BACKGROUND:Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and the leading cause of cancer-related deaths. Immune checkpoint inhibitors (ICIs) of programmed death-1 (PD-1)/programmed death ligand-1 signaling induce tumor regression in some patients with NSCLC, but most patients with NSCLC exhibit resistance to ICIs therapy. NSCLC shapes the potent tumor immunosuppressive microenvironment (TIME) that underlies tumor immune tolerance and acquired resistance. Therefore, elucidating the cellular and molecular mechanisms by which NSCLC establishes and sustains the TIME is essential for developing novel strategies to overcome immune resistance and enhance the clinical benefit of ICIs. METHODS:The correlation between sterile alpha motif domain and histidine-aspartate domain-containing protein 1 (SAMHD1) expression and ICIs was analyzed via immunohistochemistry. Cell migration assay was performed to assess the effect of SAMHD1 on macrophage recruitment. Multicolor flow cytometry was performed to analyze the effect of SAMHD1 knockdown on the tumor microenvironment. SAMHD1 regulation of the dual specificity phosphatase 6-extracellular regulated protein kinases 1/2 (DUSP6-ERK1/2) pathway was verified by RNA sequencing and western blotting. RESULTS:Here, we identify the SAMHD1 as a potential therapeutic target and a major determinant of poor response to ICIs in patients with NSCLC. Tumors with high SAMHD1 expression show resistance to anti-PD-1 antibody (αPD-1) treatment, whereas tumors with low SAMHD1 expression are highly sensitive. SAMHD1-dependent resistance to αPD-1 is characterized by increased tumor-associated macrophages (TAMs) infiltration and reduced CD8+T cell numbers. Mechanistically, SAMHD1 regulates the expression of macrophage-associated chemokines by influencing the activation of the DUSP6-ERK1/2 pathway, which contributes to TAMs aggregation within NSCLC tumors to shape an immunosuppressive microenvironment. The HIV accessory protein viral protein-x (VPX) specifically degrades SAMHD1 to promote HIV replication. Similarly, the vpx-engineered oncolytic adenovirus (oAd-vpx) targets SAMDH1 degradation to enhance oncolytic adenovirus replication and weaken the hostile immune microenvironment shaped by TAMs, thereby triggering a CD8+T-cell-dependent antitumor immune response. The combination of oAd-vpx and αPD-1 inhibits tumor growth and enhances sensitivity to ICIs in both mouse and human NSCLC. CONCLUSIONS:This research identifies a key mechanism of SAMHD1-driven immunosuppression and highlights its important role in oncolytic adenovirus therapy. This study provides a theoretical basis for targeting SAMHD1 as a drug therapy strategy in patients with NSCLC.

Osteocytes, the most abundant cells embedded in bone, possess extensive cytoplasmic extensions that form a complex network facilitating direct communication among osteocytes and with other effector cells in the bone and bone marrow. The mechanisms underlying osteocyte dendrite formation during “osteocytogenesis” remain poorly understood. MicroRNAs play a crucial role in regulating bone homeostasis. To induce osteoporosis, bilateral ovariectomy was performed on 8-week-old female C57BL/6J mice of both wild-type (WT) and miR-185 knockout (KO) genotypes. Ploton silver staining was used to visualize the effects of miR-185 KO on the osteocyte lacunar-canalicular network (LCN), while micro-computed tomography assessed bone microstructure. Bone biomechanical properties were evaluated via three-point bending tests. To investigate the mechanisms by which miR-185 regulates osteocyte LCN, miR-185 KO MLO-Y4 cells were generated using CRISPR/Cas9 technology. Bioinformatics analysis, Western blotting and dual-luciferase reporter assays were used to identify the target gene of miR-185. Co-immunoprecipitation and GST pull-down experiments were utilized to validate the interacting partner of target gene. The levels of pathway-related molecules and the activation of downstream signaling were examined using cell surface protein biotinylation, Western blotting and Lysosome immunoprecipitation. The role of miR-185 KO in osteocyte-mediated regulation of osteoblast and osteoclast differentiation was evaluated using ALP staining, alizarin red staining and TRAP staining. In this study, we demonstrated that dendrite formation and osteocyte connectivity were significantly enhanced in miR-185 KO ovariectomized (OVX) mice. Furthermore, both bone quality and mass were improved in miR-185 KO OVX mice. In miR-185 KO MLO-Y4 cells, we observed similar osteocyte morphology change. We identified Arl8b, a small GTP-binding protein that promotes lysosomal anterograde trafficking, as a target gene of miR-185. Knockdown of Arl8b significantly inhibited the enhanced dendritic process formation phenotype observed in miR-185 KO cells. Additionally, we discovered that RAB5A is a novel interacting protein of ARL8B. Downregulation of RAB5A similarly impaired osteocyte connectivity, suggesting a functional interaction between ARL8B and RAB5A. Further studies revealed that knockdown Arl8b suppressed RAB5A-mediated ITGB1 endosomal recycling, promoted its entry into lysosomes, reduced ITGB1 expression, inhibited FAK activation, and consequently diminished osteocyte dendritic process formation. Moreover, it was observed that the culture supernatant from miR-185 KO MLO-Y4 cells enhanced osteoblast mineralization and inhibited osteoclast differentiation. This study elucidates the mechanism of miR-185 in regulating osteocyte LCN and its involvement in estrogen deficiency-induced osteoporosis progression, identifying novel therapeutic targets and informing potential intervention strategies for skeletal disorders. The knockout of miR-185 upregulates ARL8B expression, enhances the interaction between ARL8B and RAB5A, and promotes endosomal recycling of ITGB1. This process suppresses ITGB1 entry into lysosomes, thereby maintaining ITGB1 protein levels and activating the ITGB1-FAK signaling pathway. Furthermore, it preserves the dendritic morphology of osteocytes, mitigates estrogen deficiency-induced damage to the osteocyte lacunar-canalicular network, and enhances bone mechanical properties. Additionally, miR-185-deficient osteocytes inhibit osteoclast differentiation and promote osteoblast mineralization by secreting key regulatory molecules, disrupting bone coupling and attenuating estrogen deficiency-induced bone loss.

TRNA splicing endonuclease subunit 15 (TSEN15) is known for its role in cell cycle regulation, but its involvement in hepatocellular carcinoma (HCC) remains underexplored. TSEN15 influences mitosis and cancer cell proliferation. Its effects on immune infiltration and prognostic value in HCC demand further study. HCC patient data were analyzed using The Cancer Genome Atlas (TCGA). TSEN15 expression in HCC tissues was significantly higher than in normal liver tissues. High TSEN15 levels were associated with advanced HCC stages, such as T3, T4, and M1. Survival analyses showed TSEN15 as an independent negative prognostic marker for overall survival. Immune profiling revealed that elevated TSEN15 expression correlates with reduced infiltration of cytotoxic immune cells, including CD8 + T cells, NK cells, and dendritic cells. Functional enrichment analyses linked TSEN15 to pathways promoting tumor progression. Results were validated through Western blotting, RT-qPCR, and cellular assays. TSEN15 serves as a novel biomarker for HCC diagnosis and prognosis, indicating its potential as a target for therapeutic interventions. Its role in promoting tumor proliferation, modulating immune infiltration, and influencing clinical outcomes underscores its clinical significance in HCC management.

Paclitaxel, a microtubule-stabilizing chemotherapy drug, can cause severe paclitaxel-induced peripheral neuropathic pain (PIPNP). The roles of transient receptor potential (TRP) ion channel vanilloid 1 (TRPV1, a nociceptor and heat sensor) and melastatin 8 (TRPM8, a cold sensor) in PIPNP remain controversial. In this study, Western blotting, immunofluorescence staining, and calcium imaging revealed that the expression and functional activity of TRPV1 were upregulated in rat dorsal root ganglion (DRG) neurons in PIPNP. Behavioral assessments using the von Frey and brush tests demonstrated that mechanical hyperalgesia in PIPNP was significantly inhibited by intraperitoneal or intrathecal administration of the TRPV1 antagonist capsazepine, indicating that TRPV1 played a key role in PIPNP. Conversely, the expression of TRPM8 protein decreased and its channel activity was reduced in DRG neurons. Furthermore, activation of TRPM8 via topical application of menthol or intrathecal injection of WS-12 attenuated the mechanical pain. Mechanistically, the TRPV1 activity triggered by capsaicin (a TRPV1 agonist) was reduced after menthol application in cultured DRG neurons, especially in the paclitaxel-treated group. These findings showed that upregulation of TRPV1 and inhibition of TRPM8 are involved in the generation of PIPNP, and they suggested that inhibition of TRPV1 function in DRG neurons via activation of TRPM8 might underlie the analgesic effects of menthol.

Granulosa cells (GCs) are essential for proper oocyte, follicular development, and steroidogenesis in the ovary. Transforming growth factor beta (TGF-beta) superfamily members are critical in regulating GCs growth and differentiation. Smad3 is known to serve as a signaling intermediate for the TGF-beta; however, the functions of Smad3 in the human GCs remain unidentified. In this study, the luteinized GCs collected from follicular aspirates from patients undergoing in vitro fertilization were cultured and engineered to overexpress and knockdown Smad3, which were validated by RT-PCR and Western blotting. Immunocytochemistry showed that Smad3 protein was strongly expressed in human ovarian luteinized GCs. EdU incorporation demonstrated that Smad3 promoted the proliferation of GCs, and the expression of PCNA was also enhanced by Smad3. ELISA analysis indicated that the secretion of both estradiol and progesterone was stimulated by Smad3. In addition, Smad3 upregulated the level of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and protein kinase A (PKA) proteins. We subsequently added special PKA inhibitor H89 into the GCs and found that the stimulating effect on the growth of GCs by Smad3 was blocked partly. The morphology of cultured GCs was changed by Smad3, and the expression level of integrin beta 1 was enhanced by Smad3. Kindlin-2, an important cellular mediating molecule of integrin beta signaling, was expressed in human ovarian luteinized GCs and was upregulated by Smad3. Our results indicated that Smad3 promoted the proliferation and steroidogenesis of human ovarian luteinized GCs, and these effects may be mediated by the FSHR/LHR-PKA signaling pathway. (C) 2014 IUBMB Life, 66(6): 424-437, 2014

目的探讨Smad3基因对大鼠卵巢颗粒细胞自噬的影响。方法 21d SD雌性大鼠腹腔注射孕马血清20 IU/只,48h后收集卵巢颗粒细胞进行原代培养。培养细胞分为3组:空白对照组:培养液中不加任何处理因素;敲低实验组:培养液中加入Smad3基因特异性的SiRNA及转染试剂RNAiMAX;过表达实验组:培养液中加入Smad3真核表达质粒及转染试剂Lipo 2000。免疫细胞化学方法鉴定培养细胞纯度;Western blotting方法检测Smad3蛋白表达变化,检测转染效率。Smad3基因敲低及过表达后,Western blotting方法检测自噬体膜形成标志蛋白LC3B及自噬调控相关蛋白Bcl-2的蛋白表达变化。结果 Smad3敲低时,LC3B蛋白Ⅱ型与Ⅰ型的比值（LC3BⅡ/Ⅰ）变化不明显,Bcl-2蛋白表达明显降低;Smad3过表达时,LC3BⅡ/Ⅰ明显增加,Bcl-2蛋白表达明显增加。结论Smad3基因特异的SiRNA及真核表达质粒可以有效地转入大鼠卵巢颗粒细胞中,Smad3基因促进大鼠卵巢颗粒细胞自噬,其机制可能与Bcl-2蛋白相关。

The function of Smad3, a downstream signaling protein of the transforming growth factor β (TGFβ) pathway, in ovarian follicle development remains to be elucidated. The effects of Smad3 on ovarian granulosa cells (GCs) in rat were studied. Female rats (21 days of age Sprague-Dawley) received i.p. injections of pregnant mare serum gonadotropin, and GCs were harvested for primary culture 48 h later. These cells were engineered to overexpress or knockdown Smad3, which were validated by immunohistochemistry and western blot. The expression of proliferating cell nuclear antigen (PCNA), cyclin D2, TGFβ receptor II (TGFβRII), protein kinase A (PKA), and FSH receptor (FSHR) was also detected by western blotting. Cell cycle and apoptosis of GCs were assayed by flow cytometry. The level of estrogen secreted by GCs was detected by ELISA. Smad3 overexpression promoted estrogen production and proliferation while inhibiting apoptosis of GCs. Reduction in Smad3 by RNAi resulted in reduced estrogen production and proliferation and increased apoptosis of GCs. Manipulation of Smad3 expression also resulted in changes in FSHR and PKA expression, suggesting that the effects of Smad3 on follicle development are related to FSHR-mediated cAMP signaling.

Paracrine and autocrine growth factors can affect many different aspects of ovarian follicle development. Many members of the transforming growth factor β (TGFβ) family of growth factors and their receptors are expressed in developing follicles. However, the presence and function of the family of the TGFβ signaling molecules known as Smads have not been evaluated during follicle development. We have demonstrated that two Smad family members that function as mediators for both activin and TGFβ are expressed in granulosa cells of preantral follicles but not in large antral follicles. Smad2 expression, but not Smad3 expression, returns in luteal cells. Both Smad2 and Smad3 are translocated to the nucleus of granulosa cells in response to treatment with either TGFβ or activin. However, Smad2 is more responsive to activin stimulation, and Smad3 is more responsive to TGFβ stimulation. Stage-specific expression and differing ligand sensitivity of signaling molecules may work together to allow different effects of TGFβ family ligands using the same signaling pathways over the course of follicular development.