The Function And Mechanism Of KIAA1199 In Gastric Cancer Progress

Gastric cancer(GC)is one of leading causes in cancer-related death worldwide and is the second leading cause of death among patients with cancer in China in 2015.The incidence and mortality of GC are slowly decreasing with the rapid development of diagnose and therapeutic technology,but GC patients are generally diagnosed to be with advanced tumor stage,which contribute to dismal prognosis and a high degree of malignancy.Therefore,identifying oncogene biomarkers and clarifying the regulatory mechanisms involved in the GC progress are important.KIAA1199 is a gene that is included in the Human Unidentified Gene-Encoded(HUGE)large protein database and is associated with nonsyndromic hearing loss.However,accumulative evidence over recent decades has suggested that KIAA1199 is related to cancer progression,metastasis,and poor prognosis and that KIAA1199 affects the proliferation,adhesion,motility,invasiveness,and the epithelial-mesenchymal transition(EMT)of cancer cells.Some researchers found that KIAA1199 was highly expressed in GC tissues and was associated with prognosis,lymph node metastasis,but the precise molecular mechanisms by which KIAA1199 is elevated and affects GC metastasis and proliferation are largely unclear.Thus,further exploring the function and mechanism of KIAA1199 in GC metastasis and proliferation and the reason for upregulation in GC,are meaningful for providing a new understanding of GC development and progression and may provide novel therapeutic strategies for GC.Objectives1.To describe the expression and clinical characteristics of KIAA1199 in GC.2.To investigate the function and corresponding mechanisms of KIAA1199 inducing GC cells proliferation.3.To clarify the function and corresponding mechanisms of KIAA1199 inducing GC metastasis in vitro and vivo.4.To elucidate the corresponding molecular mechanisms of KIAA1199 upregulation in GC.Methods1.The expression and clinical characteristics of KIAA1199 in GCHigh-throughput mRNA array analysis was used to examine the mRNA expression profiles of four pairs of primary tumor tissues and matched nontumor tissues from GC patients.IHC and qRT-PCR were performed to detect the expression of KIAA1199 in 60 paired GC and matched non-cancerous tissues and cells.The results of IHC assays were scored by Image-Pro Plus(IPP)6.0 scoring system.The relationships between KIAA1199 and clinical characteristics were analyzed with correlation coefficient analyses.The Kaplan–Meier Plotter curves were used to evaluate the correlation of KIAA1199 level with GC patients' outcomes.2.The function and corresponding mechanisms of KIAA1199 inducing GC cells proliferationKIAA1199 siRNA was used to silence the expression of KIAA1199.The full KIAA1199 sequence was ligated into the pHBLV-CMV-MCS-EF1-ZsGreen-T2A-Puro lentiviral vector to generate HBLV-GFP-Puro-KIAA1199 lentiviral vectors and achieve stable overexpression.qRT-PCR,IFC and WB were used to examine the effiency of inhibition or overexpression of KIAA1199.In vitro,CCK8 assays were performed to detect cells proliferation when the expression of KIAA1199 changed.In vivo,the xenografted tumor model was used to investigate the funtion of KIAA1199 in GC growth.After 28 days,mice were sacrificed and measured the tumor volume.All animal experiments were approved by the Animal Ethical and Experimental Committee of Army Medical University.To make clear of the mechanisms of KIAA1199 inducing GC cells proliferation,flow cytometric analysis was used to detect the change of cells apoptosis and cell cycles.Then the functional experiments were repeated.3.The function and corresponding mechanisms of KIAA1199 inducing GC metastasis in vitro and vivoIn vitro,the Transwell assays were performed to detect cells migration ablities.In vivo,abdominal dissemination model and distal lung metastasis model were used to explore the function of KIAA1199 in GC metastasis.All animal experiments were approved by the Animal Ethical and Experimental Committee of Army Medical University.The lungs were fixed with 4% paraformaldehyde before paraffin embedding,and 5?m sections were stained with hematoxylin and eosin to verify the metastases.To make clear of the mechanisms of KIAA1199 inducing GC cells matastasis,the mRNA and protein levels of EMT markers(N-cadherin and E-cadherin)were quantified by qRT-PCR and WB assays.Immunoprecipitation and MS analysis were applied to investigate the protein factors that could interact with KIAA1199.The Oncomine database was uesed to analyse the signal pathways relative to KIAA1199.And then,the expression of KIAA1199 was inhibited or overexpressed to explore the changes of downstream signaling pathway proteins by WB assays.In addition,to determine whether WBP11 and PTP4A3 were downstream effectors of KIAA1199,we knocked down WBP11 and PTP4A3 in the KIAA1199 overexpressing cells and then assessed the effects on GC cells proliferation with CCK8 assays,on GC cells migration with transwell assays and on the expression of EMT markers and key proteins of KIAA1199 downstream signaling pathways by Western blot.4.The corresponding molecular mechanisms of KIAA1199 upregulation in GCTCGA data and TargetScan data were combined to identify which KIAA1199-targeting mRNAs were simultaneously downregulated in GC.Pearson correlation analysis and linear regression analysis were used to calculate correlations between KIAA1199 and miR-29c-3p.The dual-luciferase reporter assays,qRT-PCR and WB assays were performed to verify that KIAA1199 was the targeted gene of miR-29c-3p and miR-29c-3p could regulate KIAA1199 expression.In addition,in vitro,qRT-PCR,Transwell assays and WB assays were performed to explore the founction of miR-29c-3p in GC and the relationship between miR-29c-3p and the downstream signaling pathway proteins of KIAA1199.In vivo,the xenografted tumor models,abdominal dissemination model and dista lung metastasis model were applied to investigate the function of miR-29c-3p overexpression in GC process.Two-tailed Student's t-test was used to determine the differences between groups.HE stain was used to make sure the lung metastases.IHC and qRT-PCR assays were performed to examine the expression of KIAA1199 in the lung metastases.The results of IHC assays were scored by Image-Pro Plus 6.0 scoring system.Results1.The expression and clinical characteristics of KIAA1199 in GCThe results of qRT-PCR based high-throughput mRNA array and TCGA data indicated that KIAA1199 was one of the most upregulated mRNAs in GC.We then further verified KIAA1199 upregulation in 60 pairs of GC tissues and matched adjacent nonmalignant tissues by qRT-PCR and IHC.In addition,we also examined KIAA1199 expression in GC cell lines(AGS,BGC-823,HGC-27,and SGC-7901)and in normal gastric mucosal epithelial cells(GES-1)and found that KIAA1199 was upregulated in GC cell lines(AGS,BGC-823,and SGC-7901)compared with GES-1.Next,we investigated the relationship between KIAA1199 expression and clinicopathological characteristics.KIAA1199 expression significantly correlated with the TNM stages of GC and was higher in cohorts with stage III/IV GC than in cohorts with stages I/II GC.Furthermore,KIAA1199 expression was notably increased in primary tumors that subsequently metastasized compared with those that did not metastasize.By analyzing Oncomine data,we determined that high KIAA1199 expression was related to GC postoperative lymphatic metastasis and GC recurrence after surgery(https://www.oncomine.org/),and Kaplan–Meier Plotter analyses showed that high KIAA1199 expression correlated with the poor survival of GC patients(http://kmplot.com/,excluding GSE62254).These results suggest that KIAA1199 is upregulated in GC and that this upregulation positively correlates with GC progression,lymph node metastasis,postoperative recurrence,and survival.2.The function and corresponding mechanisms of KIAA1199 inducing GC cells proliferationIn vitro,the results of CCK8 assays showed that KIAA1199 knockdown remarkably slowed AGS and BGC-823 cell proliferation,while KIAA1199 overexpression notably accelerated SCG-7901 cell proliferation.In vivo,the tumor xenograft studies indicated that the volumes of the tumors resulting from the SGC-7901-KIAA1199 overexpression vector cells were significantly larger than those resulting from the SGC-7901-scramble vector cells.Mechanistically,flow cytometry results showed that cells that were subjected to KIAA1199 knockdown increased apoptosis and that KIAA1199 knockdown could induce AGS and BGC-823 cell cycle arrest in some degree.Taken together,our data demonstrate that the expression of KIAA1199 can affect GC cell proliferation by regulating cells apoptosis.3.The function and corresponding mechanisms of KIAA1199 inducing GC metastasis in vitro and vivoIn vitro,the results of transwell assays showed the inhibition of KIAA1199 significantly reduced the migration of both AGS and BGC-823 cells.However,KIAA1199 overexpression promoted cell migration.In vivo,the peritoneal dissemination assays showed that mice injected with KIAA1199 vector-transfected SGC-7901 cells exhibited a significantly larger number of macroscopic nodules in the peritoneal cavity and with lower body weight.At the same time,we observed liver metastasis formation in the KIAA1199-overexpression group.In the pulmonary metastasis assays,the KIAA1199-overexpression group developed more lung metastases than did the scramble vector group.Mechanistically,because increased cell migration is a functional consequence of EMT,we found that the inhibition of KIAA1199 significantly decreased the expression of N-cadherin and increased the expression of E-cadherin.Conversely,KIAA1199 overexpression reduced the expression of E-cadherin and increased the expression of N-cadherin.Furthermore,we found that KIAA1199 could bind with WBP11 and PTP4A3.KIAA1199 expression level could affect the expression levels of proteins involved in the WBP11-FGFR4-?-catenin axis and the PTP4A3-EGFR axis(including p-EGFR,STAT3 and p-STAT3).More importantly,when we we knocked down WBP11 and PTP4A3 in KIAA1199 overexpression model,siRNA-mediated silencing of WBP11 and PTP4A3 weakened the influence of KIAA1199 on GC cells migration and the levels of proteins involved in the WBP11-FGFR4-?-catenin axis and the PTP4A3-EGFR axis(including p-EGFR,STAT3 and p-STAT3).In summary,these data imply that KIAA1199 regulates GC metastasis by interacting with downstream effectors(WBP11 and PTP4A3)and activating FGFR4/Wnt/?-catenin signaling and EGFR signaling.4.The corresponding molecular mechanisms of KIAA1199 upregulation in GCGiven that gene expression can be regulated by miRNAs,we combined TCGA data with TargetScan data to identify which KIAA1199-targeting mRNAs were simultaneously downregulated in GC and analyzed the correlations between the miRNAs and KIAA1199.Ultimately,we observed that reduced miR-29c-3p expression correlated with KIAA1199 overexpression in GC,which was in accordance with TCGA data.In vitro,overexpression of miR-29 c significantly suppressed migration of GC cells.In vivo,miR-29 c was able to inhibit GC growth and metastasis.MiR-29c-3p expression was higher in the lung metastases from the miR-29c-3p-transfected cell injections than in those from the NC cell injection;however,KIAA1199 expression showed the opposite pattern.Mechanistically,we found that miR-29c-3p overexpression significantly decreased the mRNA and protein levels of KIAA1199;conversely,miR-29c-3p inhibition increased the protein expression of KIAA1199.The dual-luciferase reporter assays showed that KIAA1199 was the targeted gene of miR-29c-3p.In addtion,we noticed that miR-29c-3p overexpression decreased the expression levels of N-cadherin and key proteins in the FGFR/Wnt/?-catenin and EGFR signaling pathways(WBP11,PTP4A3,FGFR4,and EGFR)and increased the expression levels of E-cadherin in AGS and BGC-823 cells and counteracted the influence of KIAA1199 on the expression of N-cadherin and E-cadherin and key proteins in the Wnt/?-catenin and EGFR signaling pathways in KIAA1199-overexpressing cells.Functionally,we found that miR-29c-3p overexpression counteracted the effects of KIAA1199 on migration.Overall,these results revealed that miR-29c-3p could suppresse GC cells migration by inhibiting KIAA1199 expression and regulating Wnt/?-catenin and EGFR signaling.Conclusions1.KIAA1199 is upregulated in GC and that this upregulation positively correlates with GC progression,lymph node metastasis,postoperative recurrence,and survival.2.The expression of KIAA1199 can affect GC cell proliferation by regulating cells apoptosis.3.KIAA1199 regulates GC metastasis by interacting with downstream effectors(WBP11 and PTP4A3)and activating FGFR4/Wnt/?-catenin signaling and EGFR signaling.4.miR-29c-3p could regulate KIAA1199 expression and suppresse GC cells migration by inhibiting KIAA1199 expression and regulating Wnt/?-catenin and EGFR signaling.SignificanceIn this research,we describes a previously unknown role for KIAA1199 in the regulation of the Wnt and EGFR signaling cascades in GC and demonstrates that KIAA1199 can be blocked by miR-29c-3p,providing a new avenue for inhibiting KIAA1199-driven GC progression and novel therapeutic strategies for GC.