LINC00461 Regulates the Recurrence of Large B Cell Lymphoma through the miR-411-5p/BNIP3 Pathway.

Objective: To analyze the mechanism of LINC00461 regulating the recurrence of diffuse large B cell lymphoma (DLBCL) through microRNA (miR)-411-5p/BCL2 interacting protein 3 (BNIP3) pathway. Methods: DLBCL samples in TCGA and GSE12453 were used for differential analysis to find long noncoding RNA (lncRNA) related to DLBCL recurrence. The 4 DLBCL data with the highest and lowest expression levels of LINC00461 in the TCGA database were selected for GSEA enrichment analysis. The targeting relationships of miR-411-5p with LINC00461 and BNIP3 were verified by the dual luciferase report. Blood samples from DLBCL patients were used to analyze the correlation between miR-411-5p and LINC00461 or BNIP3. LINC00461, miR-411-5p, or BNIP3 was overexpressed or silenced by transfection, and a tumor-bearing nude mice model was constructed to detect their effects on proliferation and apoptosis. Results: The level of LINC00461 in DLBCL was significantly higher than that in normal cases, and the level in recurrence DLBCL was significantly higher than that in nonrecurrence. The enrichment analysis results showed that the function of LINC00461 was closely related to apoptosis. The results shown that miR-411-5p bound to LINC00461 and BNIP3 and was negatively correlated with LINC00461 and BNIP3 mRNA in blood of DLBCL patients. Suppressing the level of LINC00461 inhibited cell proliferation and induced apoptosis. The inhibition of LINC00461 or overexpression of miR-411-5p reduced the expression of BNIP3 protein, thereby inducing apoptosis at the in vivo and in vitro levels. Conclusion: LINC00461 may induce miR-411-5p to "sponge," thereby increasing the expression of BNIP3 protein, and exerting the function of inhibiting apoptosis and promoting DLBCL recurrence.

Associations between Huwe1 and autophagy in rat cerebral neuron oxygen­glucose deprivation and reperfusion injury.

Autophagy and the ubiquitin proteasome system (UPS) are two major protein degradation pathways involved in brain ischemia. Autophagy can compensate for UPS impairment­induced cellular dysfunction. HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1 (Huwe1), an E3 ubiquitin ligase, serves critical roles in nervous system plasticity, regeneration and disease. However, the role of Huwe1 in autophagy in brain ischemia/reperfusion (I/R) injury remains unknown. The aim of the present study was to investigate the crosstalk between autophagy and the UPS in brain ischemia. The present study established an oxygen­glucose deprivation and reperfusion (OGD/R) model in rat primary cortex neurons in vitro. Lentiviral interference was used to silence the expression of Huwe1. An autophagy promoter (rapamycin), an autophagy inhibitor (wortmannin) and a JNK pathway inhibitor (SP600125) were also used in the current study. Cellular autophagy­related proteins, including Beclin­1, autophagy related (ATG) 7, ATG5, ATG3 and microtubule associated protein 1 light chain 3 α, and apoptosis­related proteins, such as P53, cleaved caspase 3, Bax and Bcl2, were detected via western blotting and immunocytochemistry. Neuronal apoptosis was evaluated using a TUNEL assay. The results demonstrated that silencing Huwe1 increased the expression levels of autophagy­related proteins at 24 h after OGD/R. Treatment with a JNK inhibitor or cotreatment with Huwe1 shRNA significantly increased autophagy. Rapamycin increased apoptosis under OGD/R conditions. However, treatment with Huwe1 shRNA decreased the number of TUNEL­positive cells at 24 h after OGD/R. Cotreatment with Huwe1 shRNA and wortmannin alleviated neuronal apoptosis under OGD/R conditions compared with cotreatment with DMSO. Collectively, the present results suggested that silencing Huwe1 was accompanied by a compensatory induction of autophagy under OGD/R conditions. Furthermore, the JNK pathway may be a key mediator of the interaction between Huwe1 and autophagy in response to UPS impairment.

Silencing Huwe1 reduces apoptosis of cortical neurons exposed to oxygen-glucose deprivation and reperfusion.

HECT, UBA and WWE domain-containing 1 (Huwe1), an E3 ubiquitin ligase involved in the ubiquitin-proteasome system, is widely expressed in brain tissue. Huwe1 is involved in the turnover of numerous substrates, including p53, Mcl-1, Cdc6 and N-myc, thereby playing a critical role in apoptosis and neurogenesis. However, the role of Huwe1 in brain ischemia and reperfusion injury remains unclear. Therefore, in this study, we investigated the role of Huwe1 in an in vitro model of ischemia and reperfusion injury. At 3 days in vitro, primary cortical neurons were transduced with a control or shRNA-Huwe1 lentiviral vector to silence expression of Huwe1. At 7 days in vitro, the cells were exposed to oxygen-glucose deprivation for 3 hours and reperfusion for 24 hours. To examine the role of the c-Jun N-terminal kinase (JNK)/p38 pathway, cortical neurons were pretreated with a JNK inhibitor (SP600125) or a p38MAPK inhibitor (SB203508) for 30 minutes at 7 days in vitro, followed by ischemia and reperfusion. Neuronal apoptosis was assessed by TUNEL assay. Protein expression levels of JNK and p38MAPK and of apoptosis-related proteins (p53, Gadd45a, cleaved caspase-3, Bax and Bcl-2) were measured by western blot assay. Immunofluorescence labeling for cleaved caspase-3 was performed. We observed a significant increase in neuronal apoptosis and Huwe1 expression after ischemia and reperfusion. Treatment with the shRNA-Huwe1 lentiviral vector markedly decreased Huwe1 levels, and significantly decreased the number of TUNEL-positive cells after ischemia and reperfusion. The silencing vector also downregulated the pro-apoptotic proteins Bax and cleaved caspase-3, and upregulated the anti-apoptotic proteins Gadd45a and Bcl-2. Silencing Huwe1 also significantly reduced p-JNK levels and increased p-p38 levels. Our findings show that downregulating Huwe1 affects the JNK and p38MAPK signaling pathways as well as the expression of apoptosis-related genes to provide neuroprotection during ischemia and reperfusion. All animal experiments and procedures were approved by the Animal Ethics Committee of Sichuan University, China in January 2018 (approval No. 2018013).

p57KIP2­mediated inhibition of human trophoblast apoptosis and promotion of invasion in vitro.

Placental hypoxia serves a role in the early stages of normal pregnancy and is involved in the pathophysiology of preeclampsia. Previously, it was suggested that p57kinase inhibitory protein (KIP)2 regulates the cell cycle during embryogenesis and apoptosis. Recent evidence has indicated that p57KIP2 is increased in preeclamptic placentas and absence of p57KIP2 induces preeclampsia­type symptoms in rats. However, effects of p57KIP2 on apoptosis under hypoxic conditions remain to be elucidated. In the present study, HTR­8/SVneo trophoblasts were cultured under hypoxic conditions (2% O2). Knockdown using small interfering (si)RNA and overexpression of p57KIP2 were utilized to explore the biological function of p57KIP2 in apoptosis and cell function in vitro. Furthermore, expression of p57KIP2 and apoptosis were evaluated by western blotting, flow cytometry and TUNEL assays, and the response of trophoblasts to hypoxia and the role of p57KIP2 in trophoblast migration and invasion was assessed. The role of p57KIP2 in the JNK signaling pathway in HTR­8/SVneo trophoblasts was further studies. In vitro, protein expression of p57KIP2 was increased in HTR­8/SVneo cells exposed to 2% O2. Exogenous p57KIP2 overexpression significantly decreased the expression of pro­apoptosis proteins, including p53, Bax and cleaved caspase3, under hypoxic conditions for 24 h. In addition, knockdown of p57KIP2 increased the response to apoptosis following hypoxia for 24 h. The present study revealed that overexpression of p57KIP2 decreased the levels of phosphorylated­JNK. JNK inhibitor treatment combined with the overexpression of p57KIP2 significantly decreased the levels of apoptosis and increased cell invasion and migration. Taken together, p57KIP2 knockdown significantly increased apoptosis in HTR­8/SVneo cells exposed to 2% O2, whereas overexpression of p57KIP2 had opposite effects, mediated by the JNK/stress activated protein kinase (SAPK) signaling pathway. The results indicated that hypoxia­induced expression of p57KIP2 promoted trophoblast migration and invasion by mediating the JNK/SAPK signaling pathway, which is crucial during placentation. These results may provide a novel molecular mechanism to understand the involvement of p57KIP2 in the pathogenesis of preeclampsia.

Effects of skeletal size of the lumbar spine on areal bone density, volumetric bone density, and the diagnosis of osteoporosis in postmenopausal women in China.

To understand the effects of skeletal size of the lumbar spine on areal bone mineral density (aBMD), volumetric bone mineral density (vBMD), and the diagnosis of osteoporosis in postmenopausal women, we measured the projected bone area, bone mineral content (BMC), aBMD, and vBMD at the anteroposterior and lateral lumbar spines in a population of 1081 postmenopausal Chinese women, 42 to 86 years of age. The results indicated that, at the anteroposterior and lateral lumbar spine, there were significant positive correlations between bone area and both BMC ( r = 0.606; P = 0.000 and r = 0.610; P = 0.000) and aBMD ( r = 0.270; P = 0.000 and r = 0.182; P = 0.000), but not vBMD ( r = -0.055; P = 0.000 and r = 0.000; P = 0.929). When bone area at the anteroposterior spine changed by +/-1 SD, the BMC, aBMD, and vBMD correspondingly changed by 28.2%, 10.1%, and 1.69% on the basis of their respective means. When a variation of +/-1 SD was observed in bone area at the lateral spine, BMC and aBMD, correspondingly changed by 25.9% and 6.18% on the basis of their respective means, while vBMD indicated no change. Through comparisons among large-, intermediate-, and small-bone area groups, significant differences were found in the means of subjects' heights, weights, BMC, and vBMD at the anteroposterior and lateral lumbar spines, as well as in the detection rates of osteoporosis by aBMD ( P = 0.000). Detection rates of osteoporosis by aBMD at the anteroposterior spine and by aBMD at the lateral spine, and by vBMD were 44.1%, 55.5%, and 49.7%, respectively, in the total population; 31.4%, 41.7%, and 53.7%, respectively, in the large-bone area group; 43.3%, 55.9%, and 50.5%, respectively, in the intermediate-bone area group; and 61.7%, 70.0%, and 42.5%, respectively, in the small-bone area group. No significant differences were found in the detection rates of osteoporosis by vBMD among the groups. The results of multiple linear regression revealed that the major factors influencing skeletal size and aBMD of the lumbar spine were height and weight. Therefore, in menopausal women of the same ethnic group and age, the skeletal size of the lumbar spine would have significant influence upon aBMD and the diagnosis of osteoporosis, i.e., the larger the spinal size, the greater the aBMD and the lower the osteoporosis detection rate, while, conversely, the smaller the skeletal size, the smaller the aBMD and the higher the osteoporosis detection rate. When we use aBMD of the lumbar spine to diagnose osteoporosis in a population with different body sizes, we need to take this body size difference into account. When we use vBMD to diagnose osteoporosis, the effect of body size on BMD will diminish.

[Effects of long-term replacement therapy of compound nylestriol tablet or low-dose 17 beta-estradiol on the expression of nerve growth factor in OVX rat hippocampal formation].

OBJECTIVE: To determine the effects of long-term estrogen deficiency and replacement therapy of compound nylestriol tablet or low-dose 17 beta-estradiol on the expression of nerve growth factor (NGF) in rat hippocampal formation. METHODS: Fifty 7 month-old female Sprague-Dawley rats were randomly divided into 5 groups: normal control, sham operated (SHAM), ovariectomized (OVX), OVX plus 17 beta-estradiol (OVX/ERT), and OVX plus compound nylestriol tablet (OVX/NL) groups. Immunohistochemistry of NGF was used to quantitatively determine the levels of expression of NGF using cell counting and imaging system in ovariectomized rat hippocampal formation. RESULTS: The number and optical density of NGF-positive neurons of all hippocampal subregions and dentate gyrus in OVX rats were obviously lower than those of the normal control, SHAM, OVX/NL, and OVX/ERT rats. CONCLUSION: Long-term estrogen deficiency can lead to a decrease of NGF expression in hippocampal formation, while the replacement of low-dose 17 beta-estradiol or compound nylestriol tablet can equally preserve the expression of NGF to a normal level, showing a neurotrophic effect of estrogen.

The effect of low dose nylestriol-levonorgestrel replacement therapy on bone mineral density in women with postmenopausal osteoporosis.

OBJECTIVE: Recently our studies have shown that nylestriol in combination with levonorgestrel prevented bone loss, decreased bone turnover rate and increased the maximal loading of bone without obvious side effects in retinoic acid (RA) induced osteoporotic rats. In addition to the animal experiments, we evaluate the effect of Compound Nylestriol Tablet (CNT) on bone mineral density (BMD) in women with postmenopausal osteoporosis. Compound Nylestriol Tablet, which contains 0.5 mg of nylestriol (cyclopentylethinyl estriol) and 0.15 mg of levonorgestrel per tablet, was authorized as a new anti-osteoporotic agent for clinical trial in postmenopausal osteoporosis. METHODS: One year's clinical observation was performed in 191 eligible patients who were randomly divided into two groups (A and B). In group A, 119 patients were treated for one year with CNT (one tablet per week) and in group B, 72 patients with placebo. Bone mineral density of lumbar antero-posterior spine (L1-L4), lateral spine, total hip and total forearm positions including radius+ulna at the ultra distal areas, mid areas, and one-third areas, were measured before and after treatment. Biochemical parameters and effects of CNT on uterus, and breast were observed. RESULTS: We found that patients treated with CNT had a significant decrease of bone loss in total forearm, including radius+ulna at the ultra distal, mid, and 1/3 areas compared with control subjects (all P < 0.05). An improved BMD tendency could be seen at the lumbar spine. There were no differences in the observed biochemical variables. No side-effects on uterus, or mammary glands observed. None of the patients had uterine bleeding or vertebral fractures during one year's CNT treatment. CONCLUSION: These data suggested that CNT is effective, safe and convenient in treating postmenopausal osteoporosis.

Effects of different nylestriol/levonorgestrel dosages on bone metabolism in female Sprague-Dawley rats with retinoic acid-induced osteoporosis.

OBJECTIVE: To determine an optimal dosage combination for a nylestriol/levonorgestrel (NYL/LNG) regimen in the treatment of female Sprague-Dawley (SD) rats with retinoic acid (RA)-induced osteoporosis in order to examine the rationale of NYL/LNG use for postmenopausal women. METHODS: This study included two sets of experiments; one was designed to confirm the success of the RA-induced osteoporosis model involving 48 SD rats, and the other to determine the optimal dosage combination of NYL/LNG. In the second set of experiments, a total of 160 female SD rats at 7 months of age were randomly divided according to their basal body weights into 16 groups (10 in each). Treatment dosages of NYL and LNG were arrayed in a 2 factor-4 level (2(4)) factorial design, i.e., NYL level I (N1 0.015 mg/kg), level II (N(2) 0.05 mg/kg), level III (N3 0.15 mg/kg) and level IV (N4 0.5 mg/kg) and LNG level I (L1 0.005 mg/kg), level II (L2 0.015 mg/kg), level III (L3 0.05 mg/kg) and level IV (L4 0.15 mg/kg). For 14 d, 70 mg/kg/d RA was given intragastrically to establish the osteoporotic model and NYL and LNG were then administered in different dosages on alternate days over a 3 wk period. Subsequently, body weight, uterine weight, endometrial status, Bone mineral density (BMD), bone turnover, and morphometry as well as parameters of biomechanics, serum sex hormones and lipids and estrogen receptor-alpha expression were determined for these rats. RESULTS: Uterine weights at L2, L3, and L4 dosage levels were significantly lower than those at L1 (P < 0.05). Serum estradiol at N4 and progesterone at N2, N3, and N4 had decreased. Bone mineral density at distal tibiae (R5) in L and LA, and at proximal femora (R3) in L4 had increased. The peak loading of lumbar vertebrae at N3 was higher than that at N1, N2, and N4 dosage levels and that of femora higher at N3 than that at N1 and N4. Serum alkaline phosphatase (ALP) levels at N3, N4, L2, L3, and L4 dosage levels were lower than those at N1, N2, and L1 (P < 0.05). Moreover, there were lower levels of trabecular separation (Tb.Sp) at N3 and N4. The mineral apposition rates (MAR) at N2, N3, N4, L2, L3, and L4 were lower than those at N1 or L1. Bone formation rates (BFR) at L3 and L4 had significantly decreased as compared with that at L1. Levels of serum total cholesterol (TC) at N2, N3, and N4 were lower than that at N1 (P < 0.05). Marked squamatization of uterine endometrium with an increased expression of estrogen receptor (ER)-alpha was observed at N4. CONCLUSION: The dosage of 0.15 mg/kg NYL prevented bone loss, decreased bone turnover rate and increased the maximal loading of bone without obvious side effects in RA-induced osteoporotic rats. The addition of 0.015-0.15 mg/kg of LNG (L2-L4) reduced uterine weights and serum ALP levels. Overall results showed that 0.15 mg/kg of NYL in combination with 0.015 mg/kg of LNG produced beneficial effects on bone metabolism in RA-induced osteoporotic rats.