Study On Mechanism Of Pannexin-1 Promotes The Invasion Of Pituitary Adenomas

Background:The incidence of pituitary adenoma(PA)has been increasing in recent years,and it is reported that thay maybe second one only below glioma.Although the histological features of pituitary adenomas are benign,in clinical work,about 25-55% of pituitary adenomas grow aggressively to surrounding structures,have a high recurrence rate,resemble malignant tumors,and are difficult to totally remove by surgery,they are defined as invasive pituitary adenomas(invasive PA)[1].Although some pharmacological therapies are available for the treatment of certain types of PA,the sensitivity of these drugs varies from patient to patient,and these drugs are more likely to control tumor-secreting hormones,so that it is still difficult to eradicate all kinds of PA.Therefore,controlling the aggressiveness of PA is currently a challenge in clinical treatment,and elucidating its pathogenesis will help develop new clinical treatments.Pannexin-1(Panx1)is a channel located in the cell membrane with unique conductance,ranging from non-selective ion permeability to extracellular release of signaling molecules,which plays a vital role in impacting the biological behavior of cells.According to reports,Panx1 plays an important role in pathophysiological processes of diseases such as tumorigenesis,epilepsy,ischemic and hypoxic injury [2].Interestingly,it has been found that the role of Panx1 varies in different types of tumors.For example,Panx1 plays an anti-tumor role in gliomas [3],while in cutaneous melanomas it plays a role in promoting tumorigenesis[4].Such a phenomenon may be due to the tumor origin-specific and tumor microenvironment.Panx1 exerts its physiological functions mainly through the formation of large aperture channels in the cell membrane rather than intercellular gap junction function.Opening of Panx1 channel can lead to the exchange of metabolic substances between cells and the extracellular microenvironment [5],and these substances may influence the invasive biological behavior of cells.ATP and amino acid release properties affect the biological behavior of tumor cells has been demonstrated in many reports [2-4].Moreover,in CNS,Panx1 is expressed in both neurons and glial cells and is thought to mediate intercellular connections and interactions.However,in pituitary adenomas,it is unknown whether Panx1 is associated with the development of its aggressiveness.Therefore,this study aimed to explore the expression of Panx1 in invasive and non-invasive PA and the mechanism affecting the development of pituitary adenoma aggressiveness,and to provide a new theoretical basis for the clinical treatment of invasive PA.Objective:1.Six GH-PA samples(3 cases each of invasive and non-invasive)were collected and transcriptomic sequencing was performed.2.56 PA samples(28 cases each of invasive and non-invasive)were collected,and the expression at the Panx1 m RNA level and Panx1 protein level were detected by RT-q PCR and immunohistochemistry,respectively.3.In vitro,rat pituitary adenoma cell lines GH3 and MMQ cells were used to explore the effect of Panx1 on the proliferation and invasive ability of PA cells and molecular mechanism.Results:1.Several genes were expressed differently in invasive and noninvasive GH-PA.We collected 6 samples of GH-PA and divided them into invasive and non-invasive groups according to Knosp classification.The m RNA and lnc RNA expressions of the two groups were detected by transcriptome sequencing.The results showed that several m RNAs and lnc RNAs expressed differently in invasive and non-invasive GH adenomas(invasive GH-PA and non-invasive GH-PA),which may be potential biomarkers or drug targets for the regulation of pituitary adenoma invasion.Secondly,according to transcriptomics results and database analysis,among all the differential genes,we screened the cell membrane channel PANX1 to be closely related to the aggressiveness of pituitary adenoma.2.Pannexin-1 expression was upregulated in invasive PA samples compared with noninvasive PA.In order to further verify the relationship between Panx1 and pituitary adenoma invasion,we expanded the sample number and types of pituitary adenomas,collected 56 pituitary adenoma samples,and divided them into two groups,invasive group and non-invasive group according to Knosp grade(Knosp grade over 3 is invasive PA).RT-q PCR was used to detect the expression of Panx1 in the two groups,and the results showed that the expression of Panx1 in the invasive group was significantly up-regulated compared with that in the non-invasive group.Similarly,immunohistochemical(IHC)results showed that Panx1 expression was upregulated in the invasion group.Then,after overexpression of Pannexin-1in GH3 cells,we found that Panx1 overexpression(Panx1-OE)significantly promoted the invasion of GH3 cells compared to the control group(Vector).3.Pannexin-1 overexpression promoted the invasion of GH3 cells lines by increasing ATP releasing.By detecting the extracellular ATP concentration,we found that overexpression of Panx1 would promote the release of ATP.However,after incubation with Panx1 blocker(profenecid,PBN),the extracellular ATP decreased significantly.Secondly,after adding ATPase to degrade extracellular ATP in GH3 cells of Panx1-OE group,the invasive ability was greatly weakened.Similarly,after adding ATP analogue(Bz ATP)to GH3 cells in Vector group,the invasive ability of GH3 cells was enhanced again(P < 0.05).These results suggest that Panx1 promotes the invasion of GH3 cells by increasing the release of ATP.4.Pannexin-1-mediated ATP release accelerated calcium influx by activating P2X7 receptors.When we treated GH3 cells in Panx1-OE group with non-specific P2 X receptor antagonists(PPADs)and specific P2X4 and P2X7 receptor antagonists(5-BDBD and JNJ-47965567),they all significantly inhibited the invasion of GH3 cells(P < 0.05),especially JNJ-47965567,suggesting that P2X7 plays a more important role in promoting GH3 cell invasion.In addition,through calcium imaging experiments,we found that P2X7 receptor activation increased calcium influx and significantly invaded GH3 cells,whereas P2 X receptor specific antagonist(JNJ-47965567)reversed this result.These results suggest that Panx1-mediated ATP release promotes calcium ion influx through activation of P2X7 receptors.5.Increased intracellular calcium recognized actin cytoskeleton.The state of intracellular cytoskeleton proteins was observed by adding P2X7 receptor agonist and antagonist respectively,and then the change of GH3 cell invasion was detected after interfering the actin cytoskeleton using cytochalasin B.We found that the increase of intracellular calcium ions can directly act on cytoskeleton actin or indirectly act on it through calmodulin kinase,leading to changes in its morphology and activity,and further promoting the invasion of GH3 cells.Conclusions:1.Pannexin-1 upregulated in invasive PA and promoted the invasion of GH3 cells.2.The release of ATP mediated by pannexin-1 can activated P2X7 receptor and recognized actin cytoskeleton and promote the invasion of PA cells.