Downregulation of long noncoding RNA H19 rescues hippocampal neurons from apoptosis and oxidative stress by inhibiting IGF2 methylation in mice with streptozotocin-induced diabetes mellitus.

The diabetes mellitus (DM)-induced reduction of neurogenesis in the hippocampus is consequently accompanied by cognitive decline. The present study set out to define the critical role played by long noncoding RNA H19 (lncRNA H19) in the apoptosis of hippocampal neurons, as well as oxidative stress (OS) in streptozotocin (STZ)-induced DM mice through regulation of insulin-like growth factor 2 (IGF2) methylation. The expression of lncRNA H19 in the hippocampal neurons and surviving neurons were detected. Hippocampal neurons were cultured and transfected with oe-H19, sh-H19, oe-IGF2, or sh-IGF2, followed by detection of the expressions of IGF2 and apoptosis-related genes. Determination of the lipid peroxide and glutathione levels was conducted, while antioxidant enzyme activity was identified. The IGF2 methylation, the binding of lncRNA H19 to DNA methyltransferase, and the binding of lncRNA H19 to IGF2 promoter region were detected. DM mice exhibited high expressions of H19, as well as a decreased hippocampal neurons survival rate. Higher lncRNA H19 expression was found in DM. Upregulated lncRNA H19 significantly increased the expression of Bax and caspase-3 but decreased that of Bcl-2, thus promoting the apoptosis of hippocampal neuron. Besides, upregulation of lncRNA H19 induced OS. LncRNA H19 was observed to bind specifically to the IGF2 gene promoter region and promote IGF2 methylation by enriching DNA methyltransferase, thereby silencing IGF2 expression. Taken together, downregulated lncRNA H19 reduces IGF2 methylation and enhances its expression, thereby suppressing hippocampal neuron apoptosis and OS in STZ-induced (DM) mice.

Effects of microRNA-21 on Nerve Cell Regeneration and Neural Function Recovery in Diabetes Mellitus Combined with Cerebral Infarction Rats by Targeting PDCD4.

We aimed to determine the effect and mechanism of microRNA-21 (miR-21) on nerve cell regeneration and nerve functional recovery in diabetes mellitus combined with cerebral infarction (DM + CI) rats by targeting PDCD4. A total of 125 male Wistar rats were selected for DM + CI rat model construction and assigned into the blank, miR-21 mimics, mimics control, miR-21 inhibitor, inhibitor control, miR-21 inhibitor + si-PDCD4 and si-PDCD4 groups. And, 20 healthy rats were selected for the normal group. Triphenylterazolium chloride (TTC) staining and HE staining were used for determination of the area of CI and pathological changes, respectively. Behaviors of rats in the eight groups were determined by forelimb placement test and balance beam walking test. Immunohistochemical staining, double immunofluorescence staining assay, Western blotting, and qRT-PCR were used to detect expressions of miR-21, PDCD4, HNA, Nestin, NeuN, ß-III-Tub, PTEN, FasL, and GFAP. DNA laddering and TUNEL staining was used for cell apoptosis. TTC and HE staining confirmed that 87.5% rats were induced into CI + DM models successfully. Results of forelimb placement test and balance beam walking test showed that miR-21 mimics, and si-PCDC4 improved the nerve defect of model rats. Comparing with the blank group at the same time, rats in the miR-21 inhibitor group displayed significant decrease in the forelimb placement test score, significant increase in the balance beam walking test score, and exacerbation of nerve defect, while rats in the miR-21 mimics and si-PCDC4 groups displayed significant increase in forelimb placement test score and significant decrease in the balance beam walking test score and improvement of nerve defect situation. The HNA, Nestin, and PDCD4 expressions were decreased and the NeuN, ß-III-Tub, and GFAP expressions were increased in the miR-21 mimics and si-PDCD4 groups comparing with the blank group. The results of miR-21 inhibitor group were on the contrary. In comparison to the blank group, the miR-21 mimics group and the si-PDCD4 had lower miR-21 expressions and higher expressions of PDCD4, PTEN, and FasL, while the miR-21 inhibitor group was in the opposite trend. The results of qRT-PCR were the same with Western blotting. The expressions of fluorescence in other groups were higher than the normal group; compared with the blank group, the miR-21 mimics group and the si-PDCD4 group had lower fluorescence expression and DNA ladder. However, the fluorescence expressions and DNA ladder of miR-21 inhibitor group increased markedly in contrast with the blank group. Comparing with the blank group, BrdU+/DEX+ fluorescence intensity significantly enhanced in the miR-21 mimics and si-PDCD4 groups and significantly reduced in the miR-21 inhibitor group. And, comparing with the blank group, in the miR-21 mimics group, the signal strength of luciferase carrying the wild-type PDCD4 was reduced by 25%. The present studies demonstrated that miR-21 could promote the nerve cell regeneration, suppress apoptosis of nerve cells in DM + CI rats and improves the nerve defect situation of DM + CI rats by inhibiting PDCD4.

Effect of overexpression of HOX genes on its invasive tendency in cerebral glioma.

Transcription factors encoded by HOX genes are vital in the determination of cell fate and identity during embryonic development. In certain malignancies, HOX genes also behave as oncogenes. The present study demonstrated suppression of the invasive tendency of glioblastoma multiforme U-118 and U-138 cells by the introduction of the antisense fragments of HOXA6 and B13 genes using electroporation. The invasion index indicated 79 and 72% reductions in the invasive ability of antisense HOXA6 and B13, respectively. No significant differences in the invasive index of the parental and mock cells of each HOX gene were observed (invasive index, 0.75-0.91; P=0.05). A reduction in invasion tendency was also observed following betulinic acid (BA) treatment: The results from the matrigel assay analysis clearly demonstrated a significant inhibition in the invasive behaviour of U-118 and U-138 cell lines from day 15 following BA treatment, with a maximum effect on day 30. The invasion index demonstrated 62 and 65% reductions in invasion ability in the U-118 and U-138 cell lines, respectively. The suppression of HOXC6 and B13 expression by the introduction of the corresponding antisense fragments in addition to BA reduced invasion tendency in U-118 and U-138 cell lines. The mechanism underlying the association between the HOX gene and invasive behavior in glioma cells is yet to be understood. However, the anti-invasive behavior of BA may aid understanding of the mechanism in future studies.

Honokiol induces cell cycle arrest and apoptosis via p53 activation in H4 human neuroglioma cells.

OBJECTIVE: To investigate the signal pathway of honokiol-induced apoptosis in H4 human neuroglioma cells and to evaluate whether p53 signaling and cell cycle arrest were involved in honokiol-treated H4 human neuroglioma cells. METHODS: The cell viability was detected by the CCK8 assay. The cell apoptosis was assessed by annexin V-PI double-labeling staining and hoechst 33342 staining. The protein expression of cell cycle regulators and tumor suppressors were analyzed by western blotting. RESULTS: Treatment of H4 human neuroglioma cells with honokiol induced cell death in a dose-and time-dependent manner by using CCK8 assay. Consistent with the CCK8 assay, the flow cytometry results showed that the proportion of the apoptosis cells increased after honokiol when compared with untreated group. Moreover, H4 human neuroglioma cells exposed to honokiol, resulted in an accumulation of cells in S and G2/M phase. Apoptotic bodies were clearly observed in human neuroglioma cells when treated with honokiol and then stained with Hoechst 33342. The expression of Cyclin B1, CDC2 and cdc25C were downregulated, however, the expression of p-CDC2 and p-cdc25c was significantly upregulated when the neuroglioma cells were exposed to honokiol. Moreover, p53, p21 and Bax/Bcl-2 were significantly upregulated by honokiol treatment. CONCLUSIONS: These results confirmed that honokiol could induce apoptosis in human neuroglioma cells, the underlying molecular mechanisms, at least partially, through activation p53 signaling and induction of cell cycle arrest.

Ethyl acetate extracts of Fructus Ligustri Lucide induce cell apoptosis in human neuroglioma cell.

OBJECTIVE: Previous studies have shown that Fructus Ligustri Lucide (FLL) can be used to improve the tumor cells sensitivity to chemotherapeutics and promote cell death. However, the mechanism by which FLL mediate this effect is unclear. In the present study, ethyl acetate extracts of FLL induced cell apoptosis in human neuroglioma cell was investigated. METHODS: The cell viability was detected by the CCK8 assay. The cell apoptosis was assessed by annexin V-PI double-labeling staining and hoechst 33342 staining. The protein expression of cell cycle regulators and tumor suppressors were analyzed by western blotting. RESULTS: Treatment of human neuroglioma cell with FLL induced cell death in a dose-and time-dependent manner by using CCK8 assay. Consistent with the CCK8 assay, the flow cytometry results showed that the proportion of the early and terminal phase of apoptosis cells had gained after FLL treatment as compared to untreatment group. Moreover, human neuroglioma cells were exposed to the ethyl acetate extracts of FLL for 48 h, which resulted in an accumulation of cells in G2/Mphase. Apoptotic bodies were clearly observed in human neuroglioma cells that had been treated with FLL for 48 h and then stained with Hochest 33342. The expression of Cyclin B1, CDC2 and cdc25C were downregulated upon FLL treatment in human neuroglioma cells. The expression level of Cyclin B1, CDC2 and cdc25C was negatively correlated with the time of treatment by FLL. In contrast, p53, p21 and p16 were obviously upregulated by FLL treatment in a time-dependent manner. CONCLUSIONS: These results confirmed that FLL could induce apoptosis in human neuroglioma cells, the underlying molecular mechanisms, at least partially, through activation p21/p53 and suppression CDC2/cdc25C signaling in vitro.