The Effect Of GPRC5A On The Biological Behaviors Of Gastric Cancer And Related Mechanisms

Objectives:Gastric cancer(GC)is one of the most common malignancies worldwide.At present,the incidence and mortality rate of gastric cancer ranks fifth and third,respectively.There are about 951,000 new cases and 723,000 deaths every year and most of the new cases occur in East Asia,with China accounting for 42%.In china,there are about 404,000 new cases each year with about 287,000 deaths,whose morbidity and mortality are ranked second and third respectively.The rapid development of bioinformatics technology in the field of cancer,especially the tumor-related big data research,promoted the progress of molecular typing and molecular diagnosis in various tumors.G Protein-coupled receptor class C group 5 member A(GPRC5A),as a well-characterized tumor suppressor in lung cancer,is a member of G Protein-coupled receptor superfamily and has been found to be dysregualted in various cancer types.However,its role in gastric cancer(GC)was rarelyreported.Thus,the aim of this study was to elucidate the role of GPRC5 A and related mechanisms in GC using tumor-related online databases,GC tissue bank and a series of biological experiments.Besides,STAT3(signal transducer and activator of transcription 3)is an acctrative therapeutic target for GC,which was closed associated to GPRC5 A.Thus,we also aimed to explore the synergistic anti-tumor effect of GPRC5 A suppression and STAT3-targeted inhibitor.Methods:The expression level of GPRC5 A in GC and normal adjacent tissues was evaluated using online databases(including Oncomine and GEPIA).The clinical and pathological data of patients accepted GC surgery in our hospital were collected through electronic medical record system and image system.The prognosis information of patients was obtained by approprriate follow-up.The specimens of preserved GC and adjacent normal tissues were systematically arganized.Quantitative real-time polymerase chain reaction(RT-q PCR),Western Blot and immunohistochemistry(IHC)were conducted to validated GPRC5 A expression in clinical GC tissues and cell lines.The associations between clinical characteristics and GPRC5 A expression were analyzed.Finally,Cox regression analysis was used to pick out the adverse prognosis factors of GC.Gene editing of GPRC5 A in the human GC cell lines was performed to investigate its influence in GC cells in vitro and in vivo.Related mechanisms were explored through RNA-sequence and co-expression analyses and were validated through Western Blot.Tissue microarray was chosed to explore the relationship between GPRC5 A and STAT3 expression.Furthermore,CCK8 and Western Blot were used to explore the combined anti-tumor effect and related mechanism of GPRC5 A knockdown and HJC0152 in GC cells.Results:The results from databases and the GC clinical tissue bank both indicated that GPRC5 A m RNA was significantly up-regulated in GC tissues.IHC analysis concluded that GPRC5 A protein high expression was associated with older age,tumor invaison,lymph node metastasis and TNM staging.What's more,the survival analysis found GPRC5 A highly expressed had worse overall survival,and muti-variate COX regressionanalysis indicated GPRC5 A was an indenpendent prognosis predictor of GC(HR=1.886,1.162-3.062;P=0.010).In vitro experiments showed that GPRC5 A knock-down significantly inhibited the colony-formation,migration and invasion abilities of the GC cells and GPRC5 A overexpression promoted the colony-formation,migration and invasion abilities.In vivo xenografts experiment indicated GPRC5 Aknockdown suppressed tumor growth.Mechanism analysis indicated that GPRC5 A knockdown increased E-cadherin expression and decreased the expression of ITGB1,Vimentin and Snail,thus suppresing the process of epithelial-mesenchymal transition(EMT).Besides,GPRC5 A was closely related to PI3K-Akt and MAPK signaling pathway.Morever,the resut of the tissue microarray indicated that GPRC5 A expression was closely related to STAT3 expression.GPRC5 A restrain increased the drug sensitivity of GC cell lines to HJC0152 by enhancing its inhibition of the target genes of STAT3 signaling(c-Myc and Mcl1)and the activation of the p38/JNK signaling pathway,exerting a synergistic anti-cancer role in GC.Conclusions:GPRC5A up-regulation was found in GC and was positively with older age,tumor invasion,lymph node metastasis,TNM staging and worse overall s;urvival,as well as an an indenpendent prognosis predictor of GC.GPRC5 A promotes thecolony-formation,migration and invasion abilities of the GC cells in vitro and GPRC5 A knockdown suppressed tumor growth in vivo.GPRC5 A promotes migration and invasion by regulating EMT.The tumor-promoting mechanism of GPRC5 A in gastric cancer is related to PI3K-Akt and MAPK signaling pathways in GC.GPRC5 A konckdown enhances the anti-tumor efficency of STAT3 inhibitor(HJC0152)through modulating STAT3 and p38/JNK signaling pathways.Our findings indicate that GPRC5 A was a potential biomarker for the prognosis prediction,precise diagnosis and treatment in GC patients.