Molecular Mechanisms Of Abnormal Expression And Function Of NPNT Protein In Gastric Cancer

Background:Gastric cancer?GC?is a malignant disease with high incidence and mortality rates worldwide.The latest global cancer statistical data shows GC rank the fifth common cancer and the third main cause of cancer-related death.Among the different regions around the world,the highest rates of morbidity and mortality are still seen in the east Asia including China,Japan and Korea.The symptoms of early stage GC were so latent that it is of great difficulty to be noticed.Under such circumstance,many cases of GC were diagnosed at an advanced stage,which means these patients were hardly to be cured and would have poor prognosis.Therefore,it is extremely urgent to identify meaningful biomarkers which can contribute to diagnosing GC and helping predict the prognosis of GC patients.Nephronectin?NPNT?,an extracellular matrix protein,was firstly discovered in the developing mouse kidney through functioning as integrin?8?1 ligand.NPNT was a 62kDa protein and consist of a signal peptide,five EGF-like domains,an MAM domain,and an arginylglycylaspartic acid?RGD?motif.Upon physiological function,NPNT has been involved in kidney development,osteoblast differentiation,osteogenic angiogenesis and injury repair etc.Recently,some researchers have reported the influence of NPNT on various types of cancer.Recent work by Tonje S.Steigedal has presented a comprehensive study of the expression profile and localization of NPNT in tissue microarrays from 842 cases of breast cancer and found that NPNT was significantly overexpressed in breast cancer tissues.Additionally,data from Borowsky'study indicated higher levels of NPNT in metastatic mammary tumor cells compared with non-metastatic cells in a mouse breast cancer model,suggesting that NPNT may act as a pro-metastatic factor.In contrast to above findings,studies of NPNT in melanoma detected a reduced expression of NPNT in malignant melanoma compared with primary melanocytes and found overexpression of NPNT could inhibit invasive and migratory capacity.These studies suggested that the expression profile of NPNT presented tissue-specific features among in different cancer.The variability of NPNT expression in cancers makes it interesting and meaningful to discover NPNT expression in GC.And up to now,far too little attention has been paid to discover the expression profile of NPNT in GC.In current study,we attempt to explore the expression profile of NPNT based on large scales of GC samples and investigate the function role of NPNT in GC.The findings in our study may increase the understanding of the important role of NPNT in GC as well as provide a relationship between NPNT expression and prognosis.Methods:1.Bioinformatic analysis based on public databases:The Oncomine,GEPIA?Gene Expression Profiling Interactive Analysis?,UALCAN and K-M Plotter databases were used to explore the expression profile of NPNT in GC tissues and its relationship with patients'survival.2.Clinical tissue specimens:A total of 117 formalin-fixed and paraffin-embedded?FFPE?tumor samples,together with 73 corresponding non-tumorous adjacent tissues?NATs?were obtained from GC patients who accepted curative radical gastrectomy at The First Hospital of China Medical University between2009 and 2011.Before accepting operation therapy,patients did not receive any chemotherapy or radiotherapy.For all resected tissues,two pathologists evaluated and confirmed all the pathologic diagnostic results.Tumor stage was defined according to the 8^th American Joint Committee on Cancer/International Union Against Cancer tumor-node-metastasis?TNM?classification system.All patients included in our study agreed with our study and written informed consent was obtained from them.Our study was approved by the Ethics Committee of the China Medical University.3.Immunohistochemistry:Immunohistochemistry was performed by commonly used S-P method.Semi-quantitative scoring method was used to determine the results of immunohistochemical staining.The scoring criteria are as follows:intensity of staining?0,unstained;1,weak staining;2,moderate staining;3,strong staining?,and the rate of staining area?0,?5%;1,6-25%;2,26-50%;3,51-75%;4,>75%?.The value obtained by multiplying the staining intensity score and the staining area rate score is the value of the staining score result.4.Cell culture:The normal stomach epithelial cell GES-1and gastric cancer cell lines?SGC-7901,MGC-803 and BGC-823?were purchased from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences.AGS cells were acquired from ATCC.All these cells were cultured at 37°C in RPMI 1640 medium containing 10%fetal bovine serum in a humidified incubator in an atmosphere containing 5%CO2.5.RNA extraction and quantitative RT-PCR?qRT-PCR?:Total RNA was extracted from cultured cells through using TRIzol reagent.The concentration and purity of RNA were measured through using a nano-photometer UV/Vis spectrophotometer and RNA with high purity?A260/A280 value between 1.9and 2.0?was used.Before qRT-PCR,first-strand cDNA was synthesized from 1?g of total RNA using a Reverse Transcription Kit.qRT-PCR was performed in the LightCycler?480II using TB Green.The comparative Ct method was used to calculate the relative expression of RNAs.6.Protein extraction,quantification and western blot:The whole protein of gastric cancer cells was extracted using the KeyGen protein isolation Kit,and then the protein was quantified by BCA protein quantification method.Western blot was used to detect the differential expression of proteins between the experimental groups.7.Cell Counting Kit 8?CCK8?Assay:CCK8 assay was performed to measure the proliferation capacity of GC cells.The NPNT stable overexpressed or knockdown cells were seeded into 96-well culture plates for 0,24,48,72 and 96 hours.During every time set,ten microliters of CCK8 cells were gently added into plates and incubated for 1 hour at 37°C incubator.The optical density was measured by a microplate reader at a wavelength of 450 nm.8.Transwell invasion and migration assays:Transwell assay was used to detect cell invasion ability.Matrigel and RPMI-1640 medium were diluted 1:10,and 50?L was evenly spread in the transwell chamber.After 4 hours,200?L of a suspension containing 50,000 cells was added to each well.The cells were cultured in a medium containing 10%serum,and incubated for 24 hours.After incubation,the chamber membranes were stained using hemoxylin and eosin.The use of transwell assay to detect cell migration capacity does not require matrigel.Other steps are consistent with invasion experiments.9.Statistics analysis:All statistical analyses were using SPSS V19.0 software?SPSS Inc.,Chicago,IL,USA?and GraphPad Prism V8.0 software.The abberent expression of NPNT between the tumor tissues and non-tumorous adjacent tissues was analyzed using?² test.The correlation between the expression level of NPNT and the clinicopathological data was analyzed using non-parametric test?Mann–Whitney U test for two groups and Kruskall–Wallis test for three or more groups?.Kaplan-meier method was used to calculate the overall survival rate,and the log-rank test was used to evaluate the disparities.Continuous variables were presented as mean±SD.P<0.05 was considered statistically significant.Results:1.NPNT is significantly upregulated in GC tissues:Oncomine and GEPIA databases analysis indicated that NPNT was highly expressed in GC tissues.Using KM-Plotter to analyze the relationship between the expression of NPNT and the prognosis of GC patients,it was found that the prognosis of patients was significantly correlated with NPNT expression profile.Patients with high expression of NPNT showed poor prognosis.The results of immunohistochemistry also showed that the positive expression rate of NPNT in GC tissues?40.17%,47/117?was significantly higher than that in adjacent non-tumorous tissues?10.96%,8/117?.2.The high expression of NPNT in GC is significantly associated with the poor prognosis of GC patients:Firstly,the relationship between NPNT expression and prognosis of GC patients was analyzed by KM-Plotter.The results showed that the prognosis of patients with high NPNT expression was significantly worse than that of patients with low expression.Furthermore,we analyzed the relationship between NPNT expression and prognosis in our cohort using the Kaplan-meier method,and found that there was a significant correlation between NPNT expression and prognosis?p=0.0032?.3.NPNT promotes GC cells proliferation:CCK8 was used to detect the effect of NPNT on the proliferation of GC cells by constructing stable NPNT overexpressed and knockdown cells.The results showed that the proliferation ability of NPNT knockdown cells was significantly lower than that of control cells?p<0.05?.On the contrary,overexpression of NPNT could significantly promote the proliferation of GC cells?p<0.05?.4.NPNP promotes the migration and invasion of GC cells:The transwell method was used to detect the effects of NPNT on the migration and invasion ability of GC cells.The results showed that the migrative and invasive ability of NPNT knockdown cells was significantly lower than that of the control group?migration:159.8±12.7 VS 294±20.3,p<0.001;invasion:78.2±16.7 VS 123.6±8.7,p<0.01?,in contrast,overexpression of NPNT in GC cells can significantly promote migration and invasion ability?migration:310.4±18.8 VS 137.6±14.1,p<0.001;invasion:157.6±9.4 VS 70.2±11.1,p<0.001?.5.Intervention of NPNT affects the expression of EMT-related indicators at transcriptional and translational level:Qrt-PCR results showed that knocking down NPNT in GC cells can significantly reduce the expression of mesenchymal markers N-cadherin,Vimentin,ZEB2,SNAI1 and invasion-related markers MMP9.Overexpression of NPNT up-regulated Twist1,Vimentin,ZEB2,SNAI1 MMP9expression.Western blot results showed that knocking down NPNT in GC cells can reduce the expression of mesenchymal markers N-cadherin,ZEB2,Vimentin,SNAI1and invasion-related markers MMP9 at the translational level.Overexpression of NPNT up-regulated mesenchymal-related markers N-cadherin,ZEB2,Vimentin,SNAI1 and invasion-related indicators MMP9.6.The effect of NPNT on GC cell cycle:PI staining was used to detect the proportion of cells in each cell cycle of G phase,S phase,and G2/M in the cell suspension.Statistical results show that NPNT knockdown does not significantly change cell cycle progression.In contrast,overexpression of NPNT had no significant effect on the progression of GC cell cycle.7.NPNT may regulate the biological behavior of GC cells by activating the PI3K-Akt signaling pathway:We detected changes in the expression of PI3K-Akt signaling pathway downstream marker p-Akt through intervening the expression of NPNT in GC cells.It was revealed that knockdown of NPNT can reduce the level of p-Akt.On the contrary,overexpression of NPNT can increase the expression of p-Akt in GC cells.Conclusion:1.Compared with non-cancerous tissues,NPNT showed significantly higher expression in GC tissues.2.The high expression level of NPNT in GC was positively correlated with the T stage,TNM stage and poor prognosis of GC patients.3.NPNT can promote the proliferation,migration and invasion of GC cells.4.NPNT can promote the expression of mesenchymal-related markers and the expression of invasion-related markers.5.NPNT promoted GC progression through regulating the PI3K-Akt pathway.