PPT1 Regulates The Proliferation,Invasion And Migration Of Ovarian Cancer

Objective:Ovarian cancer is the most malignant tumor in the female reproductive system.The lack of early symptoms and effective detection indicators leads to a high recurrence rate and high mortality.Although the traditional treatment of ovarian cancer can improve the disease to some extent,due to the heterogeneity of the cancer leading to complex drug resistance and recurrence,it is not effective to inhibit the progression of the disease.Protein palmitylation is the modification of protein lipids after translation,which changes its hydrophobicity,makes the protein better located on the subcell,and increases its stability,so as to play its biological function.Tumor microenvironment is the survival environment of cancer cells,composed of a variety of cells and secreted substances and extracellular matrix,etc.Tumor microenvironment plays a key role in the development of tumor.PPT1 is a lysosomal enzyme with α/ β-hydrolase structure,which can remove fatty acyl groups and depalmitylation proteins.It plays an important role in the basic cell life processes such as cell proliferation and differentiation,signal transduction,decomposition and metabolism.Current studies have found that PPT1 is highly expressed in a variety of tumors.However,whether PPT1 can promote the proliferation,invasion and migration of cancer cells in ovarian cancer and its mechanism has not been studied.In this study,the effect of PPT1 on the phenotype of ovarian cancer cells in the intracellular and tumor microenvironment was studied by the knockdown interference of PPT1 gene in ovarian cancer cells and the overexpression of PPT1 gene in 293 T cells,and the mechanism of the role of PPT1 in ovarian cancer was elucidated.Methods:1.Bioinformatics analysis: based on the ovarian cancer database in TCGA database,use the website GEPIA(http://gepia.cancer-pku.cn)The difference of PPT1 expression level between ovarian cancer tissue and non-ovarian cancer tissue was analyzed;Based on the GEO database,single-cell transcriptome sequencing analysis was performed on the selected ovarian cancer data set to detect the expression of PPT1 in various cells in the tissue;Kaplan-Meierl was used to further investigate the association between high expression of PPT1 and survival in ovarian cancer patients.2.The PPT1 gene in two kinds of ovarian cancer cells was interfered by siRNA.Western blot and qRT-PCR used to verify the interference effect.Two cell proliferation experiments were conducted to study the proliferation of cancer cells after interference.Transwell test was used to detect the invasion and migration of cancer cells after interference;3.The human embryonic kidney 293 T cells were overexpressed with PPT1 gene,and the supernatant of culture was collected.Two cell proliferation experiments were conducted to study the proliferation of cancer cells after interference,and the invasion and migration of the overexpressed supernatant on the undisturbed cancer cells after the interference was detected by Transwell test,to further verify the role of PPT1 in the microenvironment of ovarian cancer;4.Ovarian cancer patients in the TCGA data set were divided into high expression group and low expression group according to the median expression of PPT1,and the limma package of R software was used for Bayesian difference analysis.After the variance analysis of the gene,as well as a t value placed on Web Gestalt website(http://www.webgestalt.org/)for GSEA KEGG pathways analysis.Western blot analysis demonstrated the effect of siRNA interference on the expression of PI3K/AKT pathway protein and autophagy related protein in two ovarian cancer lines.Results:1.The GEPIA website showed that the expression of PPT1 in ovarian cancer tissues was higher than that in normal tissues,and the single cell transcriptome sequencing analysis showed that the expression of PPT1 was the highest in epithelial cells.Kaplan-Meierl website showed that the higher the expression of PPT1,the worse the prognosis of patients.2.After the knockdown interference of PPT1 gene in ovarian cancer cells,the proliferation of fine cells of ovarian cancer was inhibited by CCK-8 and EdU proliferation experiments,and the invasion and migration ability of ovarian cancer cells were significantly inhibited by Transwell experiments;3.After knockdown interference of PPT1 gene in ovarian cancer cells,the cell medium was collected,and Western blot results showed that PPT1 could be secreted extracellular,and compared with the control group,the secretion of PPT1 in ovarian cancer cells decreased in the knockdown group;4.After overexpression of PPT1 gene in 293 T cells,ovarian cancer cells were cultured with the cell supernatant,and the experiment was divided into control group(complete medium +50% no-load conditioned medium)and overexpression group(complete medium +50% overexpression conditioned medium).CCK-8 and EdU proliferation experiments showed that compared with the control group,Overexpressed medium can restore the proliferation of hypo ovarian cancer fine cells.Transwell experiment results showed that overexpressed medium can restore the invasion and migration ability of hypo ovarian cancer fine cells;5.Ovarian cancer cells were cultured with overexpressed medium.The results of CCK-8 showed that overexpressed medium could promote the proliferation of ovarian cancer fine cells compared with the control group;6.Pathway enrichment analysis results showed that it was highly correlated with PI3K/AKT pathway.Western blot results showed that after PPT1 gene knockdown interference in ovarian cancer cells,the protein expression of PI3K/AKT pathway was down-regulated,autophagy related protein Beclin-1 was up-regulated,and p62 protein was down-regulated.Conclusions:It was found for the first time that PPT1 gene was highly expressed in ovarian cancer tissue,and inhibition of PPT1 can significantly inhibit ovarian cancer progression.It was further found that PPT1 can be secreted in vitro,which may promote the development of ovarian cancer through paracrine effect;Moreover,inhibition of PPT1 can affect the expression of autophagy-related protein and PI3K/AKT pathway.Therefore,this study suggests that PPT1 may be a potential target for the treatment of ovarian cancer.