Study On The Role And Mechanism Of PZR In The Development Of Lung Adenocarcinoma

Protein Zero Related（PZR）is a transmembrane glycoprotein encoded by the MPZL1 gene and belongs to the immunoglobulin superfamily.PZR is a specific binding protein and substrate of Src homology2（SH2）domain-containing protein tyrosine phosphatase SHP-2.However,little is known about the biological function of PZR.Recent studies have shown that PZR tyrosine phosphorylation is associated with Noonan syndrome（NS）and LEOPARD syndrome（LS）.In addition,PZR is highly expressed and has pro-tumor activity in gallbladder cancer,ovarian cancer,hepatocellular carcinoma,and colorectal cancer,but its role in lung adenocarcinoma remains unclear.Through analysis of bioinformatics data from the Gene Expression Profiling Interactive Analysis（GEPIA）database and clinical samples from lung adenocarcinoma patients,we found that PZR is overexpressed in lung adenocarcinoma and is associated with poor prognosis.To investigate the role and mechanism of PZR in lung adenocarcinoma,we demonstrated using gene knockout cells（seeds）and animal models（soil）that PZR is a tumor promoter.The specific contents are as follows:（1）We used the CRISPR/Cas9 gene editing technology to construct a PZR knockout SPC-A1 lung adenocarcinoma cell line and characterized it.We found that PZR knockout caused the SPC-A1 cells to exhibit a cobblestone-like morphology with tighter cell-cell contacts.Colony formation assays showed that PZR knockout decreased the colony-forming ability of the cells.Wound healing assays demonstrated that PZR knockout significantly reduced the migration rate of the cells.Transwell invasion assays revealed that PZR knockout resulted in a significant decrease in the invasive capacity of the cells.To examine the in vivo tumorigenicity of SPC-A1 cells,we subcutaneously implanted PZR knockout SPC-A1 cells（seeds）into NYG mice.The results showed that tumor growth was slower in the PZR knockout group,and the tumor volume was significantly smaller than that in the wild-type group（721 mm³ vs 221mm³,p<0.001;0.68 g vs 0.2 g,p<0.0001）.H&E staining of tumor tissues revealed reduced angiogenesis and fewer Ki-67 positive cells in the PZR knockout group.In contrast,stable overexpression of PZR using the lentiviral packaging technology in SPC-A1 cells resulted in the opposite effects.These results suggest that the loss of PZR significantly inhibits the proliferation,migration,invasion,and tumorigenicity of SPC-A1 cells.（2）RNA-seq technology was used to analyze wild-type（n=3）and PZR knockout SPC-A1 cells（n=3）.The volcano plot results showed that compared with the WT group,when the cutoff was|log2FC|≥2 and Q≤0.05,226 genes were differentially expressed in PZR knockout SPC-A1 cells,including 75 upregulated genes and 151 downregulated genes.KEGG pathway analysis of 3767 genes revealed that a large number of differentially expressed genes（DEGs）were enriched in signaling pathways such as signal transduction,tumors,and the immune system.KEGG pathway classification bubble chart analysis showed that DEGs were enriched in key signaling pathways such as adhesion junctions and focal adhesions.GO enrichment of important biological processes showed that many DEGs were enriched in signaling pathways related to cell adhesion,migration,actin,and the cytoskeleton.Integrated analysis using GSEA revealed that PZR affects signaling pathways such as oxidative reductase activity,neuropeptide receptor binding,and fibronectin-binding-associated pathways.Based on the comprehensive analysis of KEGG,GO,and GSEA,we found that the knockout of PZR affects multiple pathways related to cell adhesion and migration,providing clues for exploring the molecular mechanisms in future studies.（3）Through protein-protein interaction analysis of the focal adhesion signaling pathway,we found that the m RNA levels of COL1A1（Collagen type I alpha 1）and FYN were significantly decreased in PZR knockout SPC-A1 cells,which was also confirmed by RT-q PCR.In addition,cell adhesion and detachment experiments showed that PZR knockout resulted in increased adhesion between SPC-A1 cells and the culture dish.Protein immunoblotting confirmed that the phosphorylation levels of tyrosine residues in Src,FAK,and Cortactin were positively correlated with PZR.These results indicate that PZR plays a positive role in activating FAK and c-Src.Integrated GSEA analysis suggested that there were differences in the oxidative stress signaling pathway in PZR knockout SPC-A1 cells.Reactive oxygen species（ROS）measurement results showed that ROS levels were decreased in PZR knockout SPC-A1 cells and increased in PZR-overexpressing SPC-A1 cells.Subsequent protein immunoblotting results demonstrated that PZR regulates oxidative stress mainly through SHP-2 and NOX4,and PZR expression levels were positively correlated with SHP-2 and NOX4expression levels.Treatment of PZR-overexpressing SPC-A1 cells with the ROS scavenger N-acetylcysteine（NAC）resulted in decreased ROS levels and decreased phosphorylation levels of FAK and Cortactin,indicating that PZR plays a positive role in maintaining intracellular ROS levels.（4）To investigate the role of PZR in the tumor-forming environment,we implanted Lewis lung adenocarcinoma cells subcutaneously into PZR gene knockout mice（soil）.The experiment data demonstrated that tumor growth was slower in the PZR^-/-group than the PZR^+/+group(tumor volume（1522 mm³ vs 572 mm³,p<0.001 on day 21 and1.31 g vs 0.38 g,p<0.0001,on day 21）.H/E staining of tumor tissues in the PZR^-/-group revealed an increased area of necrosis,with a large number of fragmented or disappearing nuclei,when compared to PZR^+/+group.Therefore,the absence of PZR in the growth environment can also inhibit tumor growth.It has been observed that patients with idiopathic pulmonary fibrosis（IPF）are more susceptible to non-small cell lung cancer.Furthermore,IPF itself is considered an independent risk factor for inducing lung cancer and is viewed as a precancerous lesion.Since wound healing involves tissue fibrosis,a skin wound healing model was established using PZR gene knockout mice to investigate the effects of PZR knockout on scar formation.The experimental results showed that the wound healing rate was accelerated in the PZR^-/-group of mice.H/E and Masson staining revealed an increased level of skin granulation and collagen fibers with the PZR^-/-mice at day 7 after injury,but the collagen fiber contents were comparable in both PZR^-/-and PZR^+/+groups by day 11.Thirty days after injury,no obvious scar was observed in the wound healing site of PZR^-/-group mice,while prominent scars were present in the PZR^+/+group mice.This indicates that loss of PZR promotes wound healing by first increasing skin collagen fibers,and then reducing the degree of skin collagen fibrosis to inhibit scar formation.In epithelial tumors,a common cell characteristic is tumor formation promoted by fibroblast activation.Together,the experiments above suggest that PZR may inhibit the tumorigenicity of transplanted syngeneic tumor cells in C57BL/6 mice by reducing the formation of collagen fibers.In summary,this article described successful construction of PZR knockout and overexpression SPC-A1 lung adenocarcinoma cell lines using gene editing and lentiviral packaging technology.It reveals that PZR plays a role in promoting the proliferation,migration,and invasion of SPC-A1 lung adenocarcinoma cells through a dual mechanism of activating FAK,c-Src,Cortactin,and regulating the expression levels of SHP-2 and NOX4 to maintain the intracellular ROS level.When examining the effect of PZR on the tumor-forming ability of lung adenocarcinoma cells,we applied PZR gene knockout cells and animal models for the first time,clarifying that PZR is indeed a tumor-promoting factor from the perspectives of both tumor cells（seed）and tumor growth microenvironment（soil）.Our research provides a basis for PZR as a new target for lung adenocarcinoma treatment and a prognostic biomarker.