Promotion Effect And Mechanisms Of Cholingernic Receptor M1 On Invasion And Migration Of Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma(HCC)is the second cause of cancer deaths in our country.At present,surgical resection is the main treatment for HCC.However postoperative recurrence rate is still as high as 80% within five years.Invasion and metastasis are the main factors influencing the recurrence of HCC,but its mechanism is complex and still not fully elucidated.In recent years,more and more research evidences suggest that nervous system plays an important role in the development of cancer.As we know,liver is innervated by sympathetic and parasympathetic nerves.The vagus nerve releases neurotransmitters such as acetylcholine and other neurotransmitters by specifically binding the corresponding receptors to activate them,thereby acting on various biological effects.Our previous study has proved that the regulation of vagus nerve can promote the invasion and migration of liver cancer and a variety of cholinergic receptors are expressed on liver cancer cells.In addition,it has been reported that muscarinic cholinergic receptor M1(ChRM1)acts a vital role in the development of lung cancer and prostate cancer.Therefore,the study of the role of cholinergic receptor M1 in HCC will provide a new theoretical basis for prevention and treatment of HCC recurrence and metastasis.ObjectiveInvestigate the expression of muscarinic cholinergic receptor M1 in hepatocellular carcinoma and the effect of this receptor on the invasion and migration of HCC.And explore the mechanism of M1 receptor activation to regulate invasion and migration of HCC.Methods1.Sixty-three HCC specimens were collected and the expression of ChRM1 was detected by immunohistochemistry.The relationship between the expression of ChRM1 and clinicopathological features of HCC was analyzed.2.Hep G2 and SMMC-7721 liver cells were cultured in vitro and Western blot was used to detect the expression of ChRM1 in hepatocarcinoma cells.The two kinds of cells were treated with ChRM1 agonists and antagonist respectively.Then the effects of ChRM1 agonists and antagonists on the migration and invasion of hepatoma cells were detected by Transwell invasion and migration assay.In addition,the expression of ChRM1 in hepatocarcinoma cells was silenced by interfering plasmids and the specific agonists were used to treat hepatoma cells.Transwell assay was used to test the migration and invasion capability of hepatoma cells.3.After interfering plasmid treatment of cancer cells HepG2,flow cytometry sorting was employed to sort out relatively stable expression of ChRM1 silenced cells(sh ChRM1).The normal HepG2 cells and the shChRM1 cells were injected into the liver of nude mice in situ respectively.The mice of each treatment group were divided into drinking water containing bethanechol chloride(BeCh)group and non-Be Ch group(Control group: HepG2 cells and normal water;BeCh group: HepG2 cells and water containing Be Ch(400ug/ml);BeCh+M1 shRNA group: M1 shRNA HepG2 cells and water containing Be Ch(400ug/ml);M1 shRNA group: M1 shRNA HepG2 cells and normal water).Then the intrahepatic tumorigenesis and metastasis of mice in each group were observed by small animal magnetic resonance imaging at 30 days.4.The two kinds of cells were treated with ChRM1 agonists and antagonists respectively and interfering plasmids to inhibit ChRM1 expression.Then the expression of epithelial-mesenchymal transition(EMT)markers in the two hepatocellular carcinoma cells was detected by Western Blot assay.5.The two kinds of hepatoma cells were treated with agonists and blockers,agonists were used to treat shChRM1 cells and total protein was extracted.Western Blot assay was used to detect the changes of related signal pathways after ChRM1 blockade.Results1.ChRM1 was highly expressed in HCC,and it was positively correlated with the pathological features(recurrence,vascular invasion,HCC differentiation and TNM stage)of HCC patients.Patients with high expression of ChRM1 had short survival time.2.The expression of ChRM1 was confirmed in Hep G2 and SMMC-7721 cells.ChRM1 activation with agonist promoted invasion and migration of hepatocellular carcinoma cells.Blocker and interfering plasmids pretreatment can inhibit this promoting effect.3.Animal in situ implantation tumor model showed that mice of Be Ch group had the most intrahepatic metastatic lesions and had the shortest survival time than the other three groups.While the mice of Be Ch+M1 shRNA group and M1 shRNA group had the least number of intrahepatic metastatic lesions and had a long survival time.4.In both hepatoma cells,the activation of ChRM1 could induce up-regulation of N-cadherin and down-regulation of E-cadherin.Blocker pretreatment and gene silencing ChRM1 expression can inhibit the above promotion effect of EMT process.5.The activation of ChRM1 increased the phosphorylation of Akt and expression of Slug,while did not affect p38 and ERK phosphorylation significantly.Inactivation of the receptor with antagonist and interfering plasmid inhibited the phosphorylation of Akt and expression of Slug.Conclusion1.The high expression of ChRM1 indicates high malignancy of HCC and poor clinical prognosis.2.The activation of ChRM1 can promote the epithelial-mesenchymal transition of hepatocarcinoma cells through PI3K/Akt signaling pathway and futher promote invasion and metastasis of HCC.