| Background:Hepatocellular carcinoma,the main type of liver cancer,accounts for about90% of liver cancer cases and is the fourth leading cause of cancer-related death.According to the latest statistics,the 5-year survival rate of hepatocellular carcinoma is only 18%.In the past,the main causes of hepatocellular carcinoma were HBV and HCV,but in recent years,with the improvement of living standards,non-alcoholic steatohepatitis,metabolic syndrome and diabetes-related are becoming the fastest growing causes of liver cancer,making its incidence increase worldwide.Therapeutic strategies against hepatocellular carcinoma are mainly liver resection and liver transplantation,but they are limited to patients with early-stage hepatocellular carcinoma;meanwhile the rate of tumor recurrence and metastasis is high after surgery,and almost half of liver cancer patients eventually receive systemic treatment.Even with systemic therapy,the overall median survival was only 1 year.WNT7B,an important member of WNT families,actives complex protein interaction networks and plays essential roles in development of nerve system and cancer,though both paracrine and autocrine pathways.However,the function of WNT7B in hepatocellular carcinoma remains obscure.The liver is innervated by peripheral nerves,while sympathetic and parasympathetic nerve fibers are mainly present in the liver,and sensory nerves appear in the hepatic capsule area.And in recent years,many literature reports have reported that external stress and anxiety will lead to the activation of sympathetic nerves,and meanwhile promote the occurrence and development of cancer.Malignant cells and their microenvironment release neurotrophic factors to recruit the nerves needed for their growth,and in turn nerves are involved in cancer cell growth and expansion.Herein,the nerves distributed in liver might be implicated in development of hepatocellular carcinoma when they are activated by external stress.Objective:This study compared the blocking effect on the development of hepatocellular carcinoma after inhibiting sympathetic,parasympathetic and sensory nerves respectively in chemically induced hepatocellular carcinoma mouse,aiming at uncovering the outstanding role of sympathetic nerves.Furthermore,chronic unpredictable stress was utilized to active sympathetic nerves in vivo,thus verifying the promoting role of sympathetic nerves in the development of hepatocellular carcinoma.The content of sympathetic neurotransmitter in serum of chemically induced hepatocellular carcinoma mouse was analyzed to investigate the mechanism underlying sympathetic nerves in hepatocellular carcinoma.In a word,this study aimed at exploring the role and mechanism of sympathetic nerves in the development of hepatocellular carcinoma preliminarily.Method:Firstly,we used clinical tissue samples to evaluate the expression of WNT7B and neural distribution in liver cancer tissues,and analyzed their correlation.The transcription level of WNT7B in liver cancer patients as well as its correlation with the patient’s disease-free survival was investigated in the TCGA database,and the expression levels of WNT7B in the hepatoma cell lines were analyzed in the CCLE database.Subsequently,DEN was used to coinduce hepatocellular carcinoma mouse models with CCl4,and three different drugs,capsaicin,6OHDA and Atropine,were used to inhibit sensory,sympathetic and parasympathetic nerve respectively to evaluate their role in development of hepatocellular carcinoma and relevance with WNT7B expression.Finally,we established a chronic unpredictable stress model to verify the role of sympathetic nerve activation in the progression of hepatocellular carcinoma.In addition,to further explore the mechanism underlying WNT7B-mediated interaction between hepatocellular carcinoma cells and sympathetic nerves,we constructed WNT7B-overexpressing and WNT7B-knocked down plasmids,used Crisp/Cas9 technology to construct knockout plasmids,packaged lentiviruses and finally constructed a series of stable cell lines.Result:In this study,we found that WNT7B expression was up-regulated in liver cancer tissues,and associated with poor disease-free survival of liver cancer patients;meanwhile WNT7B expression was positively correlated with nerve abundance.Furthermore,we inhibited sensory,sympathetic and parasympathetic nerve in chemically induced hepatocellular carcinoma mouse and revealed that sympathetic nerves may promote the progression of liver cancer,and inhibition of sympathetic nerves down-regulated WNT7B expression.Sympathetic nerve activation using chronic unpredictable stress model promoted the development of hepatocellular carcinoma,while the promoting effect could be blocked by 6OHDA,further verifying the positive role of sympathetic nerves in hepatocellular carcinoma progression.Analysis of sympathetic neurotransmitters by ELISA showed that sympathetic neurotransmitters were up-regulated in serum of mouse with chemically induced hepatocellular carcinoma and the up-regulation could be inhibited by6 OHDA,suggesting sympathetic nerves might release sympathetic neurotransmitters to promote hepatocellular carcinoma.In addition,we have successfully constructed a series of stable cell lines expressing different levels of WNT7B for further research.Conclusion:This study revealed the positive correlation between WNT7B expression and innervation in hepatocellular carcinoma for the first time.Furthermore,among three types of peripheral nerves,sympathetic nerves had close relevance with WNT7B and promoted the development of hepatocellular carcinoma though releasing sympathetic neurotransmitters.Therefore,our study suggested that inhibiting sympathetic nerves might be an effective therapeutic strategy against hepatocellular carcinoma. |
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