LncRNA AGAP2 antisense RNA 1 stabilized by insulin-like growth factor 2 mRNA binding protein 3 promotes macrophage M2 polarization in clear cell renal cell carcinoma through regulation of the microRNA-9-5p/THBS2/PI3K-Akt pathway.

BACKGROUND: Increasing evidence highlights the potential role of long non-coding RNAs (lncRNAs) in the biological behaviors of renal cell carcinoma (RCC). Here, we explored the mechanism of AGAP2-AS1 in the occurrence and development of clear cell RCC (ccRCC) involving IGF2BP3/miR-9-5p/THBS2. METHODS: The expressions of AGAP2-AS1, IGF2BP3, miR-9-5p, and THBS2 and their relationship were analyzed by bioinformatics. The targeting relationship between AGAP2-AS1 and miR-9-5p and between miR-9-5p and THBS2 was evaluated with their effect on cell biological behaviors and macrophage polarization assayed. Finally, we tested the effect of AGAP2-AS1 on ccRCC tumor formation in xenograft tumors. RESULTS: IGF2BP3 could stabilize AGAP2-AS1 through m6A modification. AGAP2-AS1 was highly expressed in ccRCC tissues and cells. The lentivirus-mediated intervention of AGAP2-AS1 induced malignant behaviors of ccRCC cells and led to M2 polarization of macrophages. In addition, THBS2 promoted M2 polarization of macrophages by activating the PI3K/AKT signaling pathway. AGAP2-AS1 could directly bind with miR-9-5p and promote the expression of THBS2 downstream of miR-9-5p. These results were further verified by in vivo experiments. CONCLUSION: AGAP2-AS1 stabilized by IGF2BP3 competitively binds to miR-9-5p to up-regulate THBS2, activating the PI3K/AKT signaling pathway and inducing macrophage M2 polarization, thus facilitating the development of RCC.

Culture and identification of neonatal rat brain-derived neural stem cells.

BACKGROUND: Timing of passaging, passage number, passaging approaches and methods for cell identification are critical factors influencing the quality of neural stem cells (NSCs) culture. How to effectively culture and identify NSCs is a continuous interest in NSCs study while these factors are comprehensively considered. AIM: To establish a simplified and efficient method for culture and identification of neonatal rat brain-derived NSCs. METHODS: First, curved tip operating scissors were used to dissect brain tissues from new born rats (2 to 3 d) and the brain tissues were cut into approximately 1 mm3 sections. Filter the single cell suspension through a nylon mesh (200-mesh) and culture the sections in suspensions. Passaging was conducted with TrypLTM Express combined with mechanical tapping and pipetting techniques. Second, identify the 5th generation of passaged NSCs as well as the revived NSCs from cryopreservation. BrdU incorporation method was used to detect self-renew and proliferation capabilities of cells. Different NSCs specific antibodies (anti-nestin, NF200, NSE and GFAP antibodies) were used to identify NSCs specific surface markers and muti-differentiation capabilities by immunofluorescence staining. RESULTS: Brain derived cells from newborn rats (2 to 3 d) proliferate and aggregate into spherical-shaped clusters with sustained continuous and stable passaging. When BrdU was incorporated into the 5th generation of passaged cells, positive BrdU cells and nestin cells were observed by immunofluorescence staining. After induction of dissociation using 5% fetal bovine serum, positive NF200, NSE and GFAP cells were observed by immunofluorescence staining. CONCLUSION: This is a simplified and efficient method for neonatal rat brain-derived neural stem cell culture and identification.

A diagnostic model for differentiating tuberculous spondylitis from pyogenic spondylitis: a retrospective case-control study.

The purpose of this study was to describe and compare the clinical data, laboratory examination and imaging examination of tuberculous spondylitis (TS) and pyogenic spondylitis (PS), and to provide ideas for diagnosis and treatment intervention. The patients with TS or PS diagnosed by pathology who first occurred in our hospital from September 2018 to November 2021 were studied retrospectively. The clinical data, laboratory results and imaging findings of the two groups were analyzed and compared. The diagnostic model was constructed by binary logistic regression. In addition, an external validation group was used to verify the effectiveness of the diagnostic model. A total of 112 patients were included, including 65 cases of TS with an average age of 49 ± 15 years, 47 cases of PS with an average of 56 ± 10 years. The PS group had a significantly older age than the TS group (P = 0.005). In laboratory examination, there were significant differences in WBC, neutrophil (N), lymphocyte (L), ESR, CRP, fibrinogen (FIB), serum albumin (A) and sodium (Na). The difference was also statistically significant in the comparison of imaging examinations at epidural abscesses, paravertebral abscesses, spinal cord compression, involvement of cervical, lumbar and thoracic vertebrae. This study constructed a diagnostic model, which was Y (value of TS > 0.5, value of PS < 0.5) = 1.251 * X1 (thoracic vertebrae involved = 1, thoracic vertebrae uninvolved = 0) + 2.021 * X2 (paravertebral abscesses = 1, no paravertebral abscess = 0) + 2.432 * X3 (spinal cord compression = 1, no spinal cord compression = 0) + 0.18 * X4 (value of serum A)-4.209 * X5 (cervical vertebrae involved = 1, cervical vertebrae uninvolved = 0)-0.02 * X6 (value of ESR)-0.806 * X7 (value of FIB)-3.36. Furthermore, the diagnostic model was validated using an external validation group, indicating a certain value in diagnosing TS and PS. This study puts forward a diagnostic model for the diagnosis of TS and PS in spinal infection for the first time, which has potential guiding value in the diagnosis of them and provides a certain reference for clinical work.

ROCK inhibition enhances neurite outgrowth in neural stem cells by upregulating YAP expression in vitro.

Spontaneous axonal regeneration of neurons does not occur after spinal cord injury because of inhibition by myelin and other inhibitory factors. Studies have demonstrated that blocking the Rho/Rho-kinase (ROCK) pathway can promote neurite outgrowth in spinal cord injury models. In the present study, we investigated neurite outgrowth and neuronal differentiation in neural stem cells from the mouse subventricular zone after inhibition of ROCK in vitro. Inhibition of ROCK with Y-27632 increased neurite length, enhanced neuronal differentiation, and upregulated the expression of two major signaling pathway effectors, phospho-Akt and phospho-mitogen-activated protein kinase, and the Hippo pathway effector YAP. These results suggest that inhibition of ROCK mediates neurite outgrowth in neural stem cells by activating the Hippo signaling pathway.

Effect of spinal cord extracts after spinal cord injury on proliferation of rat embryonic neural stem cells and Notch signal pathway in vitro.

OBJECTIVE: To investigate the effect of the spinal cord extracts (SCE) after spinal cord injuries (SCIs) on the proliferation of rat embryonic neural stem cells (NSCs) and the expressions of mRNA of Notch1 as well as of Hes1 in this process in vitro. METHODS: The experiment was conducted in 4 different mediums: NSCs+PBS (Group A-blank control group), NSCs+SCE with healthy SD rats (Group B-normal control group), NSCs+SCE with SD rats receiving sham-operation treatment (Group C-sham-operation group) and NSCs+ SCE with SCIs rats (Group D-paraplegic group). Proliferative abilities of 4 different groups were analyzed by MTT chromatometry after co-culture for 1, 2, 3, 4 and 5 d, respectively. The expressions of Notch1 and Hes1 mRNA were also detected with RT-PCR after co-culture for 24 and 48 h, respectively. RESULTS: After co-culture for 1, 2, 3, 4 and 5 d respectively, the MTT values of group D were significantly higher than those of group A, group B and group C (P<0.05). However, there were no significantly differences regarding MTT values between group A, group B and group C after co-culture for 1, 2, 3, 4 and 5 d, respectively (P>0.05). Both the expressions of Notch1 and Hes1 mRNA of group D were significantly higher than those of other 3 groups after co-culture for 24 h and 48 h as well (P<0.05). But there was no difference oin expressions of Notch1 and Hes1 mRNA among group A, group B and group C after co-culture for 24 h and 48 h (P>0.05). There was no difference in expressions of Notch1 and Hes1 mRNA between 24 h and 48 h treatment in group D. CONCLUSIONS: SCE could promote the proliferation of NSCs. It is demonstrated that the microenvironment of SCI may promote the proliferation of NSCs. Besides, SCE could increase the expression of Notch1 and Hes1 mRNA of NSC. It can be concluded that the Notch signaling pathway activation is one of the mechanisms that locally injured microenvironment contributes to the proliferation of ENSC after SCIs. This process may be performed by up-regulating the expressions of Notch1 and Hes1 gene.