Downregulation of LncRNA GCLC-1 Promotes Microcystin-LR-Induced Malignant Transformation of Human Liver Cells by Regulating GCLC Expression.

Microcystin-LR (MCLR) is an aquatic toxin, which could lead to the development of hepatocellular carcinoma (HCC). Long non-coding RNAs (lncRNAs) are considered important regulatory elements in the occurrence and development of cancer. However, the roles and mechanisms of lncRNAs during the process of HCC, induced by MCLR, remain elusive. Here, we identified a novel lncRNA, namely lnc-GCLC-1 (lncGCLC), which is in close proximity to the chromosome location of glutamate-cysteine ligase catalytic subunit (GCLC). We then investigated the role of lncGCLC in MCLR-induced malignant transformation of WRL68, a human hepatic cell line. During MCLR-induced cell transformation, the expression of lncGCLC and GCLC decreased continuously, accompanied with a consistently high expression of miR-122-5p. Knockdown of lncGCLC promoted cell proliferation, migration and invasion, but reduced cell apoptosis. A xenograft nude mouse model demonstrated that knockdown of lncGCLC promoted tumor growth. Furthermore, knockdown of lncGCLC significantly upregulated miR-122-5p expression, suppressed GCLC expression and GSH levels, and enhanced oxidative DNA damages. More importantly, the expression of lncGCLC in human HCC tissues was significantly downregulated in the high-microcystin exposure group, and positively associated with GCLC level in HCC tissues. Together, these findings suggest that lncGCLC plays an anti-oncogenic role in MCLR-induced malignant transformation by regulating GCLC expression.

Human Urinary Kallidinogenase Pretreatment Inhibits Myocardial Inflammation and Apoptosis after Coronary Microembolization by Activating PI3K/Akt/FoxO1 Axis.

BACKGROUND: As a fatal cardiovascular complication, coronary microembolization (CME) results in severe cardiac dysfunction and arrhythmia associated with myocardial inflammation and apoptosis. Human urinary kallidinogenase (HUK) can provide a protective function for cardiomyocytes by improving microcirculation. However, the therapeutic effects and underlying mechanisms of HUK in CME-induced myocardial injury remain unclear. AIMS: We evaluated the effect of HUK on cardiac protection in a rat model of CME and whether it could restrain myocardial inflammation and apoptosis, and alleviate CME-induced myocardial injury. METHODS: We established the CME model by injecting 42 µm inert plastic microspheres into the left ventricle of rats in advance, then the rats were randomly and equally divided into CME, CME + HUK (the dose of HUK at 0.016 PNA/kg/day), CME + HUK + LY (the dose of LY294002 at 10 mg/kg, 30 minutes before modeling), and Sham operation groups. Cardiac function, the serum levels of myocardial injury biomarkers, myocardial inflammation and apoptosis-related genes were measured; and the myocardial histopathological examination was performed at 12 h after the operation. RESULTS: The results revealed that HUK effectively reducing myocardial inflammation, apoptosis, and myocardial infarction area; and improving CME-induced cardiac injury by activating the PI3K/Akt/FoxO1 axis. In addition, these cardioprotective effects can be reduced by the PI3K specific inhibitor LY294002, suggesting that the aforementioned protective effects may be related to activation of the PI3K/Akt/FoxO1 axis. CONCLUSIONS: HUK seems to control inflammatory infiltration and cardiomyocyte apoptosis significantly to improve CME-induced cardiac injury via regulating the PI3K/Akt/FoxO1 axis.

The Glu69Asp Polymorphism of EME1 Gene is Associated with an Increased Risk of Hepatocellular Carcinoma in Guangxi Population, China.

Background: The dysfunction of Essential meiotic endonuclease 1 homolog 1 (EME1) can lead to genomic instability and tumorigenesis. Single nucleotide polymorphisms (SNPs) in the EME1 gene have been reported to be associated with the risk of several cancers, but its association with hepatocellular carcinoma (HCC) has not been investigated. This study aimed to determine the association between EME1 SNPs and the risk of HCC. Methods: This study included 645 HCC patients and 649 healthy controls from a Guangxi population of Southern China, and genotyped three functional SNPs (Glu69Asp: rs3760413A>C, Ile350Thr: rs12450550T>C, and rs11868055A>G) of the EME1 gene utilizing the Agena MassARRAY platform. Results: The rs3760413C variant genotypes (AC+CC: Glu/Asp+Asp/Asp) conferred a 1.419-fold risk of HCC compared to the AA (Glu/Glu) genotype (adjusted OR = 1.419, 95% CI = 1.017-1.980), and the allele C increased the risk of HCC in a dose-dependent manner (P trend = 0.017). Moreover, the effects of the rs3760413C variant genotypes were more pronounced in individuals who drank pond/ditch water (adjusted OR = 3.956, 95% CI = 1.413-11.076) than in those who never drank (P = 0.033). We further observed that a potential carcinogen microcystin-LR induced more DNA oxidative damages in peripheral blood mononuclear cells from the carriers of rs3760413C variant genotypes than those from the subjects with AA genotype (P = 0.006). A nomogram was also constructed combining the rs3760413A>C polymorphism and environmental risk factors for predicting HCC risk with a good discriminatory ability (concordance index = 0.892, 95% CI: 0.874-0.911) and good calibration (mean absolute error = 0.005). Conclusion: Our data suggest that the Glu69Asp missense polymorphism (rs3760413) of EME1 gene is associated with the risk of HCC, which may be a susceptible biomarker of HCC in the Guangxi population.

Genetic Variant of PP2A Subunit Gene Confers an Increased Risk of Primary Liver Cancer in Chinese.

BACKGROUND: Protein phosphatase 2A (PP2A, a serine/threonine phosphatase) is frequently inactivated in many types of cancer, including primary liver cancer (PLC). Genetic variations in PP2A subunits have been reported to be associated with the risk of many types of cancer but rarely in PLC. This study aims to assess the association between functional polymorphisms of PP2A subunit genes and the risk of PLC in Chinese. METHODS: In a case-control study with a total of 541 PLC patients and 547 controls in Guangxi province of Southern China, we genotyped six putatively functional polymorphisms (rs10421191G>A, rs11453459del>insG, rs1560092T>G, rs7840855C>T, rs1255722G>A and rs10151527A>C) of three PP2A subunit genes (PPP2R1A, PPP2R2A and PPP2R5E) using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry platform. RESULTS: The rs11453459insG variant genotypes (ins/ins+del/ins) of PPP2R1A were found to be significantly associated with an increased risk of PLC compared with the del/del genotype (adjusted OR = 1.290, 95% CI = 1.009-1.650), and the number of insert G allele worked in a dose-dependent manner (P trend= 0.007). The stratified analysis showed that the effects of rs11453459insG variant genotypes were more evident in the subgroup who drink pond-ditch water (adjusted OR = 3.051, 95% CI = 1.264-7.364) than those never drink (P = 0.041). The carriers of rs11453459 del/ins genotype had a significantly lower level of PPP2R1A mRNA expression in liver cancer tissues than those of the del/del genotype (P = 0.021). Furthermore, we used microcystin-LR, a carcinogen presents in the pond-ditch water, to treat human peripheral blood mononuclear cells and found that the cells from carriers of rs11453459insG variant genotypes induced more DNA oxidative damages than those from the del/del genotype carriers (P < 0.001). CONCLUSION: These findings suggest that the PPP2R1A rs11453459del>insG polymorphism is associated with an increased risk of PLC, especially for persons with a history of drinking pond-ditch water. This insertion/deletion polymorphism may be a susceptible biomarker for PLC in Chinese.

Specific Differentially Methylated and Expressed Genes in People with Longevity Family History.

BACKGROUND: We attempt to identify specific differentially methylated and expressed genes in people with longevity family history, it will contribute to discover significant features about human longevity. METHODS: A prevalence study was conducted during October 2017 to January 2019 in Bama County of Guangxi, China and individuals were recruited and grouped into longevity family (n=60) and non-longevity family (n=60) to identify differentially methylated genes (DMGs). The expression profile dataset GSE16717 was downloaded from the GEO database in which individuals were divided into 3 groups, namely longevity (n=50), longevity offspring (n=50) and control (n=50) for identifying differentially expressed genes (DEGs). It was considered significantly different when P or adjusted P≤0.05. RESULTS: In total, 117 longevity-related hypermethylated genes enriched in interleukin secretion/production regulation, chemokine signaling pathway and natural killer cell-mediated cytotoxicity. Another 296 significant key longevity-related DEGs primarily involved in protein binding, nucleus, cytoplasm, T cell receptor signaling pathway and Metabolic pathway, H19 and PFKFB4 were found to be both methylated and downregulated in people with longevity family history. CONCLUSION: Human longevity-specific genes involve in many immunity regulations and cellular immunity pathways, H19 and PFKFB4 show hypermethylated and suppressed status in people with longevity family history and might serve as longevity candidate genes.

Down-regulation of GCLC is involved in microcystin-LR-induced malignant transformation of human liver cells.

Microcystin-LR (MCLR) is a potent hepatotoxin which could lead to the development of hepatocellular carcinoma. However, the mechanisms of its carcinogenic action remain obscure. The catalytic subunit of glutamylcysteine ligase (GCLC) primarily regulates de novo synthesis of glutathione and is central to the antioxidant capacity of the cell, but emerging data suggest that the GCLC expression is associated with cancer development. The purpose of this study was to investigate the role and molecular mechanisms of GCLC in MCLR-induced malignant transformation of a human liver cell line WRL68. During MCLR-induced cell transformation, the expression of GCLC and activity of glutamate-cysteine ligase (GCL) decreased continuously, accompanied with consistent low levels of glutathione (GSH) but high levels of oxidative DNA damages. Furthermore, MCLR markedly inhibited protein phosphatase 2 A (PP2 A), and increased the level of GCLC phosphorylation. In contrast, overexpression of GCLC significantly enhanced the levels of GSH, inhibited oxidative DNA damages, and suppressed MCLR-induced cell invasion and migration, as well as tumor growth in nude mice. GCLC overexpression partially attenuated MCLR-induced PP2 A inhibition. Together, the current results suggest that down-regulation of GCLC is involved in MCLR-induced malignant transformation of human liver cells by inducing oxidative stress.

Functional Polymorphism in the MSI1 Gene Promoter Confers a Decreased Risk of Lung Cancer in Chinese by Reducing MSI1 Expression.

BACKGROUND: Musashi1 (MSI1) is a characteristic stem cell marker that regulates the balance between cell self-renewal and differentiation. Evidence has identified MSI1 as a pivotal oncogenic regulator in diverse malignancies. However, little evidence uncovers the role of genetic variations of MSI1 gene in cancer etiology. OBJECTIVE: The aim of this study was to investigate the association between genetic variants in the MSI1 gene and lung cancer risk. METHODS: Based on a two-stage retrospective study with a total of 1559 patients with lung cancer and 1667 healthy controls, we evaluated the relevance between three putative functional SNPs in the MSI1 promoter (i.e., -2696T>C[rs7959801], -2297T>C[rs3742038] and -1081C>T[rs34570155]) and lung cancer risk. RESULTS: We found that the SNP rs7959801T>C was significantly associated with lung cancer susceptibility. Compared to those with rs7959801TT wild-genotype, individuals with CT/CC variant genotypes exerted consistently beneficial roles in lung cancer risk in the discovery set (adjusted odd ratios [OR] = 0.67; 95% confidence interval [CI] = 0.57-0.80), and in the validation set (OR=0.69; 95%CI=0.54-0.88). Functional assays indicated that the allele transformation from T to C in rs7959801 of MSI1 gene arrestingly decreased its transcription activity in vitro. Furthermore, the expression levels of MSI1 were significantly lower in the patients with CT/CC variants than in those who were with TT genotype. CONCLUSION: Our findings suggested that the rs7959801T>C polymorphism in the MSI1 promoter conferred a decreased risk to lung cancer by reducing the expression of MSI1 and it may be a promising indicator for lung cancer predisposition.

NBS1 rs2735383 polymorphism is associated with an increased risk of laryngeal carcinoma.

BACKGROUND: Nijmegen breakage syndrome 1 (NBS1), as a key protein in the DNA double-strand breaks (DSBs) repair pathway, plays an important role in maintaining genomic stability. Although single nucleotide polymorphisms (SNPs) in NBS1 have frequently been studied in multiple cancers, the relationships of two functional NBS1 polymorphisms (rs2735383 and rs1805794) with laryngeal carcinoma are yet unclear. Therefore, in the present study, we performed a case-control study including 342 cases and 345 controls to analyze the associations between two polymorphisms of NBS1 and the risk of laryngeal carcinoma. METHODS: We used the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to determine the genotypes of the functional SNPs in NBS1 gene. RESULTS: In comparison with the homozygous rs2735383GG genotype, the CC genotype was significantly associated with an increased risk of laryngeal carcinoma (adjusted OR = 1.884, 95%CI = 1.215-2.921). The rs2735383C variant genotypes (GC + CC) conferred a 1.410-fold increased risk of laryngeal carcinoma (adjusted OR = 1.410, 95%CI = 1.004-1.980). Furthermore, when compared to rs2735383GG genotype in laryngeal carcinoma tissues, the combined GC and CC genotypes exerted a significantly lower mRNA level of NBS1 (P = 0.003). In contrast, no significant association was found between rs1805794G > C polymorphism and cancer risk (adjusted OR = 1.074, 95%CI = 0.759-1.518 for GC; adjusted OR = 1.100, 95%CI = 0.678-1.787 for CC; adjusted OR = 1.079, 95%CI = 0.774-1.505 for GC + CC). CONCLUSIONS: These findings indicate that rs2735383G > C polymorphism in NBS1 may play a crucial role in the development of laryngeal carcinoma.

Environmental Microcystin Exposure Increases Liver Injury Risk Induced by Hepatitis B Virus Combined with Aflatoxin: A Cross-Sectional Study in Southwest China.

Three liver hazards, two confirmed-hepatitis B virus (HBV) and aflatoxin (AFB), and one rarely studied in populations-microcystin (MC), simultaneously exist in tropical and humid areas; however, there are no epidemiological data on their risks in the same population. We conducted a community-based cross-sectional survey among 5493 adults in two rural towns and statistically analyzed the comparative and combinative effects of the three factors after detecting HBsAg and HBV DNA titers, determining estimated daily intakes (EDIs) of AFB1 and MC-LR and testing serum AST and ALT as liver injury markers for each participant. We observed a HBsAg(+) rate of 7.6%, a relatively high AFB1 exposure level (mean EDIAFB1 = 471.30 ng/d), and a relatively low MC-LR exposure level (mean EDIMC-LR = 228.25 ng/d). ORs for abnormal AST (2.42, 95%CI = 1.69-3.45) and ALT (2.87, 95%CI = 1.91-4.29) increased in HBV infections compared with HBV-unexposed participants but did not increase in participants with separate or combined exposure to AFB1 and MC-LR (EDIs ≥ mean). Meanwhile, after adjustment for confounding factors, means of AST and ALT and ORs of abnormal AST and ALT were successively elevated after exposure to HBV, HBV&AFB1 (or HBV&MC-LR), and HBV&AFB1&MC-LR, especially in the group with detectable HBV DNA (AST: OR = 11.38, 95%CI = 3.91-33.17; ALT: OR = 17.09, 95%CI = 5.36-54.53). Notably, ORs for abnormal AST and ALT in the HBV exposed group were not significantly different from those in HBV&AFB1 or in the HBV&MC-LR exposed group but were significantly higher in the HBV&AFB1&MC-LR exposed group (P = 0.029 and P = 0.037, respectively). Our study indicated that microcystin may have the potential to increase the risk of liver injury induced by combined exposure to HBV and aflatoxin. However, in consideration of the uncertainties in the detection of the toxins and evaluation of the EDIs, more epidemiological data are expected to determine the increasing toxic effects of microcystins.

Rare variant of MAP2K7 is associated with increased risk of COPD in southern and eastern Chinese.

BACKGROUND AND OBJECTIVE: A wide range of common loci have been extensively screened and evaluated for their associations with various complex diseases; however, the relevance of rare variants causing missense substitutions in the protein-coding genes in human diseases is still poorly understood. METHODS: In this study, we conducted a two-stage retrospective study of a total of 1791 patients with COPD and 1940 controls in southern and eastern Chinese to test relevancies of five rare variants (i.e. p.Glu116Lys, p.Asn118Ser, p.Arg138Cys, p.Ala195Thr and p.Leu259Phe) of human mitogen-activated protein kinase kinase 7 (MAP2K7) to COPD susceptibility. The effects of these loci on lung function were further estimated. RESULTS: The p.Glu116Lys rare variant had significant associations with COPD risk. Compared to individuals with Glu/Glu wild-genotype, those with 116Lys rare variants (Lys/Glu+Lys/Lys) had an increased risk of COPD (OR = 3.83, 95% CI: 2.64-5.56; P = 1.45 × 10-12 ). Meanwhile, the carriers with 116Lys rare variants (Lys/Glu+Lys/Lys) had lower pre-forced expiratory volume in 1 s (pre-FEV1 : 1.74 ± 0.70 vs 2.00 ± 0.68; P = 3.97 × 10-5 ) and lower pre-FEV1 to pre-forced vital capacity ratio (pre-FEV1 /FVC: 0.68 ± 0.14 vs 0.75 ± 0.12; P = 2.40 × 10-10 ) than those with Glu/Glu genotype. However, for other rare variants, no significant association with either COPD risk or lung function was observed. CONCLUSION: Our data strongly suggest that the p.Glu116Lys rare variant in MAP2K7 predisposes its carriers to develop COPD, which would provide a useful genetic biomarker for COPD susceptibility in Chinese.

The MKK7 p.Glu116Lys Rare Variant Serves as a Predictor for Lung Cancer Risk and Prognosis in Chinese.

Accumulated evidence indicates that rare variants exert a vital role on predisposition and progression of human diseases, which provides neoteric insights into disease etiology. In the current study, based on three independently retrospective studies of 5,016 lung cancer patients and 5,181 controls, we analyzed the associations between five rare polymorphisms (i.e., p.Glu116Lys, p.Asn118Ser, p.Arg138Cys, p.Ala195Thr and p.Leu259Phe) in MKK7 and lung cancer risk and prognosis. To decipher the precise mechanisms of MKK7 rare variants on lung cancer, a series of biological experiments was further performed. We found that the MKK7 p.Glu116Lys rare polymorphism was significantly associated with lung cancer risk, progression and prognosis. Compared with Glu/Glu common genotype, the 116Lys rare variants (Lys/Glu/+ Lys/Lys) presented an adverse effect on lung cancer susceptibility (odds ratio [OR] = 3.29, 95% confidence interval [CI] = 2.70-4.01). These rare variants strengthened patients' clinical progression that patients with 116Lys variants had a significantly higher metastasis rate and advanced N, M stages at diagnosis. In addition, the patients with 116Lys variants also contributed to worse cancer prognosis than those carriers with Glu/Glu genotype (hazard ratio [HR] = 1.53, 95% CI = 1.32-1.78). Functional experiments further verified that the MKK7 p.116Lys variants altered the expression of several cancer-related genes and thus affected lung cancer cells proliferation, tumor growth and metastasis in vivo and in vitro. Taken together, our findings proposed that the MKK7 p.Glu116Lys rare polymorphism incurred a pernicious impact on lung cancer risk and prognosis through modulating expressions of a serial of cancer-related genes.

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity.

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl dulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.

Involvement of mitogen-activated protein kinase signaling pathways in microcystin-LR-induced apoptosis after its selective uptake mediated by OATP1B1 and OATP1B3.

The serine/threonine protein phosphatase (PP) 2A inhibitor, microcystin-LR, selectively induces liver damage and promotes hepatocarcinogenesis. It is thought that microcystin-LR affects hepatocellular viability mainly through inhibition of PP2A, partially through PP1, and, in addition, by generation of reactive oxygen species (ROS). However, the molecular basis of the selective liver damage and the balance between cell death and survival remained unclear. We analyzed the cytotoxicity of low doses of microcystin-LR using HEK293 cells stably expressing the human hepatocyte uptake transporters, organic anion transporting polypeptide (OATP)1B1 (HEK293-OATP1B1 cells) and OATP1B3 (HEK293-OATP1B3 cells). HEK293-OATP1B1 (IC(50) 6.6nM) and HEK293-OATP1B3 cells (IC(50) 6.5nM) were equally very sensitive to microcystin-LR. In contrast, control-vector-transfected (HEK293-CV) cells were resistant to microcystin-LR. Using HEK293-OATP1B3 cells, the cytotoxicity was attenuated by substrates and inhibitors of OATP1B3, including bromosulfophthalein, rifampicin, and cyclosporin A. Microcystin-LR was transported into HEK293-OATP1B3 cells with 1.2 microM Km value, and its uptake was inhibited by above substances. Accumulation of microcystin-LR in the HEK293-OATP1B1 and HEK293-OATP1B3 cells was increased in a dose-dependent manner but not in HEK293-CV cells. Cellular serine/threonine PP activity of HEK293-OATP1B3 cells was decreased by microcystin-LR but not in HEK293-CV cells. Apoptotic changes were observed after incubation of the HEK293-OATP1B3 cells with microcystin-LR. We found by FACS analysis that microcystin-LR induced apoptosis but not necrosis in HEK293-OATP1B3 cells. Microcystin-LR activated several mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK, and p38 through inhibition of PP2A. In addition, the cytotoxicity of microcystin-LR was attenuated by the inhibitors of MAPK pathways, including U0126, SP600125, and SB203580. The ROS scavenger N-acetyl-L-cysteine partially attenuated the cytotoxicity of microcystin-LR. Thus, the present study demonstrates that microcystin-LR induces apoptosis through activation of multiple MAPK pathways subsequent to its selective uptake via OATP1B1 and OATP1B3 and followed by inhibition of PP2A, in addition to the ROS generation which might contribute to apoptosis.