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Microcystins (MCs) have been shown to be carcinogenic by animal and cellular experiments and found to be associated with the development of human hepatocellular carcinoma (HCC) through epidemiological studies. However, the molecular mechanism of microcystin-LR (MC-LR) induced HCC is still unclear. This study is determined to clarify the role and mechanism of LHX6 in MC-LR-induced hepatocarcinogenesis. Using the previously established MC-LR-induced malignant transformation model in L02 cells, we screened out LHX6, homeobox gene that was significantly changed. We found that LHX6 was significantly down-regulated in MC-LR treated L02 cells and the liver tissue of rats treated for 35 weeks with 10 μg/kg body weight of MC-LR. Expression of LHX6 in human tumor tissue was significantly down-regulated in high MC-LR-exposure group. LHX6 was hypermethylated in MC-LR treated L02 cells and up-regulated after treatment with 10 μM of 5-aza-2′-deoxycytidine. Furthermore, overexpression of LHX6 inhibited proliferation, invasion and migration of malignantly transformed L02 cells in vitro and in vivo, while knockdown of LHX6 resulted in an opposite phenotype. In addition, we found that up-regulation of P53 and Bax resulted in apoptosis, and that down-regulation of CTNNB1 and MMP7 led to migration of MC-LR treated L02 cells. Blockade of P53 and CTNNB1 by its inhibitor significantly diminished the effect of LHX6. These genes were working together during the process of MC-LR-induced hepatocarcinogenesis. Our study demonstrated for the first time that LHX6 gene expression is regulated by DNA methylation and can inhibit the proliferation, invasion and migration through Wnt/β-catenin and P53 signaling pathways during the MC-LR-induced hepatocarcinogenesis. This result may suggest that LHX6 gene can be used as a potential target gene and a biomarker for liver cancer treatment.

Recent studies have shown that microcystin-LR (MC-LR) is one of the principal factors that cause liver cancer. Previously we have found that Aristaless-like Homeobox 4 (ALX4) was differentially expressed in MC-LR-induced malignant transformed L02 cells. However, the expression regulation, role and molecular mechanism of ALX4 during the process of liver cancer induced by MC-LR are still unclear. The expression of ALX4 was detected by quantitative reverse-transcription PCR and Western blot in MC-LR induced malignantly transformed cell and rat models. Methylation status of ALX4 promoter region was evaluated by methylation-specific PCR and bisulfite genomic sequencing. The anti-tumor effects of ALX4 on MC-LR induced liver cancer were identified in vitro and in vivo. ALX4 expression was progressively down-regulated in MC-LR-induced malignantly transformed L02 cells and the MC-LR exposed rat models. ALX4 promoter regions were highly methylated in malignantly transformed cells, while treatment with demethylation agent 5-aza-dC significantly increased ALX4 expression. Functional studies showed that overexpression of ALX4 inhibits cell proliferation, migration, invasion and metastasis in vitro and in vivo, blocks the G1/S phase and promotes the apoptosis. Conversely, knockdown of ALX4 promotes cell proliferation, migration and invasion. Mechanism study found that ALX4 exerts its antitumor function through the P53 pathway, C-MYC and MMP9. More importantly, ALX4 expression level showed a negative relation with serum MC-LR levels in patients with hepatocellular carcinoma. Our results suggested that ALX4 was inactivated by DNA methylation and played a tumor suppressor function through the P53 pathway in MC-LR induced liver cancer.

3-methylcholanthrene (3-MCA) is a typical representative PAH. It has strong toxicity and is a typical chemical carcinogen. However, the epigenetic mechanisms underlying 3-MCA-induced tumourigenesis are largely unknown. In this study, a model of the 3-MCA-induced malignant transformation of human bronchial epithelial (HBE) cells was established successfully. The profiles of gene expression and DNA methylation and hydroxymethylation were obtained and analysed with an Illumina HiSeq 4000. A total of 707 genes were found to be significantly up-regulated, and 686 genes were found to be significantly down-regulated. Compared to control cells, 8545 mRNA-associated differentially methylated regions and 15,121 mRNA-associated differentially hydroxymethylated regions in promoters were found to be significantly altered in transformed cells. By using mRNA expression and DNA methylation and hydroxymethylation interaction analysis, 99 differentially expressed genes were identified. Among them, CA9 and EGLN3 were verified to be significantly down-regulated, and CARD6 and LCP1 were shown to be significantly up-regulated, and these genes mainly participated in cell growth, migration and invasion, indicating that these genes were key genes involved in the 3-MCA-induced malignant transformation of HBE cells. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that a large number of differentially expressed genes (DEGs) were involved mainly in RNA polymerase II transcription factor activity, chemical carcinogenesis, base-excision repair (BER), cytokine-cytokine receptor interactions, glycerolipid metabolism, steroid hormone biosynthesis, cAMP signalling pathways and other signalling pathways. Our study suggested that characteristic gene alterations associated with DNA methylation and hydroxymethylation could play important roles in environmental 3-MCA-induced lung carcinogenesis.

Backgroud: The methylcytosine dioxygenase Ten-eleven translocation 1 (TET1) is an important regulator for the balance of DNA methylation and hydroxymethylation through various pathways. Increasing evidence has suggested that TET1 probably involved in DNA methylation and demethylation dysregulation during chemical carcinogenesis. However, the role and mechanism of TET1 during lung cancer remains unclear. Methods: The gene and protein expression were detected by qRT-PCR, Western blot and immunohistochemistry during lung carcinogenesis. Methylation and hydroxymethylation status were evaluated by MeDIP-qPCR and hMeDIP-qPCR. The effect and mechanism of TET1 on lung cancer were identified both in vitro and in vivo models. Results: In this study, we found that TET1 expression was significantly down-regulated and the methylation level was significantly up-regulated in 3-MCA-induced cell malignant transformation model, rat chemical carcinogenesis model, and human lung cancer tissues. Demethylation experiment further confirmed that DNA methylation negatively regulated TET1 gene expression. TET1 overexpression inhibited cell proliferation, migration and invasion in vitro and in vivo, while knockdown of TET1 resulted in an opposite phenotype. DNA hydroxymethylation level in the promoter region of base excision repair (BER) pathway key genes XRCC1, OGG1, APEX1 significantly decreased and the degree of methylation gradually increased in malignant transformed cells. After differential expression of TET1, the level of hydroxymethylation, methylation and expression of these genes also changed significantly. Furthermore, TET1 binds to the promoter of XRCC1, OGG1, and APEX1 to maintain them hydroxymethylated. Blockade of BER pathway key gene alone or in combination significantly diminished the effect of TET1. Conclusions: Our study demonstrated for the first time that TET1 gene expression is regulated by DNA methylation and TET1-mediated hydroxymethylation regulates BER pathway to inhibit the proliferation, migration and invasion during the 3-MCA-induced lung carcinogenesis. These results may suggest that TET1 gene can be used as a potential biomarker and therapy target for lung cancer.

The super-Gaussian random vibration test has increasingly attracted attention for its existed advantages in comparison with traditional random vibration test. The super-Gaussian random signal generation principle is investigated in study based on the research about control theory for the super-Gaussian random vibration test, meanwhile, super-Gaussian random drive signal used in control system is obtained from Poisson process and filter theory. The experiment proves that high control accuracy of output power spectrum and kurtosis can be realized by taking super-Gaussian random vibration control strategy proposed in paper, which is consistent with the requirement in engineering. In the end, super-Gaussian random vibration control system is applied into the Highly Accelerated life Testing(HALT) for the incandescent lamp, and a comparison between super-Gaussian random vibration test and traditional Gaussian distribution random vibration test is carried out, the comparison results indicate that super-Gaussian random vibration excitation signal comparing with Gaussian distribution signal can markedly quicken the fatigue failure process for incandescent lamp.

Multi-exciter vibration can provide greater thrust for overweight test specimen, achieve the uniformity of vibration for the special structure of the thin specimen, and overcome the shortcomings in single-exciter test, it becomes the trend of vibration test. Based on the control theory of multi-exciter vibration test, a system equilibrium synthesis is presented to make up for deficiencies of different methods for random vibration test control algorithm. the relevant experimental study is carried out on the experimental platform, experimental results show that dynamic control range of multi-exciter random vibration control system achieves 90dB in the self closed-loop control test. high precision is reached for self-spectrum control of actual test system after establishing multiple shaker, self-spectrum of control response is controlled in the range of ± 1.5dB, which satisfies requirements in engineering test.