| BackgroudAt present, liver cancer is the second most frequent cause of cancer death in China. Although, surgical treatments including resection is the most effective treatment for HCC, the recurrent and metastasizing rate is up to 80% after undergone these treatments in 5 years, which is the primary problem for the liver surgeon. The high recurrent and metastasizing rate is due to the invasion and migration of HCC. Plenty of evidences have been discovered to prove that the emergence and development of cancer can not process without the innervation. Our previous studies had proved that the over excitation of sympathetic nerve can enhance the inflammatory reaction when the liver is injured to promote the emergence of HCC. Meanwhile in recent studies, parasympathetic nerve had been proved that it could inhibit the inflammatory reaction through “cholinergic anti-inflammatory pathway”, which is universally acknowledged. Nevertheless, there is still not any study reported that whether vagus never could influence the recurrence and migration of HCC.ObjectiveTo clarify the correlation between the distribution and regulation of vagus never and the recurrence and migration of HCC. And to discuss which neurotransmitter released by vagus nerve could influence the invasion and migration of human’s HCC, and what mechanism may be involved in this process.Methods1. The specimen of primary and recurrent HCC which came from clinical surgical resection were collected. With these specimen, immunohistochemistry and western blotting were used to detect the correlationship between vagus never and the recurrence and migration of HCC.2. SMMC-7721 and Hep G2 cell lines were cultured in vitro. To detect the expression of the vagus never neurotransmitters receptors in the two kinds of cells above, real time-polymerase chain reaction(RT-PCR) and immunofluorescence staining were utilized. And transwell was carried out to discover the effect of the vagus never neurotransmitters on the 9ability of the migration and invasion of these two kinds of cells in celluar l evel.3. SMMC-7721 and Hep G2 cell lines were cultured with the agonists and blockers of the muscarinic receptor(MR) and nicotinic receptor(NR) respectively. Transwell was used again to clarify the effect on the ability of the migration and invasion of these two kinds of cells after their MR and NR were excited or blocked. To discuss the effect of the vagus never neurotransmitters and the agonists and blockers of MR and NR on the ability of the proliferation of these two kinds of cells, Cell Titer Blue cell viability assay was applied. Finally, western blotting was carried out to detect the change of the expression of the protein which were considered as the markers of the epithelial-mesenchymal transition(EMT) after SMMC-7721 and Hep G2 cells were cultured with the vagus never neurotransmitters and the agonists and blockers of MR and NR.Results1. The vagus nerve fibers distribute in the hepatocellular carcinoma tumor tissue, and the density of distribution in tumor tissue is higher than that in tissues adjacent to cancer. Meanwhile, this phenomenon is greatly different between primary cases and recurrent cases.2. Both SMMC-7721 and HepG2 cells express neurokinin-1 receptor(NK-1R) and cholinergic receptors including muscarinic receptor and nicotinic receptor. Substance P, acetylcholine, bethanechol chloride and nicotine could promote the ability of migration and invasion of both SMMC-7721 and HepG2 cells, in which this effect of bethanechol chloride and nicotine could be inhibited by atropine and mecamylamine respectively.3. Substance P, acetylcholine, bethanechol chloride, nicotine, atropine and mecamylamine can not influence the proliferation of both SMMC-7721 and Hep G2 cells.4. Substance P,acetylcholine, bethanechol chloride and nicotine could upregulate the expression of N-Cadherin and Slug, meanwhile downregulate the expression of E-Cadherin in both SMMC-7721 and HepG2 cells, in which this effect of bethanechol chloride and nicotine could be inhibited by atropine and mecamylamine respectively.Conclusion1. There are vagus nerve fibers distributed in the hepatocellular carcinoma tumor tissue of human’s. The vagus nerve’s density of distributionis distinctly higher in tumer tissue of recurrent cases than that in primary cases, which indicates that the regulation of vagus nerve may promote the recurrence and metastasis of hepatocellular carcinoma.2. Neurokinin-1 receptor and several kinds of cholinergic receptors are expressed in human’s hepatocellular carcinoma cell lines. The vagus nerve release substance P and acetylcholine, which could combine with neurokinin-1 receptor and cholinergic receptors(muscarinic receptor and nicotinic receptor) respectively, to enhance the ability of migration and invasion of HCC cell lines through promoting their epithelial-mesenchymal transition. |
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