Autonomic Nerve Contributes To Perineural Invasion Of Salivary Adenoid Cystic Carcinoma And The Underlying Mechanisms

Salivary adenoid cystic carcinoma(SACC)is a common adenocarcinoma that most often originates from the salivary glands and accounts for ~22% of all salivary gland malignancies.A majority of tumor in the sublingual gland are diagnosed as SACC.Perineural invasion(PNI)is the most prominent biological characteristic of SACC and is thought to be associated with local recurrence and poor prognosis of SACC.However,there is no effective treatment for PNI due to insufficient understanding of the pathogenesis of PNI in SACC.The autonomic nervous system plays an important role in maintaining the normal physiological functions of the human body.Recent studies suggest that autonomic nerves and their neurotransmitters may promote the development of malignant tumors such as prostate cancer,lung cancer and breast cancer.At present,there is no other report on the distribution of autonomic nerves in SACC and its relationship with PNI,except for the preliminary study of SACC sympathetic distribution in my master's thesis.In view of the abundant autonomic nerve distribution in salivary gland,this paper intends to analyze the distribution of autonomic nerves,neurotransmitters and corresponding receptors in SACC tissues and analyze their correlations with PNI.In addition,we intend to observe the influence of autonomic neurotransmitters on PNI of SACC cells through in vitro and in vivo experiments.This study helps to elucidate the pathogenesis of SACC PNI and provides an experimental basis for the treatment of PNI.Autonomic nerves consists of sympathetic nerve and parasympathetic nerve.Therefore,the research content of this thesis includes two parts: the role of sympathetic nerve and parasympathetic nerve in promoting SACC PNI and their downstream mechanisms,respectively.Part ?: Sympathetic nerve promoted PNI of SACC and the underlying mechanisms.Purpose:1)To study the distribution of sympathetic nerve,?2-adrenergic receptor(?2-AR)and sympathetic neurotransmitter norepinephrine(NE)in SACC tissues and their correlations with PNI.To study the expression of NE in the supernatant of the dorsal root ganglia(DRG)PNI coculture models.2)To study the expression of ?2-AR in SACC-83 and SACC-LM cell lines.To clarify the effects of NE on the PNI of SACC-83 and SACC-LM cell lines.To study the influence of blocking ?2-AR or ?-AR on PNI of SACC-83 and SACC-LM cell lines.3)To study the possible mechanisms of NE/?2-AR in promoting PNI of SACC-83 and SACC-LM cell lines.Methods:1)Immunohistochemical staining(IHC)was used to observe the distribution of sympathetic nerve and ?2-AR in clinical SACC tissues and normal salivary gland(NSG)tissues,we further analyzed the correlation of their distribution with clinicopathological characteristics.NE concentrations in SACC tissues,NSG tissues and rat DRG PNI coculture models were detected by enzyme-linked immunosorbent assay(ELISA)method.2)Quantitative real-time polymerase chain reaction(qRT-PCR)and immunofluorescence staining methods were used to detect ?2-AR expression in SACC-83 and SACC-LM cell lines.Wound-healing assay and DRG PNI models were used to study the effect of NE on migration and PNI of SACC-83 and SACC-LM cell lines.In addition,we studied the effect of down-regulation of ?2-AR or ?-AR expression on PNI of SACC-83 and SACC-LM cell lines through blocking ?2-AR by specific antagonist ICI118,551 and transfection with ?2-AR siRNA or blocking ?-AR through non-selective ?-AR blocker phentolamine.3)We performed Western blot methods to explore the possible mechanisms by which NE/?2-AR promoted PNI in SACC-83 and SACC-LM cell lines.Results:1)Positive sympathetic nerve areas in PNI SACC tissues were significantly larger than in the non-PNI SACC tissues(P<0.05)and NSG tissues(P<0.01).Chi-square test analysis suggested that sympathetic innervation correlated with PNI(P=0.035)and distant metastasis(P=0.010).The positive correlation between sympathetic innervation and PNI was further confirmed by logistic regression univariate analysis(P=0.002)and multivariate analysis(P=0.042).?2-AR was highly expressed in PNI SACC tissues compared to non-PNI SACC tissues(P>0.05)and NSG tissues(P<0.05).Chi-square test results indicated that ?2-AR expression positively correlated with PNI(P=0.003)and TNM stage(P=0.035).Moreover,the positive association between ?2-AR overexpression and PNI was further confirmed by logistic regression univariate analysis(P=0.004)and multivariate analysis(P=0.076).ELISA results revealed that NE concentrations in PNI SACC tissues were markedly higher in comparison to SACC tissues without PNI(P<0.01)and NSG tissues(P<0.001).In addition,the concentrations of NE in the supernatant of DRG PNI co-culture models were higher than the supernatant of SACC cell lines or DRG alone.2)The expression level of ?2-AR was the highest among ? and ? adrenergic receptors.NE can promote the migration and PNI of SACC-83 and SACC-LM cell lines.Non-selective ?-AR blocker phentolamine failed to abrogate migration and PNI of SACC-83 and SACC-LM cell lines.In contrast,down-regulation of ?2-AR expression by specific antagonist ICI118,551 or siRNA transfection markedly inhibited NE-induced migration and PNI.3)qRT-PCR and Western blot methods demonstrated that NE/?2-AR may contribute to PNI of the SACC-83 and SACC-LM cell lines by inducing EMT and up-regulating of MMP expression.Conclusion:1)Sympathetic nerve,NE and ?2-AR were widely distributed in SACC tissues and their distributions were associated with PNI status.2)SACC-83 and SACC-LM cell lines are capable of secreting NE and expressing ?2-AR.3)NE promoted PNI of SACC-83 and SACC-LM cell lines,while blocking ?2-AR can inhibit the NE-induced PNI.4)NE/?2-AR signaling may promote PNI of SACC-83 and SACC-LM cell lines by inducing EMT and up-regulating MMP expression.Part ?: Parasympathetic nerve promoted PNI of SACC and the underlying mechanisms.Purpose:1)To study the distribution of parasympathetic nerve,M3 muscarinic receptor(M3R)and parasympathetic neurotransmitter acetycholine(ACh)in SACC tissues and their correlations with PNI.To study ACh concentration in the supernatant of the DRG PNI coculture models.2)To study the expression of M3 R in SACC-83 and SACC-LM cell lines.To clarify the effects of ACh on PNI of SACC-83 and SACC-LM cell lines.To study the influence of blocking M3 R on PNI of SACC-83 and SACC-LM cell lines.3)To study the effect of blocking M3 R on PNI of SACC-LM cell line in nude mice.4)To study the possible mechanisms of ACh/M3 R in promoting PNI of SACC-83 and SACC-LM cell lines.Methods:1)IHC was used to observe the distribution of parasympathetic nerve and M3 R in clinical SACC tissues and NSG tissues,we further analyzed the correlations of their distribution with clinicopathological characteristics.ACh concentrations in SACC tissues,NSG tissues and DRG PNI models were detected by ELISA method.2)qRT-PCR and immunofluorescence staining were used to detect expression M3 R and choline acetyltransferase(CHAT)in SACC-83 and SACC-LM cell lines.MTT proliferation assay,wound-healing assay,Transwell migration and invasion assay,Transwell PNI model and DRG PNI model were used to study the effects of ACh on proliferation,migration,invasion and PNI of SACC-83 and SACC-LM cell lines,respectively.In addition,the effects of down-regulation of M3 R expression on PNI of SACC-83 and SACC-LM cell lines were detected through blocking M3 R by either specific antagonist darifenacin(DAR)or transfection with M3 R siRNA.3)The effects of DAR on proliferation,lung metastasis and PNI of SACC-LM cell line in nude mice were performed by intraperitoneal injection of DAR.4)Western blot analysis was performed to detect the expression of p-ERK,p-GSK3?,EMT and MMP in SACC-83 and SACC-LM cell lines after incubating with ACh,DAR or transfecting with M3 R siRNA,aimed to explore the possible mechanisms by which ACh/M3 R promoted PNI in SACC-83 and SACC-LM cell lines.Results:1)Positive parasympathetic nerve areas in PNI SACC tissues were larger than in the non-PNI SACC tissues(P>0.05)and NSG tissues(P<0.001).Chi-square test analysis suggested that parasympathetic innervation positively correlated with PNI(P=0.011),tumor site(P=0.024)and recurrence(P=0.018).The correlation between parasympathetic innervation and PNI was further confirmed by logistic regression univariate analysis(P=0.015).M3 R was highly expressed in PNI SACC tissues compared to non-PNI SACC tissues(P>0.05)and NSG tissues(P<0.001).Chi-square test results indicated that M3 R expression positively correlated with PNI(P=0.019).Moreover,the positive association between M3 R overexpression and PNI was further confirmed by logistic regression univariate analysis(P=0.022).ACh concentration in PNI SACC tissues was higher than that in non-PNI SACC tissues(P>0.05)and NSG tissues(P<0.001).In addition,the concentrations of ACh in the supernatant of DRG co-culture PNI models were higher than the supernatant of SACC cell lines or DRG alone.2)The expression level of M3 R was the highest among muscarinic receptors.ACh promoted proliferation,migration,invasion and PNI of SACC-83 and SACC-LM cell lines.In contrast,down-regulation of M3 R expression by specific antagonist DAR or siRNA transfection markedly inhibited ACh-induced proliferation,migration,invasion and PNI of SACC cell lines;3)In vivo experiments showed that the weight of xenografts in DAR group were lighter than that in nude mice(P<0.05).The lung metastasis rate of SACC-LM cells in nude mice of DAR group was lower than that in control group(P=0.248).Compared with the control group,the nude mice of DAR group had shorter time of becoming left hind limb dysfunction,lower PNI rate(P=0.039),higher sciatic nerve score(P<0.001)and sciatic nerve index(P>0.05).4)ACh/M3 R may induce EMT and MMP expression in SACC-83 and SACC-LM cell lines by activating p-ERK/p-GSK3? signaling pathway,and then promote PNI of SACC cell lines.Conclusion:1)Paraympathetic nerve,ACh and M3 R were widely distributed in SACC tissues and their distributions were associated with PNI status.2)SACC-83 and SACC-LM cell lines express M3 R and CHAT and are capable of secreting ACh.3)ACh promoted PNI of SACC-83 and SACC-LM cell lines through M3 R,while blocking M3 R inhibited the ACh-induced PNI.4)Blocking M3 R inhibited proliferation,lung metastasis and PNI of SACC-LM cell line in nude mice.5)ACh/M3 R may induce EMT and MMP expression in SACC-83 and SACC-LM cell lines by activating p-ERK/p-GSK3? signaling pathway,and then promote PNI of SACC cell lines.In summary,this thesis further explores the distribution of autonomic nerves,neurotransmitters and their corresponding receptors in SACC tissues and their correlations with PNI on the basis of our previous study.Our results suggest that autonomic nerve may promote PNI of SACC,while blocking the corresponding receptors of autonomic neurotransmitters may inhibit PNI.The present study helps to elucidate the mechanisms of autonomic nerves influences on PNI and provides a possible experimental basis and molecular targets for treatment of SACC PNI.The common clinical symptoms of SACC PNI include pain,tongue numbness and facial paralysis,which suggests that SACC may invade sensory nerves and motor nerves.The release of substance P due to sensory nerve trauma is related to the transmission of pain sensation.The motor nerve regulate muscles by releasing the neurotransmitter ACh.In view of the fact that the neurotransmitters of motor nerve and parasympathetic nerve are the same,which indicates that they may exert similar effects on tumor biological behaviors.We are performing experiments to study the effects of sensory and motor nerves on PNI of SACC and we aim to improve our understanding of the function of nerves during SACC PNI.