Blockade of ZFX Alleviates Hypoxia-Induced Pulmonary Vascular Remodeling by Regulating the YAP Signaling.

High expression of the zinc finger X-chromosomal protein (ZFX) correlates with proliferation, aggressiveness, and development in many types of cancers. In the current report, we investigated the efficacy of ZFX in mouse pulmonary artery smooth muscle cells (PASMCs) proliferation during pulmonary arterial hypertension (PAH). PASMCs were cultured in hypoxic conditions. Real-time PCR and western blotting were conducted to detect the expression of ZFX. Cell proliferation, apoptosis, migration, and invasion were, respectively, measured by CCK-8, flow cytometry, wound scratchy, and transwell assays. Glycolytic ability was validated by the extracellular acidification rate and oxygen consumption rate. Transcriptome sequencing technology was used to explore the genes affected by ZFX knockdown. Luciferase and chromatin immunoprecipitation assays were utilized to verify the possible binding site of ZFX and YAP1. Mice were subjected to hypoxia for 21 days to induce PAH. The right ventricular systolic pressure (RVSP) was measured and ratio of RV/LV + S was calculated. The results show that ZFX was increased in hypoxia-induced PASMCs and mice. ZFX knockdown inhibited the proliferation, migration, and invasion of PASMC. Using RNA sequencing, we identify glycolysis and YAP as a key signaling of ZFX. ZFX knockdown inhibited Glycolytic ability. ZFX strengthened the transcription activity of YAP1, thereby regulating the YAP signaling. YAP1 overexpression reversed the effect of ZFX knockdown on hypoxia-treated PASMCs. In conclusion, ZFX knockdown protected mice from hypoxia-induced PAH injury. ZFX knockdown dramatically reduced RVSP and RV/(LV + S) in hypoxia-treated mice.

FIN56, a novel ferroptosis inducer, triggers lysosomal membrane permeabilization in a TFEB-dependent manner in glioblastoma.

Objective: To explore the anti-tumor effect of FIN56, a novel ferroptosis inducer, on glioblastoma and its underlying mechanisms. Methods: Two human glioblastoma cell lines, LN229 and U118 were applied in this study. Anti-tumor effect was measured by CCK-8 assay, EdU assay and cell cycle analysis. Fluorescent probes, immunofluorescence, plasmid transfection, shRNA knocking out, reverse transcription PCR, western blot analysis, and transmission electron microscopy were used to study the underlying mechanisms. At last, a subcutaneous nude mice model was used to study the anti-tumor effect of FIN56 in vivo. The GraphPad Prism software program was applied for statistical analysis. Results: FIN56 decreased cell viability, inhibited cell proliferation and caused cell cycle arrest on LN229 and U118 cells. Further study showed that FIN56 induced ferroptosis and induced lysosomal membrane permeabilization in a ferroptosis and transfactor EB dependent manner. Animal study demonstrated that FIN56 inhibited glioma growth and caused ferroptosis in vivo. Conclusion: FIN56 is a promising anti-tumor compound.

SNHG29 regulates miR-223-3p/CTNND1 axis to promote glioblastoma progression via Wnt/ß-catenin signaling pathway.

BACKGROUND: Glioblastoma has been seen as the most common malignancy of brain tumor. Emerging reports has claimed that SNHG29 (LRRC75A-AS1) was involved in several biological processes via modulation of signaling pathway, and served as an malignant facilitatorin osteosarcoma. However, the specific role of SNHG29 in glioblastoma remains unknown. METHODS: RT-qPCR and microarray were operated to measure genes expression. Western blot was performed to examine protein expression. CCK-8 and colony formation assays were used to evaluate cell proliferation. Cell migration was tested by transwell assay. Nuclear-cytoplasmic fractionation was conducted to locate SNHG29. The binding capacity of miR-223-3p to SNHG29 or CTNND1 3'UTR was verified by RIP and luciferase reporter assay. RESULTS: SNHG29 presented high expression in glioblastoma to boost cell proliferation, migration and EMT process. In addition, miR-223-3p was validated to bind with SNHG29 after prediction and screening. Furthermore, miR-223-3p was proved to be a negative regulator for its target CTNND1. Then, the inhibition on cell proliferation, migration and EMT process resulted from SNHG29 knockdown was recovered by CTNND1 overexpression. At last, the inhibitive impacts on cell proliferation, migration and EMT process of CTNND1 deficiency was abrogated by LiCl. CONCLUSIONS: In conclusion, SNHG29 regulates miR-223-3p/CTNND1 axis to promote glioblastoma progression via Wnt/ß-catenin signaling pathway, offering a potential therapeutic point for glioblastoma patients.

SMAD3 silencing enhances DNA damage in radiation therapy by interacting with MRE11-RAD50-NBS1 complex in glioma.

Radiotherapy is the major treatment modality for malignant glioma. However, the treatment response of radiotherapy is suboptimal due to resistance. Here we aimed to explore the effect and mechanism of Mothers against decapentaplegic homologue (SMAD3) silencing in sensitizing malignant glioma to radiotherapy. Clonogenic assay was used to evaluate the sensitivity of glioma cells to increasing doses of radiation. Glioma cells were transfected with small-interfering RNAs (siRNAs) specific to SMAD3. Overexpression of SMAD3 was achieved by transfecting expression plasmid encoding SMAD3 cDNA. Changes in MRE11-RAD50-NBS1 mRNA and protein levels were assessed through qPCR analysis and western blot analysis, respectively. Chromatin immunoprecipitation (ChIP) was used to confirm the interaction between SMAD3 and MRE11-RAD50-NBS1 (MRN) complex. Silencing of SMAD3 increased sensitivity of glioma cells to radiotherapy. MRE11, RAD50 and NBS1 were overexpressed in response to radiotherapy, which was attenuated by SMAD3 silencing while boosted by SMAD3 overexpression. ChIP analysis confirmed the interaction of SMAD3 with MRE11, RAD50 and NBS1 under radiotherapy, which was inhibited by SMAD3 silencing. SMAD3 silencing is an effective strategy for sensitizing glioma to radiotherapy, which is mediated by the interaction of SMAD3 with the MRN complex.

Enhanced radiosensitization of human glioblastoma multiforme cells with phosphorylated peptides derived from Gli2.

Glioma-Associated Oncogene Family Zinc Finger 2 (Gli2) seems to be the major nuclear effector of Sonic Hedgehog (SHH) signaling to regulate self-renewal and tumorigenic potential of Glioblastoma multiforme (GBM) cells. Three phosphorylated peptides derived from Gli2 were synthesized and combined with cell-penetrating peptide Tat-(47-57) (AYGRKKRRQRRR). Western Blot was applied to detect the phosphorylation level of Gli2 and cell division protein kinase 6 (CDK6) luciferase reporter was utilized to detect the transcriptional activator function of Gli2. Clonogenic survival assay and apoptosis assay were used to testify the radiosensitization effect. The mixed three phosphorylated peptides derived from Gli2 increased the phosphorylation level of Gli2 and decreased Gli2 transcriptional activator activity significantly than the individually used peptide. The mixed three phosphorylated peptides showed greater radiation-sensitizing effects in GBM cells in clonogenic and survival assay compared with control peptide. We present here a novel rational strategy for developing phosphorylated peptides derived from Gli2 to decrease Gli2 transcriptional activator activity and such administration could radiosensitize GBM.

Effect of low-energy extracorporeal shock wave on vascular regeneration after spinal cord injury and the recovery of motor function.

BACKGROUND: Latest studies show that low-energy extracorporeal shock wave therapy (ESWT) can upregulate levels of vascular endothelial growth factor (VEGF). VEGF can ease nervous tissue harm after spinal cord injury (SCI). This study aims to explore whether low-energy ESWT can promote expression of VEGF, protect nervous tissue after SCI, and improve motor function. METHODS: Ninety adult female rats were divided into the following groups: Group A (simple laminectomy), Group B (laminectomy and low-energy ESWT), Group C (spinal cord injury), and Group D (spinal cord injury and low-energy ESWT). Impinger was used to cause thoracic spinal cord injury. Low-energy ESWT was applied as treatment after injury three times a week, for 3 weeks. After SCI, the Basso, Beattie, and Bresnahan (BBB) scale was used to evaluate motor function over a period of 42 days at different time points. Hematoxylin and eosin (HE) staining was used to evaluate nerve tissue injury. Neuronal nuclear antigen (NeuN) staining was also used to evaluate loss of neurons. Polymerase chain reaction was used to detect messenger RNA (mRNA) expression of VEGF and its receptor fms-like tyrosine kinase 1 (Flt-1). Immunostaining was used to evaluate VEGF protein expression level in myeloid tissue. RESULTS: BBB scores of Groups A and B showed no significant result related to dyskinesia. HE and NeuN staining indicated that only using low-energy ESWT could not cause damage of nervous tissue in Group B. Recovery of motor function at 7, 35, and 42 days after SCI in Group D was better than that in Group C (P<0.05). Compared with Group C, number of NeuN-positive cells at 42 days after SCI increased significantly (P<0.05). The mRNA levels of VEGF and Flt-1 and VEGF expression at 7 days after SCI in Group D were significantly higher than those in Group C (P<0.05). CONCLUSION: Low-energy ESWT promotes expression of VEGF, decreases secondary damage of nerve tissue, and improves recovery of motor function. It can be regarded as one mode of clinical routine adjunctive therapy for spinal injury.

Small ubiquitin-like Modifier (SUMO) modification inhibits GLI2 protein transcriptional activity in vitro and in vivo.

The Gli transcription factors are key downstream mediators of the Hedgehog (Hh) signaling pathway. How the activities of Gli transcription factors are regulated by upstream Hh signaling events and protein modifications are not fully understood. Here we show that GLI2 is conjugated by small ubiquitin-like modifier (SUMO) at lysine residues 630 and 716 in the cell. The level of GLI2 sumoylation is reduced by either mutations in six serine residues that are normally phosphorylated by protein kinase A (PKA) or stimulation by HH. This suggests that PKA phosphorylation enhances GLI2 sumoylation, whereas HH signaling inhibits it. In addition, mutation of these two lysines into arginine residues significantly increases GLI2 transcriptional activity in a cell-based reporter assay. The same mutations in the GLI2 locus also result in an increase in GLI2 activity in the mouse. Interestingly, GLI2 can interact with HDAC5 (histone deacetylase 5), but the GLI2 mutant cannot. Taken together, our results suggest that SUMO modification inhibits GLI2 transcriptional activity by recruiting HDAC5.

The characteristics of acromegalic patients with hyperprolactinemia and the differences in patients with merely GH-secreting adenomas: clinical analysis of 279 cases.

OBJECTIVE: The aim of this study is to evaluate clinical data from a large cohort of acromegalic patients with and without hyperprolactinemia. DESIGN AND METHODS: Between January 2002 and June 2010, a set of data on 279 acromegalic patients undergoing transsphenoidal surgery was available. Based on preoperative GH and prolactin (PRL) levels, patients were divided into GH and GH+PRL groups. A stabilization or a further improvement of postoperative changes in clinical, hormonal, immunohistochemical, and magnetic resonance imaging parameters was observed in all patients throughout the follow-up period. RESULTS: The GH group had significantly more coarse facial features, large hands and feet, hypertension, and diabetes mellitus compared with the GH+PRL group but significantly less menstrual disorders (13.8 vs 54.3%, P<0.001) and galactorrhea (3.1 vs 22.4%, P<0.001). The GH group had a higher age at diagnosis compared with the GH+PRL group (45.6 ± 13.9 vs 40.4 ± 11.4 years, P=0.001). The GH group had a smaller mean maximal diameter of the adenomas (2.2 ± 0.9 vs 2.6 ± 1.1 cm, P=0.004). There were no significant correlations between hormone levels and the immunohistochemical results. According to the criteria for hormonal cure of acromegaly, the surgical control rates in the GH and GH+PRL groups were 68.4 and 59.7% respectively (P=0.187). Tumor size was an important factor that affected the results of the operations. The rates of surgical control in GH and GH+PRL groups were 80.7 and 69.1% respectively (P=0.037), and the recurrence rates in the two groups were 7.1 and 11.3% respectively (P=0.185). CONCLUSIONS: Compared with patients with merely GH-secreting adenomas, acromegalic patients with hyperprolactinemia are characterized by an earlier onset of disease, lesser acromegalic features, lower GH levels, but larger tumor sizes, whereas in female patients, GH-PRL secreting adenomas are associated with higher incidences of menstrual disorders and galactorrhea.

Surgical outcomes and prognostic factors of transsphenoidal surgery for prolactinoma in men: a single-center experience with 87 consecutive cases.

Context Little systematic data on male prolactinomas treated with surgery are available. Objective To clarify the clinical features and confirm the efficacy of transsphenoidal surgery for male prolactinomas and predictive factors after initial surgery. Patients and methods This retrospective study included 87 male patients with prolactinoma treated by transsphenoidal surgery at an academic medical center. Hormonal and visual status, remission rates, and the rate of tumor relapse, as well as predictive factors, were evaluated. Results Postoperative initial remission was achieved in 52.9% of patients. The remission rate was markedly higher in microadenomas (83.3%) than in macroadenomas (44.9%). Logistic regression analysis showed that the predictive factors of the early negative outcomes were high preoperative prolactin (PRL) levels and tumor invasion. After a median follow-up of 45 months, the long-term remission rate was 42.5%, and relapse of hyperprolactinemia occurred in 19.6% of the cured patients. The 5-year recurrence-free survival was 78.2% (95% confidence interval, 62.3-88.1%). When surgery was followed by adjuvant treatment in uncured and recurrent patients, 78.8% of patients in the entire group in the absence of dopamine agonists obtained biochemical remission at the end of follow-up. Conclusion Transsphenoidal surgery is a viable treatment alternative for male prolactinomas. The remission rates of male patients with microadenomas and/or intrasellar macroprolactinomas by surgery alone remain excellent, and surgery followed by adjuvant therapy as necessary is required for optimizing management of male prolactinomas, especially for extrasellar macroprolactinomas. The early negative results are associated with preoperative PRL levels and tumor invasion.

CD147 expression in pituitary adenomas and its significance for clinical outcome.

CD147, a transmembrane glycoprotein member of the immunoglobulin superfamily of receptors, is involved in invasion and angiogenesis of some types of tumors; but its roles and clinicopathologic significance in pituitary adenomas are not clear. Using immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction, we measured the expression of CD147, matrix metalloproteinase-2, and Ki-67 in 74 pituitary adenomas and evaluated the associations of CD147 with matrix metalloproteinase-2, Ki-67 labeling index, clinicopathologic characteristics, and prognosis. The CD147 protein was expressed in 35 (87.5%) of 40 invasive and in 16 (47.1%) of 34 noninvasive pituitary adenomas; and matrix metalloproteinase-2, in 32 (80.0%) and in 14 (41.2%) of 34, respectively. The Ki-67 labeling index was 3.93% +/- 2.48% for invasive samples and 1.32% +/- 1.04% for noninvasive ones. In addition, the expression of CD147 was positively correlated with matrix metalloproteinase-2, Ki-67 labeling index, or both in invasive pituitary adenomas (P< .01 and P< .01, respectively). All of the 4 recurrent adenomas were concurrently positive for CD147 and matrix metalloproteinase-2, and the Ki-67 labeling indexes of all were greater than 3%. Thus, CD147 may play a pivotal role in the development and progression of invasive pituitary adenomas and also be a useful prognostic biomarker.