Studies On The Relationship Between The Methylation Of Chemosensitivity-related Gene,LRP16 And NF-κB Activation In Gastric Carcinoma

BackgroundGastric cancer is the second leading cause of cancer-related death worldwide, with the highest incidence in Eastern Asian and European countries. Unfortunately, most patients with advanced gastric cancer (AGC) have a miserable outcome. Even after curative gastrectomy,60% of AGC patients develop local recurrences or distant metastasis. So adjuvant postoperative chemotherapy is now widely accepted. Epigenetics changes are heritable changes in gene expression that do not involve an alteration in the DNA sequence. Some researches have discovered that epigenetic changes can be a crucial driving force behind the acquisition of drug resistance. This study mainly focused on the relationship between the promoter methylation of chemosensitivity-related gene and the prognosis of gastric carcinoma patients. Thus, the alliance of pharmacodynamic and predictive epigenetic biomarkers will allow rational and efficient evaluation of novel epigenetic therapies for gastric cancer treatment.Nuclear factorκB (NF-κB)-mediated pathways have been widely implicated in cell survival, development and tumor progression. Although the molecular events of determining NF-κB translocation from cytoplasm to nucleus have been extensively documented, the regulatory mechanisms of NF-κB activity inside the nucleus are still poorly understood. Being a special member of macro domain proteins, LRP16 was previously identified as a coactivator of both estrogen receptor and androgen receptor, and probably took part in NF-κB coactivator. Here, to clearly determine whether LRP16 expression could be extended to NF-κB activity in clinical samples, human gastric carcinoma specimens were selected, instead of hormone related cancers, in order to exclude the possible competitive interaction interference of LRP16 with other transcription factors, such as ERa and AR.ObjectiveEpigenetics is a subject which focuses on gene expression, mainly including DNA methylation, histone modifications, and noncoding RNAs. Epigenetics plays an important role in gastric cancer carcinogenesis. More and more researchers start to focus on epigenetics changes of gastric cancer. The clinical value of DNA methylation changes was recongized widely. In this study, the methylation level of chemosensitivity-related gene promoter in gastric cancer tissues was detected to explore the relationship between the promoter hypermethylation of those genes and the prognosis of gastric cancer patients.NF-κB-associated pathways have been widely implicated in oncogenesis and tumor progression by stimulating cell proliferation, inhibiting apoptosis, promoting metastasis and angiogenesis. Therefore, this study was based on the previous researches. The relationship between LRP16 and NF-κB activation was investigated in gastric carcinoma, which might help to find a new potential therapeutic target for the control of excessive NF-κB activity.MethodsOne hundred and two Samples were collected, and then genomic DNA was extracted from tissues by using Phenol/CHC12, finally treated with bisulfite. The technique of methylation specific PCR (MSP) was adopted to investigate the promoter hypermethylation of CHFR, FANCF, MLH1, MGMT and RASSF1A in the specimens of gastric cancer. And the following biological markers, such as MLH1, Ki-67 and P53, were studied by immunohistochemistry (IHC). The relationship between the frequency of hypermethylation and the expression of the genes and clinicopathological data was analyzed.293T cells were cotransfected with pCDNA3.1-LRP16 or pCDNA3.1-p65 or the empty expression vector andκB-luc by SuperFect reagent. Forty-two hours after transfection, cells were simulated with TNF-αor IL-1βfor 7 h before luciferase assays was performed. Then cells were lysed for luciferase assays and western blot. Total proteins were extracted using a ReadyPrepTM protein extraction kit from gastric carcinoma samples.The DNA binding activity of NF-κB in the tumor samples was measured by using the enzyme-linked immunosorbent assay. LRP16 expression in primary tumor tissues was determined by immunohistochemistry. ResultsOnly the methylation of FANCF gene promoter was not detected in the 102 gastric cancer tissues. In these tissues, the positive methylation rates of CHFR, MGMT, MLH1 and RASSF1A were 34.3%(35/102),9.8%(10/102),21.6%(22/102) and 12.7%(13/102) respectively. Methylation of CHFR gene promoter in gastric cancer patients is correlated to lymph node metastasis (p<0.05). CHFR methylation increased resistance to docetaxel in RFS (log rank p<0.05). While the methylation of MLH1 gene in gastric cancer patients did not correlate with any clinicopathological characteristics, such as gender, age, size of tumor, differentiation level, lymph node metastasis, TNM phase, and so on. Patients without MLH1 methylation showed signifcantly better survival in overall survival (OS) (log rank p<0.05) than patients with MLH1 methylation. MLH1 methylation increased oxaliplatin resistance in RFS (log rank p<0.05). The protein expression rates of MLH1, Ki-67 and P53 were 39.2% (40/102),69.6%(71/102) and 51.0%(52/102) respectively. P53 protein expression increased oxaliplatin resistance in RFS (log rank p<0.05).We demonstrate that the ectopic expression of LRP16 increased TNF-α-or IL-1β-inducedκB-luc activity by more than 1.5 fold. Gene activation induced by p65 alone was also increased in cells in which LRP16 was overexpressed. The ectopic expression of LRP16 increased NF-κB activity in cells. A positive link between LRP16 expression intensity in nuclei of tumor cells and NF-κB activity was preliminarily established in human gastric carcinoma specimens (r=0.326,P=0.014). The level of active NF-κB in the LRP16-positive subset (OD value=0.316±0.042) was significantly higher than that in the LRP16-negative subset (OD value=0.288±0.036) (t=31.64, P=0.012). ConclusionsThe methylation of CHFR, MLH1 and the protein expression of P53 maybe play an important role in gastric cancer carcinogenesis. The detection of them will apply to clinic for gastric cancer prognosis, chemosensitivity and treatment.Our findings not only indicate that LRP16 is a crucial regulator for NF-κB activation inside the nucleus, but also suggest that LRP16 may be an important contributor to the aberrant activation of NF-κB in tumors. Aberrant nuclear accumulation of LRP16 in tumor cells might represent a therapeutic target for the control of excessive NF-kB activity.