Schwann cell-derived amphiregulin enhances nerve regeneration via supporting the proliferation and migration of Schwann cells and the elongation of axons.

Peripheral nerves have limited regeneration ability following nerve injury. Applying growth factors with neurotrophic roles is beneficial for accelerating peripheral nerve regeneration. Here we show that after rat sciatic nerve injury, growth factor amphiregulin (AREG) is upregulated in Schwann cells of sciatic nerves. Elevated AREG stimulates the proliferation and migration of Schwann cells by activating ERK1/2 cascade. Schwann cell-secreted AREG further facilitates the outgrowth of neurites and the elongation of injured axons. Administration of AREG to injured sciatic nerves stimulates the proliferation of Schwann cells to replace lost cell population, encourages the migration of Schwann cells to form cell cords, and facilitates the regrowth of axons. Overall, our results identify AREG as an important neurotrophic factor and thus provide a promising therapeutic avenue towards peripheral nerve injury.

Lobetyolin Alleviates Ferroptosis of Skeletal Muscle in 5/6 Nephrectomized Mice via Activation of Hedgehog-GLI1 Signaling.

BACKGROUND: Muscle wasting increases morbidity and mortality and is related to chronic kidney disease (CKD) and dialysis. It is still unclear whether ferroptosis occurs during this progression and whether it is a potential intervention target for the treatment of CKD-related muscle injury. PURPOSE: The objective is to identify potential compounds for treating ferroptosis and muscle wasting and explore the potential mechanisms in vivo/in vitro. METHODS: Initially, we explored whether ferroptosis is present in the skeletal muscle of 5/6 nephrectomized (NPM) mice via RNA-Seq analysis, TUNEL staining, Oil red O staining, MDA/GSH/GSSG level detection and real-time quantitative PCR (qPCR). Subsequently, utilizing our established molecular phenotyping strategy, we screened potential traditional Chinese herb-derived compounds for alleviation of muscle wasting and ferroptosis. HE staining, Oil red O staining, TUNEL staining, immunofluorescence staining, MDA/GSH/GSSG level detection, Fe level detection, western blotting and qPCR were applied to assess the effects of the identified compound on muscle wasting and ferroptosis and explore the potential mechanism. Furthermore, RNA-Seq analysis, ChIP-Seq analysis and further experiments in vitro were performed to determine the role of Hedgehog signaling in the effect of Lobetyolin (LBT) on ferroptosis. RESULTS: In NPM mice, skeletal muscle dysfunction, lipogenesis, reduced GSH/GSSG ratio, decreased GSH content, increased MDA production and and higher levels of ferroptosis markers were observed. LBT treatment (30 mg/kg or 50 mg/kg) significantly alleviates skeletal muscle injury by inhibiting ferroptosis. Additionally, in an in vitro investigation, C2C12 cells exposed to Indolyl sulfate (IS) induced ferroptosis and LBT treatment (20 µM and 50 µM) protected C2C12 from such injury, consistent with the results from the in vivo analysis. Furthermore, it was found LBT increased the levels of protein involving Hedgehog signaling pathway (SMO and GLI1), and rescue analysis revealed that this pathway played a crucial role in the regulation of ferroptosis. Further experiments demonstrated that LBT upregulated a series of suppressors of ferroptosis by activating Gli1 transcription. CONCLUSION: LBT alleviates CKD-induced muscle injury by inhibiting ferroptosis through activation of the Hedgehog signaling pathway.

Sirtuin 2 Alleviates Chronic Neuropathic Pain by Suppressing Ferroptosis in Rats.

Neuropathic pain (NP) is chronic and associated with poor effects of general analgesia. It affects patients' health and quality of life. The apoptotic process of lipid peroxidation caused by iron overload is called ferroptosis, which may be associated with nervous system disease. A recent study has found that sirtuin 2 (SIRT2) achieves a neuroprotective effect by suppressing ferroptosis. Herein, we aimed to examine whether SIRT2 regulated spared nerve injury (SNI)-induced NP by suppressing ferroptosis in rats. A rat model of NP was induced in adult male Sprague-Dawley rats weighing 200-250 g. Mechanical allodynia was observed from the first day after SNI and continued for 14 days. Compared with age-matched control rats, the expression of SIRT2 and ferroportin 1 (FPN1) decreased in the L4-6 spinal cord of the SNI-induced NP rats. In addition, we observed that the levels of both iron and anti-acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) were significantly increased in the spinal cord after SNI, while the expression of glutathione peroxidase 4 (GPX4) was decreased. Furthermore, an intrathecal injection of SIRT2 overexpressed recombinant adenovirus, which upregulated the expression of SIRT2, attenuated mechanical allodynia, enhanced the level of FPN1, inhibited intracellular iron accumulation, and reduced oxidant stress levels, thereby reversing the changes to ACSL4 and GPX4 expression in the SNI rats. This evidence suggests that SIRT2-targeted therapeutics may help relieve the symptoms of chronic NP.

[Analysis of Predictive Value of Laboratory Indexes for Malignant Lymphoma Patients with Bone Marrow Involvement].

OBJECTIVE: To analyze the influence of bone marrow involvement (BMI) in patients with malignant lymphoma (ML) on laboratory indexes, and evaluate the laboratory markers that can be used to predict/diagnose BMI. METHODS: The clinical characteristics and laboratory indexes of 137 ML patients were analyzed retrospectively, from which the indexes of BMI in ML patients was studied. The logistic regression analysis and receiver operating curve (ROC) were used to evaluate independent risk factors and predictors of BMI diagnosis in ML patients. RESULTS: Compared with non-BMI group, the red blood cell distribution width, C-reactive protein, erythrocyte sedimentation rate, D-dimer, lactate dehydrogenase, alkaline phosphatase, ß2-microglobulin, transferrin, CA153, CA125, and soluble interleukin-2 receptor (sIL-2R) levels were increased while platelet (PLT) count was decreased in BMI group, and the difference was statistically significant (P<0.05). The blood indexes related to BMI and the statistically significant indexes in the univariate regression analysis were corrected by multivariate logistic regression analysis. The corrected results showed that T cell-related non-Hodgkin lymphoma (adjusted OR=2.18, 95%CI: 1.48-4.90, P＜0.001), clinical stage Ⅲ-Ⅳ (adjusted OR=3.32, 95%CI: 2.16-5.83, P＜0.001), sIL-2R (adjusted OR=4.26, 95%CI: 2.95-12.85, P＜0.001) were the risk factors for ML patients with BMI, while PLT (adjusted OR=0.89, 95%CI: 0.55-0.96, P= 0.003) was a protective factor. ROC analysis showed that the areas under the ROC curve of PLT and sIL-2R predicting BMI in ML patients was 0.712 (95%CI: 0.646-0.776, P<0.001) and 0.796 (95%CI: 0.739-0.853, P<0.001), respectively. The best cut-off point of PLT and sIL-2R was 160×109/L and 2 568 U/ml, respectively. The diagnostic specificities of the two indexes here were both greater than 80%. CONCLUSION: PLT and sIL2R show good diagnostic value for ML patients with BMI.

Sirt2 in the Spinal Cord Regulates Chronic Neuropathic Pain Through Nrf2-Mediated Oxidative Stress Pathway in Rats.

Reduction in Nrf2-mediated antioxidant response in the central nervous system plays an important role in the development and maintenance of neuropathic pain (NP). However, the mechanisms regulating Nrf2 activity in NP remain unclear. A recent in vitro study revealed that Sirt2, a member of the sirtuin family of proteins, affects antioxidant capacity by modulating Nrf2 activity. Here we examined whether central Sirt2 regulates NP through Nrf2-mediated oxidative stress pathway. In a rat model of spared nerve injury (SNI)-induced NP, mechanical allodynia and thermal hyperalgesia were observed on day 1 and up to day 14 post-SNI. The expression of Sirt2, Nrf2 and its target gene NQO1 in the spinal cord in SNI rats, compared with sham rats, was significantly decreased from day 7 and remained lower until the end of the experiment (day 14). The mechanical allodynia and thermal hyperalgesia in SNI rats were ameliorated by intrathecal injection of Nrf2 agonist tBHQ, which normalized expression of Nrf2 and NQO1 and reversed SNI-induced decrease in antioxidant enzyme superoxide dismutase (SOD) and increase in oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the spinal cord. Moreover, intrathecal injection of a recombinant adenovirus expressing Sirt2 (Ad-Sirt2) that upregulated expression of Sirt2, restored expression of Nrf2 and NQO1 and attenuated oxidative stress in the spinal cord, leading to improvement of thermal hyperalgesia and mechanical allodynia in SNI rats. These findings suggest that peripheral nerve injury downregulates Sirt2 expression in the spinal cord, which inhibits Nrf2 activity, leading to increased oxidative stress and the development of chronic NP.

Active Retrograde Extra Backup with a Mother-and-Child Catheter to Facilitate Retrograde Microcatheter Collateral Channel Tracking in Recanalization of Coronary Chronic Total Occlusion.

OBJECTIVE: To explore the feasibility and safety of the active retrograde backup (ARB) for treatment of chronic total occlusion (CTO) during retrograde percutaneous coronary intervention (PCI). BACKGROUND: Guiding support plays an important role in guidewire and microcatheter coronary channel (CC) tracking in retrograde PCI therapy for patients with CTO. However, the feasibility and safety of retrograde active use of a mother-and-child catheter are still unclear. Patients and Methods. A total of 271 consecutive patients with CTO who underwent retrograde PCI between January 2015 and January 2020 were retrospectively analyzed. Clinical data of two groups were compared to evaluate the feasibility and safety of ARB. RESULTS: Of the 271 patients, 69.0% (187/271) underwent therapy through the septal branch, 31.0% (84/271) through the epicardial collateral channel, and 47.6% (129/271) through active retrograde extra backup with a mother-and-child catheter to facilitate retrograde microcatheter collateral CC tracking. The time of wire CC tracking was shorter in the ARB group than that in the non-ARB group (25.4 ± 8.5 vs 26.4 ± 9.7, p=0.348), but there was no significant difference. The duration of the retrograde microcatheter tracking (10.2 ± 3.8 vs 15.5 ± 6.8, p=0.012) and the retrograde approach (62.8 ± 20.3 vs 70.4 ± 24.3, p=0.026) in the ARB group was significantly shorter than that in the non-ARB group. The radiation dose (223.6 ± 112.7 vs. 295.2 ± 129.3, p=0.028), fluoroscopy time (50.6 ± 21.3 vs 62.3 ± 32.1, p=0.030), and contrast volume (301.8 ± 146.7 vs 352.2 ± 179.5, p=0.032) in the ARB group were significantly lower than that in the non-ARB group. There were no life-threatening procedural complications in either group. Complications unrelated to ARB included two cases of donor-vessel dissection, one case of CC perforation, and two cases of target-vessel perforation. There was no statistically significant difference in major adverse cardiac and cerebrovascular events between the groups during hospitalization (p > 0.05). CONCLUSION: ARB is feasible, safe, and conducive to guidewire and microcatheter CC tracking in the recanalization of coronary CTO. It improves procedural efficiency and is worthy of further promotion.

The profibrotic effects of chronic microaspiration of bile acids on lungs of rats at different stages.

This study aimed to explore the profibrotic effects of chronic microaspiration of two major bile acids, including chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA), on lungs of rats at different stages, as well as the underlying mechanisms in vivo. A rat model was induced by weekly intratracheal instillation of DCA and CDCA. Our results showed that chronic microaspiration of bile acids resulted in alveolar structure disorder, and inflammatory cells infiltration in the pulmonary interstitium at the early stage. Subsequently, numerous fibroblasts were proliferated, and collagen deposition was profoundly increased over the interstitium of the airways and vessels. Compared with control group, the expression of α-smooth muscle actin, type I collagen, hydroxyproline, transforming growth factor-ß1 (TGF-ß1), and matrix metalloproteinase-9 in the lung tissues were remarkably elevated at the 2nd week, reached the highest level at the 6th week, and maintained high at the 8th week in both DCA- and CDCA-treated groups (P < 0.05). Furthermore, chronic microaspiration of bile acids led to higher levels of glutathione and malondialdehyde, while lower level of superoxide dismutase in lung tissues compared with controls (P < 0.05), thereby resulting in the oxidant/antioxidant enzyme imbalance in the formation of fibrosis. In addition, we also found a consistent growth in the expression of farnesoid X receptor (FXR) in both DCA- and CDCA-treated groups. Our findings suggested that chronic microaspiration of bile acids could initiate the process of pulmonary fibrosis from the early phase and promote its progression in a time-dependent manner, which likely involved the TGF-ß1, oxidative stress, and FXR-related pathways.

Brozopine Inhibits 15-LOX-2 Metabolism Pathway After Transient Focal Cerebral Ischemia in Rats and OGD/R-Induced Hypoxia Injury in PC12 Cells.

The goal of this study was to elucidate the mechanisms of protection of Sodium (±)-5-bromo-2-(α-hydroxypentyl) benzoate (trade name: Brozopine, BZP) against cerebral ischemia in vivo and in vitro. To explore the protective effect of BZP on focal cerebral ischemia-reperfusion injury, we evaluated the effects of various doses of BZP on neurobehavioral score, cerebral infarction volume, cerebral swelling in MCAO rats (ischemia for 2 h, reperfusion for 24 h). In addition, the effects of various doses of BZP on OGD/R-induced-PC12 cells injury (hypoglycemic medium containing 30 mmol Na2S2O4 for 2 h, reoxygenation for 24 h) were evaluated. Four in vivo and in vitro groups were evaluated to characterize targets of BZP: Control group, Model group, BZP group (10 mg/kg)/BZP group (30 µmol/L), C8E4 group (10 mg/kg)/C8E4 group (30 µmol/L). An ELISA kit was used to determine the levels of 15-HETE (a 15-LOX-2 metabolite) in vivo and in vitro. Rat nuclear factor κB subunit p65 (NF-κB p65), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and intercellular adhesion molecule-1 (ICAM-1) were also quantified in vivo and in vitro. The results showed that BZP improved focal cerebral ischemia-reperfusion injury in rats and PC12 cells treated with Na2S2O4 in dose/concentration-dependent manners through inhibition of production of 15-HETE and expression of NF-κB, IL-6, TNF-α, and ICAM-1. In conclusion, BZP exerted protective effects against cerebral ischemia via inhibition of 15-LOX-2 activity.

CHA2DS2-VASC score as a preprocedural predictor of contrast-induced nephropathy among patients with chronic total occlusion undergoing percutaneous coronary intervention: a single-center experience.

BACKGROUND: The usefulness of the CHA2DS2-VASC risk score (CVRS) in predicting the occurrence of contrast-induced nephropathy (CIN) among patients with chronic total occlusion (CTO) undergoing percutaneous coronary intervention (PCI) remains unclear. METHOD: A total of 239 patients with CTO who underwent PCI were included in this study. They were divided into 3 groups according to the CVRS: low-risk group (1 point, n = 64), intermediate-risk group (2 points, n = 135), and high-risk group (≥3 points, n = 40). Baseline serum creatinine was determined upon admission before the procedure. The serum creatinine level was monitored for 72 h post-procedure to determine the occurrence of CIN. RESULTS: The total incidence of CIN in patients with CTO who underwent PCI was 16.3%. The average CVRS in the CIN group was significantly higher than that in the non-CIN group (3.1 ± 1.2 VS 2.1 ± 1.1, P < 0.001). The incidence of CIN in the high-risk group was 5.6 times higher than that in the low-risk group (37.5% VS 6.3%, P < 0.001). Similar to the Mehran risk score (AUC, 0.754; 95% CI, 0.698-0.810; P < 0.001), the receiver operating characteristic curve analysis showed a good diagnostic value of the CVRS in predicting CIN among patients with CTO who underwent interventional therapy for having CVRS≥3 (sensitivity, 69.2%; specificity, 78.0%; AUC, 0.742; 95% CI, 0.682-0.797; P < 0.001). The multivariate analysis showed that the higher pulse pressure and contrast volume, lower baseline glomerular filtration rate, and CVRS ≥3 were independent predictors of CIN. CONCLUSIONS: The CVRS can be used as a simple pre-procedural predictor of CIN among patients with CTO undergoing PCI.

DNA damage-inducible transcript 4 is an innate guardian for human squamous cell carcinoma and an molecular vector for anti-carcinoma effect of 1,25(OH)2 D3.

Cutaneous squamous cell carcinoma (SCC) is one of the most common non-melanoma skin cancers worldwide. While its exact tumorigenesis mechanisms is far from well-established and less satisfied therapeutic strategy can be clinically used nowadays. In this study, we intended to investigate the role of DNA damage-inducible transcript 4 (DDIT4) in human SCC. Firstly, we identified DDIT4 is significantly suppressed in human SCC tissue and cultured A431 cell line, and reduced DDIT4 accelerates keratinocytes proliferation but impedes the autophagy flux through mTORC1 pathway by affecting the downstream S6 Kinase1, 4E-BP1, Beclin1 and LC3 II/I. While 1,25(OH)2 D3 enhanced DDIT4 expression and activated autophagy and inhibit mTORC1 to take the effect of anti-proliferation and activating autophagy. Further, formation of direct vitamin D receptor (VDR)-DDIT4 transcription complex was verified by ChIP-qPCR, which showed the molecular mechanism of how 1,25(OH)2 D3 promotes DDIT4 transcription. Thirdly, xenograft tumor-bearing mice model treated by gradient concentrations of 1,25(OH)2 D3 revealed the obvious anti-carcinoma effect of 1,25(OH)2 D3 in vivo and DDIT4 acted the molecular vector of 1,25(OH)2 D3 through mTORC1. Lastly, elevated DDIT4 expression was verified in human actinic keratoses tissue, and chronic long-term ultraviolet (UV) irradiation on mouse disclosed UV could promote DDIT4 expression inside epidermis. Conclusively, our research suggested a novel molecular mechanism about the human SCC tumorigenesis and the pharmacological mechanism about how 1,25(OH)2 D3 take its anti-carcinoma role on human SCC, as well as a striking paradoxes that how UV irradiation plays the tumorigenesis effect but synchronously take a protective role in the early stage of SCC carcinogenesis.

Downregulation of BarH-like homeobox 2 promotes cell proliferation, migration and aerobic glycolysis through Wnt/ß-catenin signaling, and predicts a poor prognosis in non-small cell lung carcinoma.

BACKGROUND: Human BarH-like homeobox 2 (Barx2), a homeodomain factor of the Bar family, plays a critical role in cell adhesion and cytoskeleton remodeling, and has been reported in an increasing array of tumor types except non-small cell lung carcinoma (NSCLC). The purpose of the current study was to characterize the expression of Barx2 and assess the clinical significance of Barx2 in NSCLC. METHODS: Quantitative real-time polymerase chain reaction, immunohistochemistry and western blot analysis were used to examine mRNA and protein expression, respectively. The relationships between Barx2 expression and clinicopathological variables were analyzed. Cell Counting Kit-8 and plate colony formation assay were used to detect cell proliferation. Transwell assay was used to examine cell migration ability. Glucose uptake, lactate, adenosine triphosphate, and lactate dehydrogenase assays were used to detect aerobic glycolysis. RESULTS: Barx2 is downregulated in NSCLC tissues compared with para-carcinoma. Furthermore, Barx2 expression shows a negative correlation with advanced TNM stage and a high level of Ki-67. Survival analysis reveals that Barx2 level is an independent prognostic factor for NSCLC patients. The Barx2 (low) Ki-67 (high) group had the worst prognosis. Furthermore, the data indicate that downregulation of Barx2 expression promotes cell proliferation, migration, and aerobic glycolysis, including increased lactate dehydrogenase activity, glucose utilization, lactate production, and decreased intracellular adenosine triphospahte level. Furthermore, Barx2 acts as a negative regulator of the canonical Wnt/ß-catenin pathway. Reactivation of Wnt/ß-catenin pathway by LiCl can reverse the inhibiting effect of Barx2. CONCLUSIONS: These findings reveal that Barx2 serving as a tumor suppressor gene could decrease cell proliferation, migration, and aerobic glycolysis through inhibiting the Wnt/ß-catenin signaling pathway, and predicts a good prognosis in NSCLC.

Omega-3 polyunsaturated fatty acids ameliorate ethanol-induced adipose hyperlipolysis: A mechanism for hepatoprotective effect against alcoholic liver disease.

Alcohol exposure induces adipose hyperlipolysis and causes excess fatty acid influx into the liver, leading to alcoholic steatosis. The impacts of omega-3 polyunsaturated fatty acids (n-3 PUFA) on ethanol-induced fatty liver are well documented. However, the role of n-3 PUFA in ethanol-induced adipose lipolysis has not been sufficiently addressed. In this study, the fat-1 transgenic mice that synthesizes endogenous n-3 from n-6 PUFA and their wild type littermates with an exogenous n-3 PUFA enriched diet were subjected to a chronic ethanol feeding plus a single binge as model to induce liver injury with adipose lipolysis. Additionally, the differentiated adipocytes from 3T3-L1 cells were treated with docosahexaenoic acid or eicosapentaenoic acid for mechanism studies. Our results demonstrated that endogenous and exogenous n-3 PUFA enrichment ameliorates ethanol-stimulated adipose lipolysis by increasing PDE3B activity and reducing cAMP accumulation in adipocyte, which was associated with activation of GPR120 and regulation of Ca2+/CaMKKß/AMPK signaling, resultantly blocking fatty acid trafficking from adipose tissue to the liver, which contributing to ameliorating ethanol-induced adipose dysfunction and liver injury. Our findings identify that endogenous and exogenous n-3 PUFA enrichment ameliorated alcoholic liver injury by activation of GPR120 to suppress ethanol-stimulated adipose lipolysis, which provides the new insight to the hepatoprotective effect of n-3 PUFA against alcoholic liver disease.

Hypermethylation reduces the expression of PNPLA7 in hepatocellular carcinoma.

Liver cancer has a high morbidity and mortality rate, and is one of the most common types of cancer in men. PNPLA7 is a member of the patatin-like phospholipase domain-containing protein family which is involved in triglyceride hydrolysis, energy metabolism and lipid droplet metabolism. The liver is the most important energy metabolism organ; whether PNPLA7 is deregulated in liver cancer has not been previously reported. In the present study, reverse transcription-quantitative polymerase chain reaction and subsequent methylation analysis provided evidence that PNPLA7 is down-regulated in hepatocellular carcinoma (HCC) cell lines and tissue samples, via the mechanism of transcriptional silencing by promoter hypermethylation. These results may provide novel insights for HCC diagnosis.

An aptamer targets HBV core protein and suppresses HBV replication in HepG2.2.15 cells.

Hepatitis B virus (HBV)-related hepatitis is a major health concern worldwide. As current anti-HBV therapies are limited, it is essential to develop new strategies. Aptamer, a newly developed adaptive molecule (single-strand DNA or RNA also known as nucleotide antibody), is a new strategy for clinical diagnosis and therapy due to its high affinity and specificity. In the present study, by systematic evolution of ligand by exponential enrichment (SELEX), aptamers were screened against the core protein of HBV (HBc) from a random ssDNA library. Quantitative PCR (qPCR) results showed that the binding proportions of the SELEX-enriched aptamer pools were increased with the SELEX rounds, until round seven. Thus, the pool of round seven was cloned. Following the sequence analysis of a total of 90 clones by Macaw software, five aptamer candidates were selected and their affinity to HBc was tested by dot blot. Apt.No.28, which had sequence replicates in the clones, was shown to have a high affinity. Furthermore, by agarose gel electrophoresis-capillary transfer-blotting and qPCR, Apt.No.28 was shown to inhibit the assembly of the nucleocapsid, reducing extracellular HBV DNA whose synthesis relied on the formation of the nucleocapsid, indicating its role in suppressing HBV replication. The results provided a new ideal targeting molecule and may facilitate the strategy for targeted therapy as well as drug development of HBV-related diseases.

Association study of miR­149 rs2292832 and miR­608 rs4919510 and the risk of hepatocellular carcinoma in a large­scale population.

Polymorphisms in pre­microRNAs (miRNAs) or mature miRNAs may influence miRNA processing or target binding, thus contributing to tumorigenesis and cancer development. The present study aimed to evaluate whether miR­149 rs2292832 (C>T) and miR­608 rs4919510 (G>C) are associated with the risk and clinical characteristics of hepatocellular carcinoma (HCC) in a large­scale population. miR­149 rs2292832 and miR­608 rs4919510 were genotyped in a total of 993 patients with HCC and 992 unrelated healthy subjects by Sequenom MassARRAY. The results showed that, compared with the reference CC genotype, the TC+TT genotype of miR­149 was more highly associated with HCC [CC vs. TC+TT: Odds ratio (OR)=1.384, 95% confidence interval (CI)=1.013­1.892, P=0.041], and was also associated with an increased risk of hepatitis B virus (HBV)­associated HCC (CC vs. TC+TT: OR=1.453, 95% CI=1.034­2.042, P=0.031). However, no significant association between miRNA­608 rs4919510 and the risk of HCC/HBV­associated HCC was found. In addition, these two SNPs were shown not to be correlated with a range of clinical characteristics. The present study may provide an indicator for identification of the high risk of HCC in patients.

The identification of an ESCC susceptibility SNP rs920778 that regulates the expression of lncRNA HOTAIR via a novel intronic enhancer.

Long noncoding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR), which could induce genome-wide retargeting of polycomb-repressive complex 2, trimethylates histone H3 lysine-27 (H3K27me3) and deregulation of multiple downstream genes, is involved in development and progression of esophageal squamous cell carcinoma (ESCC). We hypothesized that the functional single nucleotide polymorphisms (SNP) in HOTAIR may affect HOTAIR expression and/or its function and, thus, ESCC risk. Therefore, we examined the association between three haplotype-tagging SNPs (htSNP) across the whole HOTAIR locus and ESCC risk as well as the functional relevance of an ESCC susceptibility SNP rs920778. Genotypes were determined in three independent case-control sets consisted of 2098 ESCC patients and 2150 controls. The allele-specific regulation on HOTAIR expression by the rs920778 SNP was investigated in vitro and in vivo. We found that the HOTAIR rs920778 TT carriers had a 1.37-fold, 1.78-fold and 2.08-fold increased ESCC risk in Jinan, Shijiazhuang and Huaian populations, respectively, compared with the CC carriers (P = 0.003, 7.7 × 10(-4) and 5.9 × 10(-4)). During inspecting functional relevance of the rs920778 SNP, we identified a novel intronic HOTAIR enhancer locating between +1719bp and +2353bp from the transcriptional start site through reporter assays. Moreover, there is an allelic regulation of rs920778 on HOTAIR expression via this enhancer in both ESCC cell lines and normal esophageal tissue specimens, with higher HOTAIR expression among T allele carriers. These results demonstrate that functional genetic variants influencing lncRNA regulation may explain a fraction of ESCC genetic basis.

A cell-based single-stranded DNA aptamer specifically targets gastric cancer.

Gastric cancer is one of the most prevailing cancers with high morbidity and mortality. Limitations in the current diagnosis and therapy, specially lacking of specific molecular therapeutic targets, ask for the development of new strategies. Aptamer, a newly developed adaptive molecule, could be used in clinical detection and therapy because of its high affinity and specificity. As no aptamer has ever been developed in preventing gastric cancer so far, we were the first who cloned such an aptamer specifically targeting gastric cancer. The aptamer was selected by systematic evolution of ligands by exponential enrichment with gastric cancer cell-line HGC-27 as target cell line and immortalized gastric epithelial cell-line GES-1 as control cell line. The affinity and specificity of candidate aptamers were examined by flow cytometry, confocal imagining and aptamer-based histochemistry staining. After 19 cycles of systematic evolution of ligands by exponential enrichment and subsequent cloning and sequencing, an aptamer with the highest affinity and specificity (nominated as AGC03) among candidates was screened out from a random single-stranded DNA pool. Moreover, AGC03 could not only specifically bind to gastric cancer cells (the equilibrium dissociation constant value was 16.49 ± 0.40 nM) in vitro, but also recognize cancer cells in human cancer tissue. Our most important finding is that AGC03 could even be internalized into cells automatically. In conclusion, we obtained a novel aptamer specifically targeting gastric cancer,which is an effective tool for both gastric cancer diagnosis and drug delivery.

Screening and verification of ssDNA aptamers targeting human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most malignant cancers in the world. Molecular probes that can recognize biomarkers specific for HCC are urgently needed to improve the sensitivity and specificity of early diagnosis. A recent study has applied the method of systematic evolution of ligands by exponential enrichment and produced several aptamers that can bind specifically to mouse liver cancer cells and tissues. However, the binding affinity to human liver cancer has not been fully identified. Using human-derived hepatoma cell line HepG2 as positive target cell line and normal hepatocyte cell line HL-7702 as negative one, we obtained an aptamer HA09 that could specifically bind to human liver cancer cells with Kd in the nanomolar range and recognize paraffin-embedded human HCC tissues. This aptamer may facilitate the discovery of novel biomarkers and serve as an ideal molecular probe for intracellular delivery with both diagnostic and therapeutic implications.

The down-regulation of GNAO1 and its promoting role in hepatocellular carcinoma.

GNAO1 (guanine nucleotide-binding protein, α-activating activity polypeptide O) is a member of the subunit family of Gα proteins, which are molecular switchers controlling signal transductions and whose deregulation can promote oncogenesis. HCC (hepatocellular carcinoma) is one of the malignant tumours around the world, which summons novel biomarkers or targets for effective diagnosis and treatments. The present study was aimed to investigate the expression of GNAO1 in HCC patient tissues and the possible mechanisms by which it took effects. The expression of GNAO1 was detected by IHC (immunohistochemistry) and real-time qPCR (quantitative PCR). Cell proliferation test and cell senescence test were then performed to explore the role of GNAO1 in the occurrence and development of HCC. It was revealed that the level of GNAO1 was comparably less in HCC tissues than in the adjacent tissues. Furthermore, down-regulation of GNAO1 increased cell proliferation, while suppressing the senescence of HCC cells. In conclusion, our findings revealed and confirmed the importance of GNAO1 in HCC, indicating that GNAO1 is a potential biomarker as well as a promising therapeutic target for HCC.

Association of a genetic variation in a miR-191 binding site in MDM4 with risk of esophageal squamous cell carcinoma.

As an oncoprotein, MDM4 plays a key part in P53 tumor suppressor pathway through negatively regulating P53 function. It has been reported that an rs4245739 A>C polymorphism locating in the MDM4 3'-untranslated region creates a miR-191 target site and results in decreased MDM4 expression. Therefore, we investigated the association between this polymorphism and esophageal squamous cell carcinoma (ESCC) risk as well as its biological function in vivo. Genotypes were determined in two independent case-control sets consisted of 1128 ESCC cases and 1150 controls from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. The impact of the polymorphism on MDM4 expression was examined with esophagus tissues. Our results demonstrated that the MDM4 rs4245739 AC and CC genotypes were significantly associated with decreased ESCC risk compared with the AA genotype in both case-control sets (Jinan set: OR = 0.54, 95% CI = 0.35-0.82, P = 0.004; Huaian set: OR = 0.68, 95% CI = 0.45-0.99, P = 0.049). Stratified analyses revealed that a multiplicative interaction between rs4245739 and smoking or drinking was evident (Gene-smoking: P(interactioin) = 0.022; gene-drinking: P(interactioin) = 0.032). After detecting In vivo MDM4 mRNA expression, we found that the rs4245739 AC and CC genotype carriers had significantly decreased MDM4 expression in normal esophagus tissues compared with AA genotype carriers, indicating a consistent genotype-phenotype correlation. Our results elucidate that the MDM4 rs4245739 polymorphism contributes to susceptibility of ESCC and support the hypothesis that genetic variants, interrupting miRNA-mediated gene regulation, may modify cancer risk.