MicroRNA-29a-3p Regulates SH-SY5Y Cell Proliferation and Neurite Growth through Interaction with PTEN-PI3K/AKT/mTOR Signaling Pathway.

The effects of microRNA-29a-3p in the proliferation process of nerve cells are unclear. The purpose of this study is to delve into the regulatory role of microRNA-29a-3p, via interaction with phosphatase and tension homolog (PTEN), in the SH-SY5Y cell proliferation process. Different expressions of microRNA-29a-3p in the SH-SY5Y cells were constructed by transfected miRNA-29a-3p mimic and inhibitor. The effects of cell transfection and the mRNA expressions of PTEN, Akt, and mTOR were detected by qPCR. The expressions of PTEN, Akt, and mTOR protein and the phosphorylation levels of Akt and mTOR were examined using Western blotting. Nerve cell proliferation activity and neurite length of each group were measured and examined by the use of 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2Htetrazolium bromide (MTT), and morphological examination. We observed that the levels of PTEN mRNA and protein were distinctly decreased in the microRNA-29a-3p mimic group, but the expressions of the phosphorylated Akt and mTOR mRNA and protein were distinctly upregulated. In the transfected miRNA-29a-3p inhibitor SH-SY5Y cells, the expressions of miRNA-29a-3p were significantly suppressed; however, the expressions of PTEN gene and protein were significantly enhanced. The expressions of phosphorylated Akt and mTOR in the downregulated microRNA-29a-3p group distinctly were suppressed. The SH-SY5Y cell proliferation activity and neurite length in the upregulated microRNA-29a-3p group increased significantly. Our findings revealed that microRNA-29a-3p could enhance the proliferation activity of SH-SY5Y cells and promote neurite growth by inhibiting the expression of PTEN and regulating PI3K/Akt/mTOR signaling pathway.

The Neurotrophic Effects and Mechanism of Action for FK1706 in Neurorrhaphy Rat Models and SH-SY5Y Cells.

FK1706 is a novel non-immunosuppressive immunophilin ligand with neurotrophic activity and exerts its neurotrophic effect through NGF. The present study aimed to elaborate on the neurotrophic activity and the mechanism of action of FK1706 in end-to-side neurorrhaphy rats and SH-SY5Y cells. In the regenerating nerves of neurorrhaphy rats, FK1706 increased the thickness of myelin sheath and the level of nerve regeneration-related proteins. The mechanism of action of FK1706 on neurite regrowth was elucidated in vitro by incubating SH-SY5Y cells in different conditions (Control, NGF, FK1706, NGF + FK1706, NGF + FK1706 + geldanamycin). Under the conditions where NGF was used, the phosphorylation level of major proteins (Raf-1 and ERK) in the Ras/Raf/MAPK/ERK signaling pathway related to SH-SY5Y cell proliferation was significantly enhanced following the application of FK1706. The number of viable cells, cell viability and neurite length of SH-SY5Y cells was maximal when NGF and FK1706 were used simultaneously. The binding level of HSP90 and Raf-1 in FK1706 group was the highest. These results indicated that FK1706 could significantly promote nerve regeneration after neurorrhaphy. The putative mechanism of action stated that FK1706 could promote the binding of HSP90 and Raf-1, make Raf-1 continue to be activated, thereby affecting key proteins in the Ras/Raf/MAPK/ERK signaling pathway related to the neurotrophic effects of NGF to promote the proliferation and neurite regrowth of nerve cells.

Development and Validation of a Novel Recurrence Risk Stratification for Initial Non-Muscle Invasive Bladder Cancer in the Han Chinese Population.

Background: Some classification models for determining the risk of recurrence after transurethral resection of bladder tumor (TURBT) in patients with non-muscle invasive bladder cancer (NMIBC) had some shortcomings in clinical applications. This study aimed to investigate whether the European Organization for Research and Treatment of Cancer (EORTC) risk stratification was useful to predict the recurrence of NMIBC in the Han Chinese population. In addition, we developed and validated a novel risk stratification method for recurrence prediction of NMIBC. Methods: Excluding cases who do not meet the inclusion criteria, 606 patients with NMIBC from the First Affiliated Hospital of Zhengzhou University were included in the testing and validation groups. The recurrence-free survival (RFS) curve according to the EORTC risk classifications was calculated by the Kaplan-Meier and the log-rank test methods. Receiver operating characteristic (ROC) curve analysis was used to estimate the diagnosis value for recurrence. We built a logistic regression model for recurrence in NMIBC patients combining the independent recurrence prognostic factors. One external validation group including 166 patients with NMIBC from the Zhongnan Hospital of Wuhan University was also used to assess the logistic regression model. Results: There was no significant difference in RFS rates between the groups grouped according to EORTC. We constructed a novel risk model to predict recurrence by classifying patients into two groups using ten independent prognostic factors [bladder cancer-specific nuclear matrix protein 4 (BLCA-4), bladder tumour antigen (BTA), nuclear matrix protein 22 (NMP22), carcinoembryonic antigen (CEA), body mass index, smoking, family history of bladder cancer, occupational exposure to aromatic amine chemicals, number of tumours, bladder instillation of chemotherapeutic agents] to predict tumour recurrence based on logistic regression analyses (testing group). According to the novel recurrence risk classification, there was a significant difference in 5-year RFS rates between the low-risk group and the high-risk group (Validation group and the external validation group). Conclusions: Our novel classification model can be a useful tool to predict recurrence risk in the Han Chinese population with NMIBC.

Synergistic antitumour effects of triptolide plus 10-hydroxycamptothecin onbladder cancer.

BACKGROUND: Hydroxycamptothecin (HCPT) is used as a clinical chemotherapy regimen to treat bladder cancer, but more efficacious novel combination treatments are needed. METHODS: Cultured bladder cancer cell lines EJ and UMUC3 were treated with triptolide (TPL) and/or HCPT. A flow cytometry approach was used to detect cell cycle phase, apoptosis and reactive oxygen species. Western blotting was used to measure CDK4, CDK6, CyclinD1, catalase, Caspase8 and Bcl-xl protein levels in control, TPL treatment, HCPT treatment and TPL plus HCPT treatment bladder cancer cells. AKT pathway proteins, including AKT and p-AKT, were also detected by western blotting. UMUC3 cells treated with DMSO, HCPT, TPL and HCPT plus TPL were injected subcutaneously into mice (n = 3 per group). RESULTS: The flow cytometry and western blot results indicated that compared to TPL or HCPT treatment alone, combination treatment of HCPT and TPL significantly induced cell cycle arrest at the G1 phase via suppressing CDK4, CDK6 and CyclinD1 in the EJ and UMUC3 bladder cancer cell lines. HCPT and TPL combination treatment also significantly increased the apoptosis rate and apoptosis-related protein levels (Caspase8 and Bcl-xl). Levels of the AKT pathway-related proteins AKT/p-AKT were significantly lower in EJ and UMUC3 cells treated with TPL and UMUC3 than in those cells treated with TPL or HCPT alone. TPL plus HCPT treatment significantly reduced bladder tumour sizes in vivo on the seventh and tenth days. CONCLUSIONS: Compared to TPL or HCPT treatment alone, TPL plus HCPT combination treatment had significantly better anticancer effects.

Genotype-phenotype correlation of patients with tuberous sclerosis complex-associated renal angiomyolipoma: a descriptive study.

TSC2 gene mutation was repeatedly reported to be associated with a more severe phenotype in patients with tuberous sclerosis complex (TSC). Our current study aims to further explore whether there is such a correlation in patients with TSC-associated renal angiomyolipoma (TSC-RAML). TSC1/TSC2 gene mutation was screened by high-throughput sequencing in 25 TSC-RAML patients from 2 medical centers. Clinical data were also carefully collected. Linear regression analysis and Student t-test were conducted by IBM SPSS Statistics Version 21.0 to analyze the genotypic-phenotypic relationship. The results indicated a high level of TSC gene mutation (80%; 20/25) in TSC-RAML patients, with higher frequency of TSC2 mutation (68%; 17/25) than TSC1 mutation (12%; 3/25). Seven novel mutation sites were detected in this study. In general, there were no significant correlations between tumor size and age (r = 0.134, P = .522), hemoglobin (r = 0.255, P = .219), and serum creatinine (r = 0.043, P = .839). Patients with larger tumor size have higher risk of bleeding. Specially, it was higher hemoglobin level in patients with TSC1 mutation than ones with TSC2 mutation and without TSC mutation (P < .05). However, no difference was found in either tumor size or serum creatinine by TSC mutation genes (P > .05). Furthermore, no difference was found in tumor size, hemoglobin, and serum creatinine by TSC mutation types (P > .05). In conclusion, TSC-RAML is TSC2 mutation dominant, with the individual differences varying greatly. No definite genotype-phenotype correlation exists in patients with TSC-RAML, and it needs to be further explored.

Single-plane retroperitoneoscopic adrenalectomy: a new operative procedure for benign adrenal disease.

To evaluate the therapeutic effect of single-plane retroperitoneoscopic adrenalectomy. From February 2014 to March 2017, 251 patients underwent single-plane retroperitoneoscopic adrenalectomy, and their operative outcomes were compared with those of 98 patients who underwent anatomical three-plane retroperitoneoscopic adrenalectomy. Among 35 patients with a body mass index (BMI) of ≥30 kg/m2, their operative outcomes were compared between two operative procedures. The demographic data and perioperative outcomes of the patients were statistically analysed. The single-plane and three-plane groups were comparable in terms of estimated blood loss, time to oral intake, hospital stay, and incidence of complications among patients with similar baseline demographics. The single-plane group had a significantly shorter operation time (46.9 ± 5.8 vs 54.8 ± 7.0 mins, P < 0.0001) and lower analgesia requirement (56/251 vs 33/98, p = 0.03). For obese patients with a BMI of ≥30 kg/m2, single-plane adrenalectomy was also associated with a significantly shorter operation time(48.1 ± 6.2 vs 64.1 ± 5.1 mins, p < 0.0001). Single-plane retroperitoneoscopic adrenalectomy is feasible, safe, and effective in the treatment of adrenal masses <5 cm in size and provides a shorter operation time and better pain control than anatomical retroperitoneal adrenalectomy, especially in obese patients.

Down-regulation of miR-210-3p encourages chemotherapy resistance of renal cell carcinoma via modulating ABCC1.

BACKGROUND: ATP-binding cassette transporter super-family including ABCC1 and MDR-1 were involved in multi-drug resistance (MDR) of renal cell carcinoma (RCC) patients. Several miRNAs were confirmed to promote the MDR and the survival of tumor cells. METHODS: The RCC cell lines Caki-2 with vinblastine-resistant (Caki-2/VBL) or doxorubicin-resistant (Caki-2/DOX) were constructed, respectively. The expressions of miR-210-3p, ABCC1 and MDR-1 protein were determined by qRT-PCR and Western blot assays. The viability of RCC cells was assessed by MTT assay. The regulatory relationship between miR-210-3p and ABCC1 was analyzed by Dual Luciferase assay. The effect of miR-210-3p in vivo was investigated with a tumor xenograft model in mice. RESULTS: MiR-210-3p expression was observed to significantly decrease in Caki-2/VBL and Caki-2/DOX cells. Meanwhile, ABCC1 and MDR-1 were significantly increased in Caki-2/VBL and Caki-2/DOX cells. ABCC1 was a novel target of miR-210-3p and negatively regulated by miR-210-3p. And miR-210-3p improved drug-sensitivity of RCC cells. Down-regulation of ABCC1 could reverse the effect of miR-210-3p knockdown on the drug-resistance and the level of MDR-1 in drug-sensitive RCC cells. CONCLUSION: We confirmed that down-regulation of miR-210-3p increased ABCC1 expression, thereby enhancing the MRP-1-mediated multidrug resistance of RCC cells.

The effects of FK1706 on nerve regeneration and bladder function recovery following an end-to-side neurorrhaphy in rats.

BACKGROUND: Immunophilin ligands are neuroregenerative agents binding to FK506 binding proteins, by which stimulate recovery of neurons in a variety of injury nerves. FK1706 is a novel immunophilin ligand which has neuroprotective and neuroregenerative effects but without immunosuppressive activity. At present, most reports about FK1706 in ameliorating nerve injury and functional recovery are limited to cavernous nerve injury and erectile function recovery. This study aimed to demonstrate the effects of FK1706 on nerve regeneration and bladder function recovery following an end-to-side neurorrhaphy in rat models. METHOD: The numbers of regenerated myelinated axons of the pelvic parasympathetic nerve (PPN) in the three groups' rats (FK1706 + ETS, ETS and control groups) were evaluated. Their intravesical pressure (IVP), S100ß and growth associated protein 43 (GAP43) expressions were also compared. RESULTS: In FK1706 + ETS group, 90% the rats showed that the frequency of FG labeled neurons was larger than the 3.5 cutoff value, 100% the rats showed that the frequency of FG-FB double-labeled neurons was larger than the 5.5 cutoff value. The average maximum of IVP in FK1706 + ETS group reached 76.3% of the value in control group. Their average number of myelinated axons of regenerated PPN reached 80% of the amount in control group. The nerve regeneration-associated markers data indicated that the expression level of S100ß and GAP43 in FK1706 + ETS group was approximately 2-fold higher than that of ETS group (P < 0.05). CONCLUSIONS: After end-to-side neurorrhaphy, FK1706 effectively enhanced the nerve regeneration and bladder function recovery.

End-to-side neurorrhaphy for nerve repair and function rehabilitation.

BACKGROUND: End-to-side neurorrhaphy is a promising procedure for nerve repair in peripheral nerve injury. However, in previous studies, this technique was limited to somatic nerves. The present study was designed to investigate the feasibility of nerve regeneration after end-to-side neurorrhaphy between autonomic nerve and somatic nerve. MATERIALS AND METHODS: Thirty adult male Sprague-Dawley rats were randomly divided into the following three groups (n = 10 per group) for different treatments: (1) end-to-side neurorrhaphy group, the left L6 and S1 spinal nerves were transected in the dura, and the distal stump of L6 ventral root (L6VR) was sutured to the lateral face of L4 ventral root (L4VR) through end-to-side coaptation; (2) no repair group, the rats received the same operation as the end-to-side neurorrhaphy group but without coaptation; (3) control group, the rats received the same operation as the end-to-side neurorrhaphy group but the L6VR was preserved. After 4 month, the origin and mechanism of nerve regeneration were evaluated by retrograde nerve tracing. Morphologic and functional properties of the regenerated nerve were investigated by morphologic examination and intravesical pressure measurement. RESULTS: Retrograde nerve tracing indicated that the new neural reflex pathway was successfully established, and the main regeneration mechanism was axon collateral sprouting. Morphologic examination and intravesical pressure measurement indicated prominent axonal regeneration and good bladder functional rehabilitation in the neurorrhaphy group. Wet weight and morphology of left extensor digitorum longus muscles appeared no detrimental effect on the donor nerve. CONCLUSIONS: These results indicated that the somatic motor axons growth into autonomic nerve may be achieved through axon collateral sprouting for nerve repair and function rehabilitation after end-to-side neurorrhaphy of autonomic nerve and somatic nerve without apparent impairment of the donor somatic nerve.

Erectile functional restoration with genital branch of genitofemoral nerve to cavernous nerve transfer after bilateral cavernous nerve resection in the rat.

OBJECTIVE: To investigate the feasibility of erectile function restoration by genital branch of genitofemoral nerve (GN) to cavernous nerve (CN) transfer in rats. MATERIALS AND METHODS: Thirty adult (3 months) male Sprague-Dawley rats were divided into 3 groups (n = 10 per group). Rats in sham group underwent sham operation, rats in nerve resection (NR) group underwent bilateral GN and CN resection to make a 2.5-mm gap, and rats in nerve transfer (NT) group underwent nerve anastomosis bilaterally between proximal stump of GN and distal stump of CN after nerve resection. RESULTS: Three months postoperatively, mating test observed 70% rats with intromission behaviors in NT group but only 10% rats in NR group. Electrostimulating the GN of NT group rats resulted in a significant increase in intracavernous pressure, and the ratio of intracavernous pressure increase to mean arterial pressure in NT group was significantly higher than that in NR group. Seven days after Fluoro-Gold injection into the penile crus, Fluoro-Gold-labeled neurons were found in ventral horn of L1 and L2 in NT group, indicating that a new erectile efferent pathway might be established. Axon counting and ultrastructure observation confirmed axonal regeneration in NT group. Furthermore, NT group had a higher expression of nitric oxide synthase in the dorsal penile nerve than that in NR group. CONCLUSION: The results have demonstrated that nerve regeneration can be obtained, and erectile function may be restored after GN to CN nerve transfer in bilateral CN resection rats, which provides an innovative and promising treatment for neurogenic erectile dysfunction.

Estimation of net greenhouse gas balance using crop- and soil-based approaches: two case studies.

The net greenhouse gas balance (NGHGB), estimated by combining direct and indirect greenhouse gas (GHG) emissions, can reveal whether an agricultural system is a sink or source of GHGs. Currently, two types of methods, referred to here as crop-based and soil-based approaches, are widely used to estimate the NGHGB of agricultural systems on annual and seasonal crop timescales. However, the two approaches may produce contradictory results, and few studies have tested which approach is more reliable. In this study, we examined the two approaches using experimental data from an intercropping trial with straw removal and a tillage trial with straw return. The results of the two approaches provided different views of the two trials. In the intercropping trial, NGHGB estimated by the crop-based approach indicated that monocultured maize (M) was a source of GHGs (-1315 kg CO2(-eq)ha(-1)), whereas maize-soybean intercropping (MS) was a sink (107 kg CO2(-eq)ha(-1)). When estimated by the soil-based approach, both cropping systems were sources (-3410 for M and -2638 kg CO2(-eq)ha(-1) for MS). In the tillage trial, mouldboard ploughing (MP) and rotary tillage (RT) mitigated GHG emissions by 22,451 and 21,500 kg CO2(-eq)ha(-1), respectively, as estimated by the crop-based approach. However, by the soil-based approach, both tillage methods were sources of GHGs: -3533 for MP and -2241 kg CO2(-eq)ha(-1) for RT. The crop-based approach calculates a GHG sink on the basis of the returned crop biomass (and other organic matter input) and estimates considerably more GHG mitigation potential than that calculated from the variations in soil organic carbon storage by the soil-based approach. These results indicate that the crop-based approach estimates higher GHG mitigation benefits compared to the soil-based approach and may overestimate the potential of GHG mitigation in agricultural systems.

Reconstruction of anorectal function through end-to-side neurorrhaphy by autonomic nerves and somatic nerve in rats.

BACKGROUND: End-to-side nerve repair is a new tool in managing certain nerve injuries. In previous studies, it was limited to somatic nerves. Herein, we evaluate the feasibility of anorectal reinnervation after end-to-side coaptation of autonomic nerve to somatic nerve. MATERIALS AND METHODS: Forty adult male Sprague-Dawley rats were randomly divided into three groups: end-to-side coaptation group (n = 16), the left L6 and S1 spinal nerves were transected, and the distal stump of L6 ventral root (L6VR) was sutured to L4VR (L4VR) through end-to-side neurorrhaphy; no coaptation group (n = 12), rats received the same operation as the end-to-side coaptation group but without coaptation; and control group (n = 12), rats received the same operation as the end-to-side coaptation group but the L6VR was preserved. At 16 wk, using double retrograde tracing and histomorphological technique and anorectal manometry, morphological and functional properties of regenerated nerve were investigated. RESULTS: Retrograde tracing indicated that the new neural pathway was established and the main nerve regeneration mechanism was axon collateral sprouting. Histology showed good axonal regeneration with end-to-side neurorrhaphy. The wet weight and morphology of left tibialis anterior muscles appeared no detrimental effect on donor nerve. Anorectal manometry showed good anorectal functional recovery. CONCLUSIONS: These results suggest that the somatic motor axon ingrowth into autonomic nerve could be through collateral sprouting after end-to-side coaptation of autonomic nerve to somatic nerve. Our innovative technique of end-to-side coaptation may be of great value in anorectal reinnervation without functional impairment of the donor somatic nerve.

Re-innervation of the bladder through end-to-side neurorrhaphy of autonomic nerve and somatic nerve in rats.

End-to-side neurorrhaphy is widely used in the peripheral nervous system for nerve repair; however, the application of this technique has been limited to somatic nerves. The feasibility of nerve regeneration through end-to-side neurorrhaphy between autonomic and somatic nerves with different characteristics in the peripheral nervous system is still undetermined. In this study, rats were divided into three groups for different treatments (n=10 per group). In the end-to-side neurorrhaphy group, left L6 and S1 were transected in the dura, and the distal stump of L6 ventral root was sutured to the lateral face of L4 ventral root through end-to-side coaptation. In the no repair group, the rats did not undergo neurorrhaphy. In the control group, the left L6 dorsal root and S1 roots were transected, respectively, but the L6 ventral root was kept intact. After 16 weeks, the origin and mechanism of nerve regeneration was evaluated by retrograde double labeling technique as well as histological examination and intravesical pressure measurement. Retrograde double labeling indicated that the reconstructed reflex pathway was successfully established and the primary regeneration mechanism involved axon collateral sprouting. Morphological examination and intravesical pressure measurement indicated prominent nerve regeneration and successful re-innervation of the bladder in the neurorrhaphy group, compared with the "no repair" group (p<0.05). No significant changes were observed in the histology of the donor nerve and the bilateral extensor digitorum longus muscles in the neurorrhaphy group. Nerve regeneration may be achievable for nerve repair through end-to-side neurorrhaphy between autonomic and somatic nerves without apparent impairment of donor somatic nerve.

Long-term safety, tolerability, and efficacy of α1-adrenergic blocker in young men with primary bladder neck obstruction: results from a single centre in China.

OBJECTIVE: Primary bladder neck obstruction (PBNO) is a nonneurogenic voiding disorder and frequently overlooked in young men. Prior studies have reported the efficacy of α-blockers only in the short-term for male patients with PBNO. We hereby report our long-term results using α1-blocker therapy in young men with PBNO. METHODS: Between January 2005 and December 2009, PBNO was diagnosed in 30 young men (mean age 27.3 years, range 18-35) at our institution. Doxazosin 4 mg once daily was administered for at least 12 months. Safety and tolerability were assessed, and efficacy was evaluated from International Prostate Symptom Score (I-PSS), Quality of Life (QOL), uroflowmetry, and post-void residual following 3- and 12-month treatment. Successful treatment was defined as at least 3 ml per second increase in the maximum flow rate and more than a 40% decrease in I-PSS. RESULTS: In all 30 patients, Mean symptom duration was 26.4 (3-65) months. The most common symptoms were hesitancy (93.3%), weak stream (76.7%), and frequency (66.7%). A total of 24 patients (80%, 24/30) successfully completed the 12 month of treatment. The medication period was 15.2 months, and follow-up duration was 16.3 months. Doxazosin was safe and well tolerated. The efficacy of doxazosin was maintained over the 12-month treatment period. Relative to baseline, there were reductions in the number of mean I-PSS (from 17.7 ± 4.2 to 10.4 ± 4.8), mean QOL (from 4.2 ± 1.1 to 2.4 ± 1.3), and mean post-void residual urine (from 79.3 ± 33.4 to 47.1 ± 21.3), and an increase in mean maximum flow rate (from 11.4 ± 2.9 to 15.1 ± 3.2 ml) after 12-month treatment. Treatment was successful in 16 patients (66.7%, 16/24) according to the improvement in both symptoms and maximum urine flow. CONCLUSIONS: α1-blocker therapy displayed a favorable safety, tolerability, and efficacy profile during 12-month treatment in young male patients with PBNO.