Experimental cell research最新文献

Hypoxia-caused spermatogenesis impairment may contribute to male infertility. FOXA2 has been found to be abundant in spermatogonial stem cells and critical for spermatogenesis. Here we aimed to explore the roles of FOXA2 in regulating spermatogonial cells against hypoxia stimulation. Our results showed that FOXA2 expression was downregulated in hypoxia-stimulated spermatogonial cells. Overexpression of FOXA2 prevented hypoxia-induced endoplasmic reticulum (ER) stress with decreased expression levels of associated markers including GRP78, CHOP, and ATF-4. FOXA2 overexpression caused a decrease in MDA content and an increase in activities of SOD, CAT, and GSH-Px in spermatogonial cells under hypoxic conditions, implying its inhibitory effect on oxidative stress. Besides, cell apoptosis under hypoxic conditions was also prevented by FOXA2 overexpression, as shown by reduced apoptotic rate and caspase-3 activity. Moreover, we found that hypoxia stimulation inactivated the Nrf2 pathway, which could be prevented by FOXA2 overexpression. Nrf2 knockdown attenuated the effects of FOXA2 overexpression on hypoxia-induced ER stress, oxidative stress, and apoptosis in spermatogonial cells. In conclusion, FOXA2 exerted protective effects on spermatogonial cells against hypoxia-induced ER stress, oxidative stress, and apoptosis via regulating Nrf2/HO-1 signaling. These findings suggested that FOXA2 might be a therapeutic target for treating hypoxia-induced spermatogenesis impairment.

Spermatogenesis and sperm maturation are complex biological processes that involve intricate cellular and molecular interactions. The Aldh2 gene is involved in the metabolism of specific aldehydes generated by oxidative stress. Aldh2 is abundantly expressed in the testis and epididymis; however, the specific role of Aldh2 in regulating spermatogenesis and sperm maturation remains unclear. In the present study, we generated Aldh2 knockout (Aldh2^-/-) mice by using CRISPR/Cas9 technology. Aldh2 gene knockout decreased the fertility of male mice. Compared to the control group mice, Aldh2^-/- mice showed a significant decrease in the thickness of the seminiferous tubules and the number of germ cells. Further investigation revealed that the meiosis of spermatocytes and acrosome formation in sperm were disrupted in Aldh2^-/- mice, leading to oligoasthenoteratozoospermia in male mice. However, the caput epididymis and cauda epididymis in Aldh2^-/- mice showed identical proportions of morphologically abnormal sperm. Mechanistically, 4-hydroxynonenal, 3-nitro-L-tyrosine, and malondialdehyde levels were significantly elevated in both the testis and epididymis of Aldh2^-/- mice, thus indicating increased oxidative stress in the reproductive system. Collectively, our findings demonstrate that Aldh2 plays a critical role in spermatogenesis by regulating oxidative stress in mice.

Endothelial inflammation caused by hyperglycemia contributes to cardiovascular complications in patients with diabetes. Diabetic kidney injury (DKI) is one of the most significant manifestations of diabetes-related renal damage, encompassing both acute and early chronic kidney injury. DKI involves pathological mechanisms linked to inflammatory responses and early renal damage, which, if left unchecked, may progress to diabetic kidney disease. Previous research indicates that both P300 and Ese-1 play pivotal roles in hyperglycemia-induced endothelial inflammation. This study suggests that P300 modulates Ese-1 expression, promoting hyperglycemia-mediated vascular endothelial inflammation and thereby contributing to the occurrence and progression of DKI. Our findings revealed increased levels of tumor necrosis factor α (Tnf-α), p65 phosphorylation, and monocyte chemotactic proteins Mip-1β and Mip-2 in the kidney tissues of diabetic mice and hyperglycemic human renal glomerular microvascular endothelial cells (HRGECs). Additionally, hyperglycemia orchestrated endothelial inflammation through the upregulation of Ese-1 expression in vitro. Furthermore, P300 was found to be upregulated both in vitro and in vivo. Moreover, silencing P300 reduced hyperglycemia-induced inflammatory effects, which could be reversed by overexpressing Ese-1 in HRGECs. Further, P300 was observed to interact with the Ku protein family (Ku70/Ku86), which were downregulated in the kidney tissues of diabetic mice and hyperglycemic HRGECs. siKu70 and siKu86 intensified hyperglycemia-induced endothelial inflammation, an effect counteracted by P300 silencing. In essence, the Ku protein family interacts with P300 to modulate Ese-1 expression in HRGECs, thereby participating in hyperglycemia-induced endothelial inflammation.

Pellino1 (PELI1) is a key regulator of inflammatory and autoimmune diseases. The role of PELI1 in juvenile idiopathic arthritis (JIA) is unclear. The correlation between serum PELI1 mRNA levels and clinical indicators of JIA patients was evaluated by Pearson correlation analysis. The percentage of Th1, Th2, Th17 and Treg cells was analyzed by flow cytometry. ELISA kits were used to detect cytokine levels in serum and cell supernatants. Co-immunoprecipitation experiments were performed to validate PELI1 and TCF-1 interactions. The protein and ubiquitination levels of TCF-1 were detected by western blot. The results showed that JIA patients have high serum PELI1 levels. PELI1 levels were positively correlated with erythrocyte sedimentation rate, C-reactive protein levels and JADAS27 scores in JIA patients. Interfering with PELI1 promoted naïve CD4⁺ T cell differentiation to Th2 and Treg cells and increased IL-4 and IL-10 levels, while inhibiting their differentiation to Th1 and Th17 cells and decreasing IFN-γ and IL-17 levels. PELI1 increased TCF-1 ubiquitination levels and accelerated its degradation. Inhibition of TCF-1 reduced the effects of interfering with PELI1 on cell differentiation and cytokine levels. In conclusion, Silencing of PELI1 facilitated the naïve CD4⁺ T cell differentiation into Th2 and Treg cells by TCF-1.

Background: Promoting muscle regeneration through stem cell therapy has potential risks. We investigated the effect of umbilical cord mesenchymal stem cells (UMSCs) Exosomes (Exo) Follistatin on muscle regeneration.

Methods: The Exo was derived from UMSCs cells and was utilized to affect the mice muscle injury model and C2C12 cells myotubes atrophy model. The Western blot, qRT-PCR and IF were utilized to determine the effects of Exo on the levels of Follistatin, MyHC, MyoD, Myostatin, MuRF1, MAFbx, α-SMA, Collagen I, Smad2, and AKT. In addition, HE and Masson staining were used to assess muscle tissue damage in mice.

Results: The level of Follistatin in Exo was significantly higher than that in UMSCs. UMSCs-Exo increased the levels of Follistatin, MyHC, MyoD, and p-Smad2 and decreased the levels of Myostatin, MuRF1, MAFbx, α-SMA, Collagen I, p-AKT, and p-mTOR in mice or C2C12 cells. In addition, UMSCs-Exo decreased levels of inflammation and fibrosis in mice. However, UMSCs-Exo-si-Follistatin reversed the effect of UMSCs-Exo. Transfection of oe-Smad2 up-regulated the protein levels of Collagen I, α-SMA, and changed the ratio of p-Smad2/Smad2 expression to 0.33, and 0.34, 0.73. LY294002 decreased the levels of MyHC, MyoD, and the ratio of p-AKT/AKT and p-mTOR/mTOR expression to 0.12, 0.17, 0.33, and 0.41, increased the levels of MuRF1 and MAFbx to 0.36 and 0.34.

Conclusion: This study demonstrated that Follistatin in UMSCs-Exo inhibits fibrosis and promotes muscle regeneration in mice by regulating Smad and AKT signaling.

Hepatocellular carcinoma (HCC), the most common primary liver cancer, is marked by a high mortality rate, with the misregulation of long non-coding RNAs (LncRNAs) playing a key role in its development. Here, we studied the role of LINC02987 in HCC. We employed bioinformatics tools to identify LncRNAs and miRNAs that exhibit differential expression in HCC. Quantitative real-time reverse transcription PCR (RT-qPCR) and Western blot analysis were utilized to quantify gene and protein expression levels. The interaction between miR-338-3p and LINC02987 or ATG12 was confirmed through dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. We observed that LINC02987 was overexpressed in HCC tumor tissues and cell lines. Silencing of LINC02987 led to a reduction in cell viability, diminished clonogenic potential, and attenuated invasive and migratory capabilities. Also, decreasing protein level and fluorescence intensity of the autophagy-associated LC3 I/II. In HCC, miR-338-3p expression was downregulated, while inversely correlates with the overexpression of the autophagy protein ATG12. Mimicking miR-338-3p suppresses the activity of both LINC02987 and ATG12, as evidenced by reduced luciferase signals in corresponding reporter assays. Mimicking miR-338-3p suppresses the activity of both LINC02987 and ATG12, as evidenced by reduced luciferase signals in reporter assays. Transfection with si-LINC02987 decreased ATG12 expression, an effect that was partially reversed by miR-338-3p knockdown. Inhibition of miR-338-3p or overexpression of ATG12 increased LC3 I/II protein levels. Our results indicate that LINC02987 sequesters miR-338-3p, leading to increased ATG12 and promoting autophagy in HCC cells. These results highlight the potential of LINC02987 as a therapeutic target for the treatment of HCC.

SMAD3 plays a crucial role in TGF-β, regulating various normal developmental mechanisms and disease pathogenesis. Here, we report that SMAD3 directly interacts with Nucleostemin (NS), leading to nuclear translocation and affecting SMAD3 activity after TGF-β1 stimulation. Moreover, NS acts as a competitor, preventing PPM1A from recognizing and dephosphorylating SMAD3. Experimental investigations have demonstrated that NS significantly enhances cellular migration and invasion by promoting the EMT mechanism in vitro. NS knockdown notably suppresses tumor metastasis in the lungs and liver in vivo. Importantly, NS expression is significantly elevated in numerous human malignancies, correlating with a poorer prognosis. The collective evidence from these studies suggests that NS exhibits oncogenic characteristics, supporting further exploration of NS as a potential target for tumor treatment.

Keloids are disfiguring proliferative scars, and their pathological mechanisms are still unclear. We have previously established that FoxC1 plays a significant role in rheumatoid arthritis and osteoarthritis, but its molecular mechanisms in pathological scar formation remain elusive. In this study, we analyzed keloid tissue characteristics using HE staining and immunohistochemistry, revealing abnormal expression of FoxC1 and Notch3 in keloids. Lentiviral modulation of FoxC1 and Notch3 demonstrated that they promote the expression of α-SMA, fibronectin, collagen I, and Hes-1, enhancing the proliferation, migration, invasion, and cytokine production of keloid fibroblasts (KFs) while inhibiting apoptosis. Co-immunoprecipitation (CO-IP), dual-luciferase reporter assays, and chromatin immunoprecipitation (ChIP) confirmed that FoxC1 can directly bind to the Notch3 promoter and enhance its transcription. Additionally, in vivo, overexpression of FoxC1 and Notch3 promoted keloid formation. In summary, our research highlights the critical regulatory role of FoxC1 in keloid formation through Notch3 activation, potentially offering new therapeutic targets for preventing scar formation.

Hepatocellular carcinoma (HCC) is a global health issue due to its late diagnosis and high recurrence rate. The early detection and diagnosis of HCC with specific and sensitive biomarkers and using novel treatment approaches to improve patient outcomes are essential. Glypican-3 (GPC-3) is a cell surface proteoglycan that is overexpressed in many tumors, including HCC. GPC-3 could be used as a specific biomarker for HCC early detection and could be a potential target for precise therapeutic strategies. Effective identification of GPC-3 could improve both diagnosis and targeted therapy of HCC. Moreover, targeted therapy using GPC-3 could result in a better treatment outcome. Recently, GPC3-targeted therapies have been used in different investigational therapeutic approaches like bi-specific/monoclonal antibodies, peptide vaccines, and CAR T cell therapies. This study aims to highlight the theranostic potential of GPC-3 as a novel biomarker for early detection and as a potential molecular target for HCC treatment as well.

Intrahepatic cholangiocarcinoma (ICC) is a primary invasive malignant tumor. This study was conducted to explore the role of methyltransferase-like 3 (METTL3)-mediated m6A modification in ICC cells and provide novel targets for ICC treatment. Levels of METTL3/YTH N6-methyladenosine RNA binding protein 2 (YTHDF2)/Nedd4 family interacting protein 1 (NDFIP1) in cells were determined. Cell viability, proliferation, invasion, and migration were evaluated. The enrichments of METTL3, YTHDF2, and m6A on NDFIP1 mRNA were analyzed. The mRNA stability was determined. Inhibition of YTHDF2 or NDFIP1 was combined with si-METTL3 to confirm the mechanism. The role of METTL3 in vivo was verified. METTL3 was overexpressed in ICC cells. METTL3 silencing suppressed ICC cell malignant behaviors, which were reversed by METTL3 overexpression. METTL3 increased m6A modification on NDFIP1 mRNA, facilitated YTHDF2 recognition of m6A, and promoted NDFIP1 mRNA degradation, thereby suppressing NDFIP1 expression. YTHDF2 inhibition increased NDFIP1 mRNA levels. NDFIP1 downregulation partially reversed the inhibitory effects of si-METTL3 on ICC cell behaviors, while NDFIP1 overexpression partially reversed the promotive effects of METTL3 on ICC cell behaviors. METTL3 downregulation suppressed ICC growth by increasing NDFIP1 expression. In conclusion, METTL3 aggravates ICC cell proliferation, invasion, and migration by promoting the degradation of NDFIP1 mRNA in a YTHDF2-dependent manner.