The Function And Mechanism Of PHACTR1 In The Inhibition Of Lung Adenocarcinoma

Objective:We used expression data,clinical data,immune infiltration data,and methylation data from public databases to clarify the basic expression,prognostic value,functional enrichment network,immune infiltration characteristics,and DNA methylation modification changes of the PHACTR(Phosphatase and Actin Regulator,PHACTR)family in lung adenocarcinoma(Lung adenocarcinoma,LUAD).Methods:1.Use the public database websites UALCAN and UCSC XENA to clarify the expression level of the PHACTR family in LUAD.2.Use Kaplan-Meier plotter and GEPIA database to evaluate the prognostic impact of PHACTR family on LUAD patients,and perform ROC prognostic value evaluation and analysis on positive results.3.Metascape functional enrichment analysis was performed on the co-expressed genes of the PHACTR family to determine the co-acting pathway.Furthermore,we further refined the GSEA pathway analysis of the differential genes of each member of the PHACTR family to clarify the functional characteristics of individual members.4.Using CancerSEA single cell sequencing data to identify the most relevant tumor single cell status in the PHACTR family.5.The TIMER database was used to clarify the characteristics of immune cell infiltration associated with the PHACTR family.We calculated the corresponding immune infiltration score of the PHACTR family in LUAD patients based on the Estimate algorithm.6.Use GeneMANIA to explore the interaction networks of molecules related to the PHACTR family.7.MethSurv and UALCAN were used to analyze the DNA methylation modification characteristics of the PHACTR family in LUAD.Results:1.The expression levels of PHACTR1/2/3 in LUAD tissue were lower than that in normal tissue,while the expression level of PHACTR4 in LUAD tissue was higher than that in normal tissue.And the low expression group of PHACTR1 was directly related to the poor prognosis of LUAD patients.2.The PHACTR family affected the genetic stability of the LUAD genome and the process of immune signal transmission through bidirectional regulation of cell cycle,DNA damage,cell apoptosis,and epigenetics related to mRNA,DNA,and histone methylation.3.PHACTR1 was mainly negatively correlated with EMT,invasion,metastasis,and angiogenesis;PHACTR2 was mainly positively correlated with metastasis,angiogenesis,stem cell sex,EMT,differentiation,and inflammatory response;PHACTR4 was negatively correlated with metastasis,angiogenesis,EMT,differentiation,quiescence,and stem cells.However,there was no significant correlation between PHACTR3 and the important single cell functional state in LUAD.4.In LUAD samples,the expression levels of PHACTR1/2 were strongly positively correlated with the abundance of infiltration of B cells,CD8+T cells,CD4+T cells,macrophages,neutrophils,and dendritic cells;PHACTR4 was positively correlated with the abundance of CD4+T cells,macrophages,neutrophils,and dendritic cells;PHATCR3 had no significant statistical relationship with the infiltration abundance of six immune cells.Based on the Estimate score,PHACTR1/2 were positively correlated with anti-tumor immunity in LUAD patients,while PHACTR4 was negatively correlated with anti-tumor immunity.5.The DNA methylation levels of PHACTR1/2/3/4 in LUAD tissue were higher than that in normal control group,and the hypermethylation of the cg15542608 site of PHACTR1 was related to the good prognosis of LUAD patients;The hypermethylation of the cg06759993 site in PHACTR2 was associated with poor prognosis in LUAD patients;The hypermethylation of the cg20357628 site in PHACTR3 was associated with a good prognosis in LUAD patients;The hypermethylation of the cg00956142 site in PHACTR4 was associated with a good prognosis in LUAD patients.Conclusions:1.PHACTR1/2/3/4 had differential expression in LUAD,and PHACTR1 can be an important prognostic biomarker for LUAD.2.PHACTR1 inhibited EMT,invasion,metastasis,and angiogenesis of LUAD cells,positively regulated anti-tumor immune infiltration,and played an anti-tumor role in LUAD.The functional state of LUAD cells regulated by PHACTR2/4 was opposite to the direction of tumor immune infiltration,and played a dual role in promoting and suppressing cancer in the development of LUAD.3.The hypermethylation of PHACTR1 was associated with a good prognosis in LUAD patients.Objective:In the Part Ⅰ,PHACTR1 was determined to have strong diagnostic and prognostic value in public databases through bioinformatics methods,and can be used as an important prognostic biomarker for LUAD.PHACTR1 inhibited the malignant state of LUAD cells,positively regulated anti-tumor immune infiltration,and played an inhibitory role in lung adenocarcinoma.This section aims to further validate the role and pathway regulation mechanism of PHACTR1 in LUAD through in vivo,in vitro experiments,and clinical tissue samples.Methods:1.Using RT-qPCR(Real Time Quantitative)and Western Blot methods to detect the mRNA and protein expression levels of PHACTR1 in bronchial epithelial cells(BEAS-2B)and LUAD cells(A549,H1299,PC9).Collect postoperative cancer tissue and adjacent tissue samples from LUAD patients,and use immunohistochemical staining to detect the expression level of PHACTR1 in LUAD tissue and adjacent tissue.2.Transfer plasmids to overexpress PHACTR1 in A549 and PC9 cells,and detect cell proliferation ability using cell proliferation toxicity assay(CCK8)and cell proliferation imaging(EDU);The invasive ability of cell migration was detected by scratch test and Transwell test;Western blot detection of EMT related protein changes;γ-H2AX immunofluorescence assay was used to detect the degree of DNA damage;Cell cycle changes were detected by flow cytometry.3.Western Blot verifies the changes in key proteins in the main pathways regulated by PHACTR1(TGF-β pathway and ERK pathway).4.Using the stable Lentivirus strain overexpressing PHACTR1 to construct the subcutaneous tumor model of nude mice,and compare the growth rate and quality of the subcutaneous tumor between the control group and the overexpressing PHACTR1 group.Immunohistochemistry(IHC)staining was performed on subcutaneous tumor sections to clarify the expression location and relative expression level of PHACTR1.Differences in proliferation indicators(Ki-67),pathway critical proteins(P-ERK),and immune indicators(CD8,CD44)between the two groups were observed.5.The tail vein metastatic tumor model was constructed with Lentivirus stable strain overexpressing PHACTR1,and the difference of fluorescence imaging of pulmonary metastatic tumor between the control group and the high expression PHACTR1 group was compared.The metastatic tumor sections were stained with hematoxylin eosin(HE)to clarify the histological characteristics of the metastatic tumor.Results:1.The expression of PHACTR1 in normal bronchial epithelial cells(BEAS-2B)was significantly lower than that in LUAD cells(A549,H1299,PC9),with both A549 and PC9 cells having relatively lower expression levels.In clinical tissue samples,PHACTR1 was less expressed in lung adenocarcinoma tissues than in adjacent tissues.2.After overexpression of PHACTR1 in A549 and PC9 cells,the cell proliferation ability significantly decreased;The ability of cell migration and invasion was significantly reduced,the marker of inhibiting EMT(E-cadherin)was increased,and the marker of promoting EMT(N-cadherin,Vimentin,Snail)was reduced;The degree of DNA damage in cells was amplified;Cell cycle was blocked in G1 phase.3.When PHACTR1 was overexpressed in A549 and PC9 cells,there was no significant change in the key proteins of the TGF-β pathway(TGF-β,TGF-βR),while the key proteins of the ERK pathway(total ERK1/2 remained unchanged,P-ERK1/2 was downregulated).4.The subcutaneous tumor formation model of nude mice showed that compared with the control group,the tumor growth rate of the overexpression PHACTR1 nude mouse group was slower and the tumor size was smaller at the same time.Compared with the control group,the overexpression of PHACTR1 in subcutaneous tumor tissue showed a decrease in Ki-67 expression,indicating inhibition of proliferative activity;The expression of P-ERK1/2 was reduced in the overexpression group of PHACTR1,which was verified in vivo to negatively regulate the ERK signaling pathway;In the overexpression group of PHACTR1,the expression of CD44,a tumor promoting immune indicator,decreased,while the expression of CD8,an anti-tumor immune indicator,increased.In vivo experiments have confirmed that PHACTR1 can positively regulate anti-tumor immunity.5.The tail vein metastasis model showed that compared to the control group,the overexpression group had a lower intensity of lung fluorescence concentration,indicating that compared to the control group,the overexpression group had significantly fewer metastatic lesions in the lung tissue of nude mice at the same time.Conclusions:1.PHACTR1 was low expressed in LUAD.2.PHACTR1 acted as a tumor suppressor gene to inhibit LUAD proliferation,invasion and metastasis,and EMT transformation,amplify LUAD DNA damage,induce LUAD cell cycle G1 phase arrest.3.PHACTR1 negatively regulated the ERK pathway and inhibited the progression of LUAD.4.PHACTR1 positively regulated anti-tumor immunity.