KLK5 activity and selectivity over a range of serine proteases.

Design and development of a series of borocycles as selective, covalent kallikrein 5 inhibitors.

The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.

Kallikrein 5 inhibitors identified through structure based drug design in search for a treatment for Netherton Syndrome.

Netherton syndrome (NS) is a rare and debilitating severe autosomal recessive genetic skin disease with high mortality rates particularly in neonates. NS is caused by loss-of-function SPINK5 mutations leading to unregulated kallikrein 5 (KLK5) and kallikrein 7 (KLK7) activity. Furthermore, KLK5 inhibition has been proposed as a potential therapeutic treatment for NS. Identification of potent and selective KLK5 inhibitors would enable further exploration of the disease biology and could ultimately lead to a treatment for NS. This publication describes how fragmentation of known trypsin-like serine protease (TLSP) inhibitors resulted in the identification of a series of phenolic amidine-based KLK5 inhibitors 1. X-ray crystallography was used to find alternatives to the phenol interaction leading to identification of carbonyl analogues such as lactam 13 and benzimidazole 15. These reversible inhibitors, with selectivity over KLK1 (10-100 fold), provided novel starting points for the guided growth towards suitable tool molecules for the exploration of KLK5 biology.

KLK5, a novel potential suppressor of vaginal carcinogenesis.

Vaginal cancer is rare and largely unexplored. We found here that kallikrein-related peptidase 5 (KLK5) is coordinately expressed along with other KLKs in all stratified epithelia, including vagina, pointing to potential role(s) in differentiation. Further, we propose that KLK5 could be implicated in vaginal cancer development based on the fact that Klk5-/- mice are prone to develop vaginal tumors when exposed to 7,12-dimethylbenz[a]anthracene. Nf-κb activation is markedly enhanced in Klk5-/-, leading to increased resistance to apoptosis of mutated vaginal cells. This explains the higher tumor numbers observed in Klk5-/- compared to wildtype. Thus, KLK5 may represent a putative suppressor of vaginal cancer.

The Cytolethal Distending Toxin Subunit CdtB of Helicobacter Induces a Th17-related and Antimicrobial Signature in Intestinal and Hepatic Cells In Vitro.

Enterohepatic Helicobacter species are associated with several digestive diseases. Helicobacter pullorum is an emerging human foodborne pathogen, and Helicobacter hepaticus is a mouse pathogen; both species are associated with intestinal and/or hepatic diseases. They possess virulence factors, such as cytolethal distending toxin (CDT). Data indicate that CDT may be involved in chronic inflammatory responses, via its active subunit, CdtB. The proinflammatory properties of the CdtB of H. pullorum and H. hepaticus were assessed on human intestinal and hepatic epithelial cells in vitro. Interleukin 8 expression was evaluated by using wild-type strains and their corresponding CdtB isogenic mutants and by delivering CdtB directly into the cells. Nuclear factor κB nuclear translocation and transcriptomic characteristics in response to CdtB were also evaluated. The CdtB of these Helicobacter species induced nuclear factor κB nuclear translocation and exhibited proinflammatory properties, mainly the expression of T-helper type 17-related genes and genes encoding antimicrobial products also involved in cancer. The Histidine residue in position 265 of the CdtB catalytic site appeared to play a role in the regulation of most of these genes. As for flagellin or lipopolysaccharides, CdtB also induced expression of inflammation-associated genes related to antimicrobial activity.

Cathelicidin, kallikrein 5, and serine protease activity is inhibited during treatment of rosacea with azelaic acid 15% gel.

BACKGROUND: Excess cathelicidin and kallikrein 5 (KLK5) have been hypothesized to play a role in the pathophysiology of rosacea. OBJECTIVE: We sought to evaluate the effects of azelaic acid (AzA) on these elements of the innate immune system. METHODS: Gene expression and protease activity were measured in laboratory models and patients with rosacea during a 16-week multicenter, prospective, open-label study of 15% AzA gel. RESULTS: AzA directly inhibited KLK5 in cultured keratinocytes and gene expression of KLK5, Toll-like receptor-2, and cathelicidin in mouse skin. Patients with rosacea showed reduction in cathelicidin and KLK5 messenger RNA after treatment with AzA gel. Subjects without rosacea had lower serine protease activity (SPA) than patients with rosacea. Distinct subsets of patients with rosacea who had high and low baseline SPA were identified, and patients with high baseline exhibited a statistically significant reduction of SPA with 15% AzA gel treatment. LIMITATIONS: Study size was insufficient to predict clinical efficacy based on the innate immune response to AzA. CONCLUSIONS: These results show that cathelicidin and KLK5 decrease in association with AZA exposure. Our observations suggest a new mechanism of action for AzA and that SPA may be a useful biomarker for disease activity.

Bioinformatics analysis of human kallikrein 5 (KLK5) expression in metaplastic triple-negative breast cancer.

Background: Metaplastic breast carcinoma (MBC) is a rare breast cancer subtype; most cases are triple-negative breast cancers (TNBCs) and are poorly responsive to conventional systemic therapy. Few potential diagnostic and prognostic markers for distinguishing between metaplastic TNBC and nonmetaplastic TNBC have been discovered. We performed bioinformatic analysis to explore the underlying mechanism by which metaplastic TNBC differs from nonmetaplastic TNBC and provides potential pathogenic genes of metaplastic TNBC. Methods: Differentially expressed genes (DEGs) in metaplastic tumors and nonmetaplastic tumors from TNBC patients were screened using GSE165407. The GSE76275 data set and The Cancer Genome Atlas (TCGA) database were used to screen DEGs in TNBC and non-TNBC. Metascape and DAVID were used for the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and Gene Ontology (GO) analysis of DEGs. Online databases, including UALCAN, GEPIA, HPA, Breast Cancer Gene-Expression Miner, and quantitative PCR and western blot, were used to examine KLK5 messenger RNA and protein expression in breast cancer. Analysis of KLK5­associated genes was performed with TCGA data, and the LinkedOmics database was used to detect the genes co-expressed with KLK5. STRING (Search Tool for the Retrieval of Interacting Genes) and Cytoscape were used to screen for hub genes. Kaplan­Meier plotter was used for survival analysis. Results: KLK5 was identified among the DEGs in nonmetaplastic TNBC and metaplastic TNBC. The KLK5 gene was overexpressed in nonmetaplastic TNBC but downregulated in metaplastic TNBC. KEGG and GO analyses revealed that epithelial-to-mesenchymal transition was a pathogenic mechanism in metaplastic TNBC and an important pathway by which KLK5 and its associated genes DSG1 and DSG3 influence metaplastic TNBC progression. Prognosis analysis showed that only low expression of KLK5 in metaplastic TNBC had clinical significance. Conclusion: Our research indicated that KLK5 may be a pivotal molecule with a key role in the mechanism of tumorigenesis in metaplastic TNBC.

Nymphoides peltata Root Extracts Improve Atopic Dermatitis by Regulating Skin Inflammatory and Anti-Oxidative Enzymes in 2,4-Dinitrochlorobenzene (DNCB)-Induced SKH-1 Hairless Mice.

Nymphoides peltata is widely used pharmacologically in Traditional Chinese Medicine and Ayurvedic medicine as a diuretic, antipyretic, or choleretic and to treat ulcers, snakebites, and edema. Previous studies have shown that phytochemicals from N. peltata have physiological activities such as anti-inflammatory, anti-tumor, and anti-wrinkle properties. Nevertheless, research on the anti-atopic dermatitis (AD) effect of N. peltata extract is limited. This study was undertaken to assess the in vitro and in vivo anti-atopic and antioxidant activities of a 95% EtOH extract of N. peltata roots (NPR). PI-induced RBL-2H3 cells and two typical hapten mice (oxazolone-induced BALB/c mice and 2,4-dinitrochlorobenzene (DNCB)-induced SKH-1 hairless mice) were used to investigate the effect of NPR extract on AD. The expressions of AD-related inflammatory cytokines, skin-related genes, and antioxidant enzymes were analyzed by ELISA, immunoblotting, and immunofluorescence, and skin hydration was measured using Aquaflux AF103 and SKIN-O-MAT instruments. The chemical composition of NPR extract was analyzed using an HPLC-PDA system. In this study, NPR extracts were shown to most efficiently inhibit IL-4 in PI-induced RBL-2H3 cells and AD-like skin symptoms in oxazolone-BALB/c mice compared to its whole and aerial extracts. NPR extract markedly reduced DNCB-induced increases in mast cells, epidermal thickness, IL-4 and IgE expressions, and atopic-like symptoms in SKH-1 hairless mice. In addition, NPR extract suppressed DNCB-induced changes in the expressions of skin-related genes and skin hydration and activated the Nrf2/HO-1 pathway. Three phenolic acids (chlorogenic acid, 3,5-dicaffeoylquinic acid, and 3,4-dicaffeoylquinic acid) were identified by HPLC-PDA in NPR extract. The study shows that NPR extract exhibits anti-atopic activities by inhibiting inflammatory and oxidative stress and improving skin barrier functions, and indicates that NPR extract has potential therapeutic use for the prevention and treatment of AD.

Corrigendum: A Novel Mechanism of Carvedilol Efficacy for Rosacea Treatment: Toll-Like Receptor 2 Inhibition in Macrophages.

[This corrects the article DOI: 10.3389/fimmu.2021.609615.].

Murine models of rosacea: a review.

Rosacea is a chronic inflammatory disease characterized by facial flushing, erythema, telangiectasia, papules, and pustules. Its pathogenesis has not been fully understood. In 2017, the global ROSacea COnsensus (ROSCO) panel updated the diagnosis, classification, and assessment of rosacea. Phenotype-based treatments and long-term managements have also been recommended. Murine models are a powerful tool in unveiling and dissecting the mechanisms of human diseases. Here, we summarized murine models of rosacea developed or used in previous research, including LL-37 intradermal injection model, KLK-5-induced inflammation model, croton oil inflammation model, 12-O-Tetradecanoylphorbol-13-acetate inflammation model, arachidonic acid inflammation model, RTX-induced vasodilation model, and UVB-induced model. LL-37 injection model has become the most intensively used model in rosacea research. Each model could show the pathophysiological and clinical features of rosacea to some extent. However, no model can show the full picture of the characteristics of rosacea. Improving existed murine models, developing new murine models, and applying them to pathogenesis and treatment research on rosacea are highly warranted in the future.

IGF2BP3 promotes progression of gallbladder carcinoma by stabilizing KLK5 mRNA in N6-methyladenosine-dependent binding.

Background: Recent studies have reported that IGF2BP3 is linked to the pathogenesis of various malignancies. Since IGF2BP3 is associated with poor outcomes of gallbladder carcinoma (GBC), we aimed to explore the association between its N6-methyladenosine (m6A) RNA methylation and GBC progression. Methods: Bioinformatic analysis of GSE136982, GSE104165, and RNA-seq was performed. In vitro and in vivo gain- and loss-of-function assays were done. qPCR, Western blotting, and IHC were conducted in cells or in collected clinical tissue samples. RNA immunoprecipitation, RNA stability measurement, methylated RNA immunoprecipitation, and dual-luciferase reporter assays were performed in this study. Results: The expression of IGF2BP3 was higher in GBC tissues than in peritumoral tissues. Functions such as cell proliferation and migration, both in vitro and in vivo, were inhibited by downregulation of IGF2BP3. The analysis of RNA-seq indicated that KLK5 was a downstream target of IGF2BP3. The expression of KLK5 was measured in GBC cells and tumor samples. It was found to be positively correlated with IGF2BP3 level. Upon IGF2BP3 depletion, ectopic expression of KLK5 could rescue cell function in part. Mechanistically, we found that IGF2BP3 directly binds to KLK5 mRNA and regulates its stability in an m6A-dependent manner. As a result, inhibition of KLK5 decreased the expression of PAR2, and deregulated phospho-Akt. Using bioinformatic prediction combined with miRNA microarray analysis, we identified that let-7g-5p is an inhibitor of IGF2BP3, and let-7g-5p expression was negatively correlated with IGF2BP3. Overexpression of let-7g-5p affected the aggressive phenotype of GBC cells by deregulating IGF2BP3, and inhibiting the KLK5/PAR2/AKT axis. Conclusions: Our data showed that IGF2BP3 is associated with the aggressive phenotype of GBC. Mechanistically, IGF2BP3 activated the PAR2/AKT axis by stabilizing KLK5 mRNA in an m6A-dependent manner. The loss of let-7g-5p enhanced the expression of IGF2BP3 and improved GBC progression. Thus, IGF2BP3 plays a crucial role in GBC, and the let-7g-5p/IGF2BP3/KLK5/PAR2 axis may be a therapeutic target for GBC.