Genome-wide analysis of two different regions of brain reveals the molecular changes of fertility related genes in rln3a-/- mutants in male Nile tilapia (Oreochromis niloticus).

Relaxin3 (rln3) has been associated with various emotional and cognitive processes, including stress, anxiety, learning, memory, motivational behavior, and circadian rhythm. Notably, previous report revealed that Rln3a played an indispensable role in testicular development and male fertility in Nile tilapia (Oreochromis niloticus). However, the underlying molecular mechanisms remain largely unknown. We found that Rln3a is expressed exclusively in the diencephalon* (Di*) of the brain. Deficiency of Rln3a resulted in a significant increase in serum dopamine level and an upregulation of gene expression of gnrh1 and kisspeptin2. To further elucidate the role of Rln3a in fish fertility, we collected two different regions of Di* and hypothalamus (Hyp) tissues for subsequent RNA-seq analysis of both wild-type (rln3a+/+) and rln3a-/- male tilapia. Upon the transcriptomic data, 1136 and 755 differentially expressed genes (DEGs) were identified in the Di* and Hyp tissues, respectively. In Di*, the up-regulated genes were enriched in circadian rhythm, chemical carcinogenesis, while the down-regulated genes were enriched in type II diabetes mellitus, dopaminergic synapse, and other pathways. In Hyp, the up-regulated genes were enriched in circadian rhythm, pyrimidine metabolism, while the down-regulated genes were enriched in type I diabetes mellitus, autoimmune thyroid disease, and other pathways. Subsequently, the results of both qRT-PCR and FISH assays highlighted a pronounced up-regulation of core circadian rhythm genes, cry1b and per3, whereas genes such as clocka, clockb, and arntl exhibited down-regulation. Furthermore, the genes associated with dopamine biosynthesis were significantly increased in the Hyp. In summary, the mutation of rln3a in male tilapia resulted in notable changes in circadian rhythm and disease-linked signaling pathways in the Di* and Hyp. These changes might account for the fertility defects observed in rln3a-/- male mutants in tilapia.

Identification and Quantification of Human Relaxin Proteins by Immunoaffinity-Mass Spectrometry.

The human relaxins belong to the Insulin/IGF/Relaxin superfamily of peptide hormones, and their physiological function is primarily associated with reproduction. In this study, we focused on a prostate tissue-specific relaxin RLN1 (REL1_HUMAN protein) and a broader tissue specificity RLN2 (REL2_HUMAN protein). Due to their structural similarity, REL1 and REL2 proteins were collectively named a 'human relaxin protein' in previous studies and were exclusively measured by immunoassays. We hypothesized that the highly selective and sensitive immunoaffinity-selected reaction monitoring (IA-SRM) assays would reveal the identity and abundance of the endogenous REL1 and REL2 in biological samples and facilitate the evaluation of these proteins for diagnostic applications. High levels of RLN1 and RLN2 transcripts were found in prostate and breast cancer cell lines by RT-PCR. However, no endogenous prorelaxin-1 or mature REL1 were detected by IA-SRM in cell lines, seminal plasma, or blood serum. The IA-SRM assay of REL2 demonstrated its undetectable levels (<9.4 pg/mL) in healthy control female and male sera and relatively high levels of REL2 in maternal sera across different gestational weeks (median 331 pg/mL; N = 120). IA-SRM assays uncovered potential cross-reactivity and nonspecific binding for relaxin immunoassays. The developed IA-SRM assays will facilitate the investigation of the physiological and pathological roles of REL1 and REL2 proteins.

Relaxin-2 is a novel biomarker for differentiated thyroid carcinoma in humans.

Relaxin's role in differentiated thyroid cancer (DTC) has been suggested but its characterization in a large clinical sample remains limited. We performed immunohistochemistry for relaxin-2 (RLN2), CD68 (total macrophages), CD163 (M2 macrophages) on tissue microarrays from 181 subjects with non-distant metastatic DTC, and 185 subjects with benign thyroid tissue. Mean pixels/area for each marker was compared between tumor and adjacent tissue via paired-t test and between DTC and benign subjects via t-test assuming unequal variances. RNA qPCR was performed for expression of RLN2, RLN1, and RXFP1 in cell lines. Amongst 181 cases, the mean age was 46 years, 75 % were females. Tumoral tissue amongst the DTC cases demonstrated higher mean expression of RLN2 (53.04 vs. 9.79; p < 0.0001) compared to tumor-adjacent tissue. DTC tissue also demonstrated higher mean expression of CD68 (14.46 vs. 4.79; p < 0.0001), and CD163 (23.13 vs. -0.73; p < 0.0001) than benign thyroid. These markers did not differ between tumor-adjacent and benign thyroid tissue groups; and amongst cases, did not differ by demographic or clinicopathologic features. RLN1 and RXFP1 expression was detected in a minority of the cell lines, while RLN2 was expressed by 6/7 cell lines. In conclusion, widespread RLN2 expression in DTC tissue and most cell lines demonstrates that RLN2 acts in a paracrine manner, and that RLN1 and RXFP1 are probably not involved in thyroid cancer cell signaling. RLN2 is a biomarker for thyroid carcinogenesis, being associated with but not secreted by immunosuppressive macrophages. These findings will guide further investigations for therapeutic avenues against thyroid cancer.

Programmed Death-Ligand (PD-L1), Epidermal Growth Factor (EGF), Relaxin, and Matrix Metalloproteinase-3 (MMP3): Potential Biomarkers of Malignancy in Canine Mammary Neoplasia.

Gene expression has been suggested as a putative tool for prognosis and diagnosis in canine mammary neoplasia (CMNs). In the present study, 58 formalin-fixed paraffin-embedded (FFPE) paraffined canine mammary neoplasias from 27 different bitches were included. Thirty-seven tumours were classified as benign, whereas thirty-one were classified as different types of canine carcinoma. In addition, mammary samples from three healthy bitches were also included. The gene expression for vascular endothelial growth factor-α (VEGFα), CD20, progesterone receptor (PGR), hyaluronidase-1 (HYAL-1), programmed death-ligand 1 (PD-L1), epidermal growth factor (EGF), relaxin (RLN2), and matrix metalloproteinase-3 (MMP3) was assessed through RT-qPCR. All the assessed genes yielded a higher expression in neoplastic mammary tissue than in healthy tissue. All the evaluated genes were overexpressed in neoplastic mammary tissue, suggesting a role in the process of tumorigenesis. Moreover, PD-L1, EGF, relaxin, and MMP3 were significantly overexpressed in malignant CMNs compared to benign CMNs, suggesting they may be useful as malignancy biomarkers.

Specific expression and blood kinetics for relaxin 2, lipocalin 2, and tissue factor pathway inhibitor 2 at the canine placenta and pregnant bloods.

In general, humoral factors released from the placenta influence pregnancy progression, but the involvement of the canine placenta is often unidentified. We investigated specific genes in canine placentas and analyzed the blood dynamics of the translated proteins. Furthermore, RNAs are known to be released from placentas embedding in exosomes, a type of extracellular vesicles. Here, the presence of cell-free RNAs in pregnant serums was also confirmed. RNA specimens were purified from the normal healthy dog placentas and applied to RNA-Seq analysis. Expressions of frequent genes were confirmed by RT-PCR using placentas from other individuals and breeds. Relaxin (RLN) 2, lipocalin (LCN) 2, and tissue factor pathway inhibitor (TFPI) 2 were selected as high-expressed and placenta-specific genes. By western blot, the three factors were clearly detected in the pregnant serums. Quantitative analysis revealed that the amount of RLN2 increased significantly from non-pregnancy to day 41 of pregnancy. Regarding LCN2 and TFPI2, the protein serum levels elevated during pregnancy, but the statistical differences were not detected. Exosomes were found in all pregnant serums; however, the percentage was less than 6% in total extracellular vesicles. The cell-free RNA related to RLN2 was detected, but no elevation was confirmed during pregnancy. We found specific genes in the canine placenta and the transition of their translated protein into the blood. These factors may become useful tools for research on canine pregnancy and monitoring of reproductive management. Exosomes and cell-free RNA could not be found to be valid in canine reproduction.

Integrative stemness characteristics associated with prognosis and the immune microenvironment in lung adenocarcinoma.

BACKGROUND: To comprehensively analyze the stemness characteristics related to prognosis and the immune microenvironment in lung adenocarcinoma (LUAD). METHODS: The OCLR machine learning method was used to calculate the stemness index (mRNAsi) of the LUAD samples. DEGs common between the low mRNAsi, normal, and high mRNAsi groups were screened and the immune-stemness genes were obtained. Then the PPI network was created and enrichment analyses were performed. Moreover, different subtypes based on immune-stemness genes associated with prognosis were identified, and the relationships between LUAD stemness and TIME variables were systematically analyzed, followed by TMB analysis. RESULTS: Patients in the high mRNAsi groups with poor prognosis were screened along with 144 immune-stemness genes. IL-6, FPR2, and RLN3 showed a higher degree in the PPI network. A total of 26 immune-stemness genes associated with prognosis were screened. Two clusters were obtained (cluster 1 and cluster 2). Survival analysis revealed that patients in cluster 2 had a poor prognosis. A total of 12 immune cell subpopulations exhibited significant differences between cluster 1 and cluster 2 (P < 0.05). A total of 10 immune checkpoint genes exhibited significantly higher expression in cluster 1 (P < 0.05) than in cluster 2. Further, the TMB value in cluster 2 was higher than that in cluster 1 (P < 0.05). CONCLUSION: Immune-stemness genes, including L-6, FPR2, and RLN3, might play significant roles in LUAD development via cytokine-cytokine receptor interaction, neuroactive ligand‒receptor interaction, and the JAK‒STAT pathway. Immune-stemness genes were related to tumor-infiltrating immune cells, TMB, and expression of immune checkpoint gene.

Changes in microRNA Expression Profiles in Diabetic Cardiomyopathy Rats Following H3 Relaxin Treatment.

ABSTRACT: MicroRNAs (miRNAs) are noncoding RNAs that play an important role in the mechanisms of diabetic cardiomyopathy (DCM); however, whether human recombinant relaxin-3 (H3 relaxin) inhibits myocardial injury in DCM rats and the underlying mechanisms involving miRNAs remain unknown. miRNA expression profiles were detected using miRNA microarray and bioinformatics analyses of myocardial tissues from control, DCM, and H3 relaxin-administered DCM groups, and the regulatory mechanisms of the miRNAs were investigated. A total of 5 miRNAs were downregulated in the myocardial tissues of DCM rats and upregulated in H3 relaxin-treated DCM rats, and 1 miRNA (miRNA let-7d-3p) was increased in the myocardial tissue of DCM rats and decreased in H3 relaxin-treated DCM rats as revealed by miRNA microarray and validated by real-time polymerase chain reaction. Important signaling pathways were found to be triggered by the differentially expressed miRNAs, including metabolism, cancer, Rap1, PI3K-Akt, and MAPK signaling pathways. The study revealed that H3 relaxin improved glucose uptake in DCM rats, potentially via the regulation of miRNA let-7d-3p.

Relaxin increased blood pressure and sympathetic activity in paraventricular nucleus of hypertensive rats via enhancing oxidative stress.

Relaxin, an ovarian polypeptide hormone, is found in the hypothalamic paraventricular nucleus (PVN) which is an important central integrative site for the control of blood pressure and sympathetic outflow. The aim of this study was to determine if superoxide anions modulate the effects of relaxin in the PVN. Experiments were performed in normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Relaxin mRNA and protein, and its receptor, relaxin family peptide receptor 1 (RXFP1) levels in PVN were 3.24, 3.17, and 3.64 times higher in SHRs than in WKY rats, respectively. Microinjection of relaxin-2 into the PVN dose-dependently increased mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA) and heart rate (HR) in both WKY rats and SHRs, although the effects on MAP (16.87 ±â€¯1.99 vs. 8.97 ±â€¯1.48 mm Hg in 100 nmol), RSNA (22.60 ±â€¯2.15 vs. 11.77 ±â€¯1.43 % in 100 nmol) and HR (22.85 ±â€¯3.13 vs. 12.62 ±â€¯2.83 beats/min in 100 nmol) were greater in SHRs. Oxidative stress level was enhanced after relaxin-2 microinjection into the PVN. Pretreatment with superoxide anion scavengers or NADPH oxidase inhibitor blocked, and superoxide dismutase inhibitor potentiated the effects of relaxin-2 on MAP, RSNA and HR. RXFP1 knockdown significantly attenuated the blood pressure of SHRs, and inhibited the increases of atrial natriuretic peptide, brain natriuretic peptide, collagen I, collagen III and fibronectin in the heart of SHRs. These results demonstrated that relaxin is expressed in the PVN, and contributes to hypertension and sympathetic overdrive via oxidative stress. Down-regulation of RXFP1 in the PVN could attenuate hypertension and cardiac remodeling.

Identification of Potent and Long-Acting Single-Chain Peptide Mimetics of Human Relaxin-2 for Cardiovascular Diseases.

The insulin-like peptide human relaxin-2 was identified as a hormone that, among other biological functions, mediates the hemodynamic changes occurring during pregnancy. Recombinant relaxin-2 (serelaxin) has shown beneficial effects in acute heart failure, but its full therapeutic potential has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. In this study, we report the development of long-acting potent single-chain relaxin peptide mimetics. Modifications in the B-chain of relaxin, such as the introduction of specific mutations and the trimming of the sequence to an optimal size, resulted in potent, structurally simplified peptide agonists of the relaxin receptor Relaxin Family Peptide Receptor 1 (RXFP1) (e.g., 54). Introduction of suitable spacers and fatty acids led to the identification of single-chain lipidated peptide agonists of RXFP1, with sub-nanomolar activity, high subcutaneous bioavailability, extended half-lives, and in vivo efficacy (e.g., 64).

Relaxin gene delivery modulates macrophages to resolve cancer fibrosis and synergizes with immune checkpoint blockade therapy.

Cancer fibrosis serves as a major therapeutic barrier in desmoplastic tumors. Relaxin (RLN; a systemic hormone) is efficacious to decrease fibrosis, but the in vivo mechanism of action is not clear. Considering the localization of relaxin family peptide receptor type 1 (RXFP1), the receptor for RLN, on macrophages, we hypothesize that macrophages can be modulated by RLN to ameliorate cancer fibrosis. Using KPC mouse model of pancreatic ductal adenocarcinoma (PDAC), here, we report locally expressed RLN with targeted gene delivery induces increased F4/80+CD206+ macrophages originating from Ly6C+ monocytes, promoting fibrosis depletion and cytotoxic T cell infiltration. Moreover, RLN gene delivery synergizes with PD-L1 blockade for tumor inhibition by enhancing T cell-mediated tumor cell killing and macrophage phagocytosis. Collectively, our results reveal previously unidentified insights into the modulation of macrophages to regulate tumor-associated fibrosis, providing a feasible strategy to reverse the immunosuppressive environment and improve the therapeutic outcome of checkpoint immunotherapies.

Hepatic macrophages act as a central hub for relaxin-mediated alleviation of liver fibrosis.

Relaxin is an antifibrotic peptide hormone previously assumed to directly reverse the activation of hepatic stellate cells for liver fibrosis resolution. Using nanoparticle-mediated delivery, here we show that, although relaxin gene therapy reduces liver fibrosis in vivo, in vitro treatment fails to induce quiescence of the activated hepatic stellate cells. We show that hepatic macrophages express the primary relaxin receptor, and that, on relaxin binding, they switch from the profibrogenic to the pro-resolution phenotype. The latter releases exosomes that promote the relaxin-mediated quiescence of activated hepatic stellate cells through miR-30a-5p. Building on these results, we developed lipid nanoparticles that preferentially target activated hepatic stellate cells in the fibrotic liver and encapsulate the relaxin gene and miR-30a-5p mimic. The combinatorial gene therapy achieves synergistic antifibrosis effects in models of mouse liver fibrosis. Collectively, our findings highlight the key role that macrophages play in the relaxin-primed alleviation of liver fibrosis and demonstrate a proof-of-concept approach to devise antifibrotic strategies through the complementary application of nanotechnology and basic science.

BRAFV600E, hypothyroidism, and human relaxin in thyroid carcinogenesis.

PURPOSE: BRAFV600E, a major driver of thyroid cancer, evaluated in the context of thyroid hormones and human relaxin. METHODS: Immunohistochemical expressions of BRAFV600E, TSH, TSH receptor (TSHR), T4, T3 receptor (T3R), RLNH2, and its receptor, RXFP1, were evaluated in thyroid tumors from a retrospective U.S. population of 481 cancer cases diagnosed in 1983-2004. RESULTS: BRAFV600E was expressed in 52% of all thyroid tumors; expression of other markers ranged from 25% for T4 to 98% for RLNH2. Tumors predominantly exhibited hypothyroid-like conditions characterized by elevated TSH and TSHR and reduced T4. BRAFV600E prevalence was significantly higher in tumors expressing TSH, TSHR, T3R, and RXFP1 and lower in tumors expressing T4. The proportion of BRAFV600E mutation in classic papillary tumors significantly increased from 56 to 72% over the 21-year period of diagnoses, while expression of RXFP1, TSH, TSHR, and T3R decreased in non-tumor. Racial/ethnic differences were observed in thyroid hormone marker expression. Non-tumor expression of TSH, TSHR, and T3R were each associated with shorter overall survival, but did not remain significant after adjustment for demographic and clinical factors. CONCLUSIONS: Our study provides the first evidence of the potential interaction of BRAFV600E mutation, relaxin, and thyroid hormones in thyroid carcinogenesis. Moreover, our results suggest that hypothyroidism, influenced by RLNH2 activity, may underlie the development of the majority of thyroid cancers and mediate the role of BRAFV600E in thyroid carcinogenesis. BRAFV600E mutation is increasing in papillary thyroid cancers and may be contributing to the rising incidence of this malignancy.

A novel G protein-coupled receptor for starfish gonadotropic hormone, relaxin-like gonad-stimulating peptide.

Gonadotropic hormones play important regulatory roles in reproduction. Relaxin-like gonad-stimulating peptide (RGP) is a gonadotropin-like hormone in starfish. However, a receptor for RGP remains to be identified. Here, we describe the identification of an authentic receptor for RGP (RGPR) in the starfish, Patiria pectinifera. A binding assay using radioiodinated P. pectinifera RGP (PpeRGP) revealed that RGPR was expressed in ovarian follicle cells. A RGPR candidate was identified by homology-searching of transcriptome data of P. pectinifera follicle cells. Based on the contig sequences, a putative 947-amino acid PpeRGPR was cloned from follicle cells. Like the vertebrate relaxin family peptide receptors (RXFP 1 and 2), PpeRGPR was a G protein-coupled receptor that harbored a low-density lipoprotein-receptor class A motif and leucine-rich repeat sequences in the extracellular domain of the N-terminal region. Sf9 cells transfected with Gαq16-fused PpeRGPR activated calcium ion mobilization in response to PpeRGP, but not to RGP of another starfish Asterias amurensis, in a dose-dependent fashion. These results confirmed the species-specific reactivity of RGP and the cognate receptor. Thus, the present study provides evidence that PpeRGPR is a specific receptor for PpeRGP. To the best of our knowledge, this is the first report on the identification of a receptor for echinoderm RGP.

Mapping Cell Types and Efferent Pathways in the Ascending Relaxin-3 System of the Nucleus Incertus.

Relaxin-3 (Rln3) is an insulin-family peptide neurotransmitter expressed primarily in neurons of the nucleus incertus (NI) of the pontine tegmentum, with smaller populations located in the deep mesencephalon (DpMe) and periaqueductal gray (PAG). Here, we have used targeted recombination at the Rln3 gene locus to generate an Rln3Cre transgenic mouse line, and characterize the molecular identity and axonal projections of Rln3-expressing neurons. Expression of Cre recombinase in Rln3Cre mice, and the expression of Cre-mediated reporters, accurately reflect the expression of Rln3 mRNA in all brain regions. In the NI, Rln3 mRNA is expressed in a subset of a larger population of tegmental neurons that express the neuropeptide neuromedin-b (NMB). These Rln3-expressing and NMB-expressing neurons also express the GABAergic marker GAD2 but not the glutamatergic marker Slc17a6 (VGluT2). Cre-mediated anterograde tracing with adeno-associated viruses (AAVs) shows that the efferents of the Rln3-expressing neurons in the DpMe and PAG are largely confined to the brain regions in which they originate, while the NI-Rln3 neurons form an extensive ascending system innervating the limbic cortex, septum, hippocampus, and hypothalamus. Viral anterograde tracing also reveals the potential synaptic targets of NI-Rln3 neurons in several brain regions, and the distinct projections of Rln3-expressing and non-expressing neurons in the pontine tegmentum. Rabies virus (RV)-mediated transsynaptic retrograde tracing demonstrates a probable synaptic link between NI-Rln3 neurons and GABAergic neurons in the septum, with implications for the modulation of neural activity in the septo-hippocampal system. Together, these results form the basis for functional studies of the NI-Rln3 system.

Tissue-specific relaxin-2 is differentially associated with the presence/size of an arterial aneurysm and the severity of atherosclerotic disease in humans.

Circulating or tissue-related biomarkers are of clinical value for risk stratification in patients with abdominal aortic aneurysms. Relaxin-2 (RL2) has been linked to the presence and size of arterial aneurysms, and to the extent of atherosclerosis in human subjects. Here, we assessed the expression levels of RL2 in aneurysmal (AA, n = 16) and atherosclerotic (ATH, n = 22) arteries, and established the correlation between RL2 levels and the presence/size of AA and the clinical severity of atherosclerosis. The expression levels of metalloproteinases (MMPs) and endothelial nitric oxide synthetase (eNOS) were also detected for correlations with different phenotypes of atherosclerosis and AA. Temporal artery biopsy specimens (n = 6) and abdominal aortic tissues harvested from accident victims during autopsy (n = 10) were used as controls. Quantitative tissue biomarker analysis revealed that tissue-specific RL2 was increased in patients with larger or symptomatic AA compared to subjects with atherosclerotic disease and healthy controls. In situ RL2 levels were proportional to the size and the severity of aneurysmatic disease, and were substantially elevated in patients with symptomatic aneurysm of any diameter or asymptomatic aneurysm of a diameter >350% of that of the normal artery. In contrast, tissue RL2 was inversely associated with the clinical severity of atherosclerotic lesions. Correlation between RL2 and MMP2 was different between ATH1 and ATH2, depending on atherosclerosis grade. Overall, tissue RL2 is differentially associated with discrete phenotypes of arterial disease and might exert multipotent biological effects on vascular wall integrity and remodeling in human subjects.

Effect of Relaxin Expression from an Alginate Gel-Encapsulated Adenovirus on Scar Remodeling in a Pig Model.

PURPOSE: Relaxin (RLX) is a transforming growth factor-ß1 (TGF-ß1) antagonist that is believed to function as a potent collagen re-arranger and a major suppressor of extracellular matrix components. Adenoviruses (Ads) are accepted vectors for cancer gene therapy. However, repeated treatments of Ad are limited by short-term biological activity in vivo. The efficacy of sustained RLX expression to scar remodeling was assessed using an injectable alginate gel-matrix system. MATERIALS AND METHODS: Pig scar tissue was treated with relaxin-expressing Ad loaded in alginate gel (gel/Ad-RLX). Surface areas, color, and pliability of scars were compared, and various factors influencing scar formation and collagen arrangement were analyzed. RESULTS: Gel/Ad-RLX decreased scar size, color index, and pliability. Immunohistochemistry showed decreased levels of major extracellular matrix proteins in the gel/Ad-RLX-treated group. Furthermore, treatment with gel/Ad-RLX reduced expression of tissue inhibitor of metalloproteinase-1 and alpha-smooth muscle actin and markedly increased expression of matrix metalloproteinase-1 in pig scar tissues. Gel/Ad-RLX also significantly downregulated TGF-ß1 and upregulated TGF-ß3 mRNAs in pig scar tissues. CONCLUSION: These results support a prominent role for RLX in scar remodeling and suggest that gel/Ad-RLX may have therapeutic effects on scar formation.

Targeted genetic screening in mice through haploid embryonic stem cells identifies critical genes in bone development.

Mutagenic screening is powerful for identifying key genes involved in developmental processes. However, such screens are successful only in lower organisms. Here, we develop a targeted genetic screening approach in mice through combining androgenetic haploid embryonic stem cells (AG-haESCs) and clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. We produced a mutant semi-cloned (SC) mice pool by oocyte injection of AG-haESCs carrying constitutively expressed Cas9 and an single guide RNA (sgRNA) library targeting 72 preselected genes in one step and screened for bone-development-related genes through skeletal analysis at birth. This yielded 4 genes: Zic1 and Clec11a, which are required for bone development, and Rln1 and Irx5, which had not been previously considered. Whereas Rln1-/- mice exhibited small skeletal size only at birth, Irx5-/- mice showed skeletal abnormalities both in postnatal and adult phases due to decreased bone mass and increased bone marrow adipogenesis. Mechanistically, iroquois homeobox 5 (IRX5) promotes osteoblastogenesis and inhibits adipogenesis by suppressing peroxisome proliferator activated receptor γ (PPARγ) activation. Thus, AG-haESC-mediated functional mutagenic screening opens new avenues for genetic interrogation of developmental processes in mice.

Relaxin gene delivery mitigates liver metastasis and synergizes with check point therapy.

Activated hepatic stellate cell (aHSC)-mediated liver fibrosis is essential to the development of liver metastasis. Here, we discover intra-hepatic scale-up of relaxin (RLN, an anti-fibrotic peptide) in response to fibrosis along with the upregulation of its primary receptor (RXFP1) on aHSCs. The elevated expression of RLN serves as a natural regulator to deactivate aHSCs and resolve liver fibrosis. Therefore, we hypothesize this endogenous liver fibrosis repair mechanism can be leveraged for liver metastasis treatment via enforced RLN expression. To validate the therapeutic potential, we utilize aminoethyl anisamide-conjugated lipid-calcium-phosphate nanoparticles to deliver plasmid DNA encoding RLN. The nanoparticles preferentially target metastatic tumor cells and aHSCs within the metastatic lesion and convert them as an in situ RLN depot. Expressed RLN reverses the stromal microenvironment, which makes it unfavorable for established liver metastasis to grow. In colorectal, pancreatic, and breast cancer liver metastasis models, we confirm the RLN gene therapy results in significant inhibition of metastatic progression and prolongs survival. In addition, enforced RLN expression reactivates intra-metastasis immune milieu. The combination of the RLN gene therapy with PD-L1 blockade immunotherapy further produces a synergistic anti-metastatic efficacy. Collectively, the targeted RLN gene therapy represents a highly efficient, safe, and versatile anti-metastatic modality, and is promising for clinical translation.