The Pseudogene PTENP1 Regulates Smoothmuscle Cells As Acompeting Endogenous RNA

Background and ObjectionLncRNA participates in the transcription,post-transcription,and epigenetic regulation,and then affects cell proliferation,apoptosis functions,participates in the occurrence and development of various diseases ultimately.LncRNA PTENP1 is a pseudogene of the tumor suppressor gene PTEN.PTEN has been shown to be involved in SMC proliferation,apoptosis,and aortic aneurysm(AA)expansion.PTENP1 can be used as a competitive endogenous RNA(ceRNA)to target miR-21,release miR-21's inhibition of PTEN,up-regulate PTEN expression,and then affect the downstream pathway of PTEN.However,it is unclear whether and how PTENP1 affects the proliferation and apoptosis of human aortic smooth muscle cells(HASMCs),thereby promoting the development of AA or aortic dissection(AD).In this study,we tried to demonstrate that PTENP1 as a ceRNA regulates PTEN expression,affects the proliferation and apoptotic functions of HASMCs,and clarified that PTENP1 is a potential target for AA or AD intervention.Methods1.Detection of PTENP1 and PTEN in human normal arterial tissues and ADtissues for differential expressionPathological analysis was performed on human AD and normal arterial tissues,and the expression of PTENP1 and PTEN in tissues was detected by RT-PCR,WB,immunohistochemistry,and in situ hybridization.2.Explore the effect of PTENP1 on the proliferation and apoptotic function of HASMCs(1)After knocking down PTENP1,the cell proliferation level was detected by immunoconfocal experiments.(2)Construction of PTENP1 overexpression adenovirus vector,and transfection of HASMCs to detect apoptosis by RT-PCR,WB,TUNEL,Annexin V-FITC/PI double staining3.Explore the impact of PTENP1 on the AAA modelThe PTENP1 lentiviral vector was constructed,transfected into C57BL/6J mice,implanted with Ang ? pump,and induced the AAA model.After the model was established,the AA diameter and aneurysm formation rate of the mice were measured,and the vascular wall were analyzed pathologically.4.Exploring the mechanism of PTENP1 regulating HASMCs function(1)By RNA pull-down,luciferase,and immunofluorescence experiments,explore how PTENP1 targets miR-21 through the ceRNA mechanism and regulates PTEN expression.(2)Overexpressedor knocked down PTENP1 respectively,to explore how PTENP1 affects the PI3K/Akt signaling pathway and cell cycle by regulating PTEN expression.Result1.PTENP1,PTEN expression is up-regulated in human AD tissuesPCR and WB experiments showed that both PTENP1 and PTEN were up-regulated in human AD tissues,and in situ hybridization showed that PTENP1 was mainly expressed in smooth muscles in the middle layer of the aortic wall.The expression levels of SMC biomarkers such as a-SMA,MYH11 and SM22 in human AD tissues were significantly lower than those in normal aortic tissues.2.PTENP1 regulates the proliferation and apoptosis of HASMCsTUNEL and flow cytometry tests showed that compared with the control group,overexpression of PTENP1 significantly increased the expression of the apoptotic factor cleaved-casp3 and promoted apoptosis.The knockdown of PTENP1 attenuated H2O2-induced HASMCs apoptosis.Immunofluorescence experiments showed that compared with the control group,knocking down PTENP1 significantly increased the expression of proliferation-related factors phospho-H3 and Ki-67,and promoted cell proliferation.3.Overexpression of PTENP1 promotes development of Ang ?-induced AAA modelWe successfully overexpressed the PTENP1 virus in an Ang?-induced C57BL/6J mouse AAA model.Compared with the empty virus injection group,the maximum diameter of abdominal aorta and the incidence of AAA in the over-expressing PTENP1 virus group were significantly increased.Further pathological analysis results showed that overexpression of PTENP1 promoted SMC apoptosis and degradation of elastic fibers in the aortic wall of mice,and exacerbated the formation of AAA induced by Ang?.4.PTENP1 targets miR-21 by ceRNA mechanism,up-regulates PTEN expression,and then regulates the function of HASMCsIn order to further analyze the mechanism,we performed RNA pull-down experiments and performed a series of luciferase experiments with miR-21 mimics or inhibitors.It was proved that PTENP1 competitively binds miR-21 with PTEN transcripts through the ceRNA mechanism,directly "Sponge" adsorbs miR-21,reduces its inhibition of PTEN,and up-regulates PTEN expression.Immunofluorescence experiments further supported this finding:miR-21 mimics were used on the basis of over-expressing PTENP1,PTEN expression was down-regulated,and apoptosis was reduced.The results of PTEN rescue experiments showed that knocking down PTEN significantly reduced apoptosis induced by overexpression of PTENP1,proving that PTEN is an essential link in the PTENP1 regulatory pathway.Finally,WB showed that overexpression of PTENP1 in HASMCs significantly up-regulated PTEN expression and reduced Akt phosphorylation and levels of cyclinD1 and cyclinE;knocking down PTENP1 was the opposite.ConclusionThe pseudogene PTENP1 targets miR-21 through the ceRNA mechanism to up-regulate PTEN expression,thereby inhibiting its downstream PI3K/Akt pathway conduction,inhibiting the expression of cyclinDl and cyclinE,arresting the cell cycle,and ultimately regulating the apoptosis and proliferation of HASMCs and promoting formation and development of AA or AD.