| ObjectHCC ranks fifth in the malignant tumor, became a major hazard to human health. According to the latest report announced by WHO, the world’s liver cancer incidence and mortality in men are two to three times than women. Previous studies confirm that the liver is a type of endocrine organ. It seems that physiological functions of liver is dependent on hormone signal. However, the mechanism of hepatocellular carcinoma (HCC) gender bias is still unclear. Clinical tries in liver cancer patients are taking pills, chemotherapy, surgery, radiation therapy and other methods. Meanwhile, treatment of hormone drugs come to our eyes, but the effects are not very satisfactory. As side effects of drugs, a higher rate of recurrence after surgery, radiotherapy and chemotherapy patients with compromised immune problems are still prominent, it takes our ability to elucidate the molecular mechanism of liver cancer, especially, to look for new treatment methods in gender differences signaling networks.The current opinion is that androgen plays a catalytic role in liver cancer, while estrogen plays an inhibitory effect, which is the leading cause of HCC gender bias. In recent years, the interaction between hormones and pattern recognition receptors (PRR) attract scholars’attention. Early in the 20th, studies shows TLR4、MyD88 and estrogen signal make the gender differences in liver cancer. In 2015, the latest study found that estrogen though ER stimulating MAPK pathway activation on the biological characteristics of HCC cell lines. In this process of change, NLRP3 is indeed important. PRR contains TLR, RLR and NLR, our previous study focus on the TLR4 molecule. In my research, I care about the most classic molecules NODI and NOD2 of The NLR family. Wherein, TLR4 molecules mainly activated by the gut of harmful ingredients lipopolysaccharide (LPS). The intestinal microflora though enterohepatic axis activate TLR4 molecule on liver cells, and promote the occurrence of liver fibrosis and cirrhosis, HBV and liver cancer. Whether TLR4 and androgen receptor exist "cross-talk", co-promote cancer has not been reported so far.Based on the phenomenon above, we carried out research in two parts. We established HCC inducing model using DEN/CCL4, combined with hepatoma cell lines experiment in vitro. On the one hand, the cross-talk between AR and TLR4 underway explored. They regulate of liver cancer malignant proliferation, migration, invasion, may be a new target for HCC and provide a new direction for clinical liver cancer hormone therapy. On the other hand, I focus my attention on AR through NODI and its downstream pathways that regulate liver cancer. For the first time, the role of NODI and NOD2 in the liver has been clarified, moreover the internal mechanism of NOD in HCC gender bias.MethodsPart Ⅰ AR aggravated TLR4-induced HCC1. Using Real-time PCR and FACs to detect the basis of AR and TLR4 expression in male and female HCC induced mice. Immunofluorescence was used to detect the expression of TLR4 and AR in HCC cell lines.2. HCC cell lines were used to detect the TLR4 expression when stimulating hormone by Western Blot.3. MTT assay, clone assay was used to detect DHT and LPS on hepatocellular carcinoma cell proliferation. Human and murine hepatoma cell line stimuli with DHT and LPS, moreover cycle and apoptosis were detected by PI staining, Annexin V/PI staining.4. Immunohistochemistry was used to detect the expression of Ki67 in WT type and TLR4-/- mice. The expression of Ki67, PCNA, Acta2 and the AFP mRNA in liver parenchyma were detected by Real-time PCR.5. To verify AR’s effect, we silence AR by siRNA to detect proliferation, clone formation, cycle and invasion capacity.PartⅡ NOD1 promoted AR effects in hepatocarcinogenesis1. In DEN/CCL4-induced C57BL/6 mice, the expression levels of liver parenchyma Ki67, PCNA, Acta2 and AFP mRNA were detected by Real Time-PCR. HE staining of liver tissue were determined for inflammation disseminated status. IHC was used to detect the expression of Ki67 in male and female mice.2. Real Time-PCR was used for detecting NLRs gender basis in hepatic cell. IHC experiments was used to measure the expression of N0D1/RIP2. Their functional expression was detected by Western Blot.3. The basic mRNA level of NLRs, AR and ER in HCC cells were determined by Real-Time PCR. Western-blot and Real-Time PCR were used to detect the functional expression of NOD1/NOD2 in HCC cell lines.4. NOD agonist and hormones were used on hepatoma cell line, migration, invasion and proliferation were used to detect their function. Hormones and NOD agonist were injected in DEN/CCL4 induced HCC model, then Real Time-PCR and IHC were used to detect Ki67, PCNA, Acta2 and AFP expression levels.5. PI staining and Annexin V/PI staining were employed to detect cell cycle and apoptosis after NOD agonist and hormones stimuli.6. Immunohistochemistry was used to detect the expression of NOD1 in cancer tissue site and adjacent detection. Consistently, NODI, NOD2, RIP2 expression in Liver cancer patients at early stage was detect by IHC in cancerous and normal parts of the site, analyzed correlation of male and female patients.ResultsPart Ⅰ AR aggravated TLR4-induced HCC1. TLR4 and AR were expressed in both sex mice, while TLR4 and AR mRNA and protein levels were higher in DEN/CCL4 induce male mice. TLR4 protein was observed in HCC cell lines. AR was expressed high in HepG2 and HepG2.2.15, while expressed low in Hep 1-6.2. DHT can significantly increase the expression of TLR4, while E2 is able to inhibit the expression of TLR4.3. LPS significantly enhanced AR expression in hepatoma cells.4. Proliferation of HepG2 can be up-regulate obviously after LPS and DHT stimulating. LPS and DHT also can significantly co-promote hepatoma cell lines cell cycle transition from G1 to S phase, but had no effect on apoptosis.5. Under the combination of LPS and DHT, hepatoma cell would healing and migration increased significantly.6. Ki67 protein levels expressing lower in TLR4-/- DEN/CCL4 induced mice.7. TLR4 and AR expression is higher in HCC tissue samples than adjacent tissues.PartⅡ NOD1 promoted AR effects in hepatocarcinogenesis1. The male mice expressed Ki67, PCNA, Acta2 and AFP mRNA significantly higher than female mice after DEN/CCL4 induce. Male murine bears more inflammatory conditions, by contrast to female mice.2. NLRP3 and AIM2 mRNA levels did not change, but NOD1 was upregulated in the induced male mice while NOD2 molecular change is the opposite.3. NOD1 molecule mRNA and protein levels increased in the mid-late male mice, while NOD2 changes in the opposite way.4. Hepatoma cell lines expressed NOD 1, NOD2, AR and ER.5. NOD1 is hormone dose-dependent. DHT stimulate NODI expressed higher, meanwhile E2 down-regulated the expression of NODI and RIP2, indicating sex hormones involved in the regulation of NOD molecules in HCC.6. The migration of DAP combined with DHT group is obvious, and the mice model were also found DHT and DAP stimulated group expressing higher malignant proliferation joint indicators.7. DHT and NOD agonists have no effect on apoptosis.8. NOD1 and AR expressed higher in patients with liver cancer.Conclusion1. This is the first discovery about TLR4 with AR can be coordinately regulated liver cancer. Liver cancer patients treated with hormone drugs combined with TLR4 or other small molecule antagonists may enhance the therapeutic effect of liver cancer;2. The study on hepatoma cell function of NOD1/NOD2 was the first one in the world and set out its internal mechanism that NOD1 regulated the development of liver cancer though AR. |
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