RCN3 Promotes Tumor Progression And Cisplatin Resistance By Targeting IP3R1 In Esophageal Squamous Cell Carcinoma

BackgroundEsophageal squamous cell carcinoma(ESCC)is one of the most prevalent cancers worldwide.In China,the incidence and mortality rate of esophageal cancer are ranks the top five in all cancers.Poor prognoses of esophageal cancer are attributed to early metastasis and chemotherapy resistance.Thus,it is very urgent to find more sensitive and specific molecular targets for ESCC diagnosis and treatment.It is also of great significance to explore the molecular mechanism underlying ESCC progression and chemotherapy resistance which may effectively improve the treatment and reduce the mortality of esophageal cancer patients.The tandem mass tag(TMT)-based quantitative proteomic approach was applied to screen the differentially expressed proteins(DEPs)between 3 cases of ESCC tumor samples and paired normal tissues.We found protein—Reticulocalbin 3(RCN3)that highly expressed in esophageal cancer tissues.Few studies on RCN3 have been reported in tumors.The role and molecular mechanism of RCN3 in the occurrence and progression of esophageal cancer are unclear.Therefore,this study will explore the function of RCN3 in ESCC.Methods1.The tandem mass tag-based quantitative proteomic approach was applied to screen the differentially expressed proteins(DEPs)between 3 cases of ESCC tumor samples and paired normal tissues(ratio>2.0).Then 5 DEPs were validated in human ESCC tissues using western blot assays and GEPIA database respectively.2.The expression level of RCN3 was detected in 161 cases of ESCC tissues and paired normal tissues by IHC.We further analyze the relationship between RCN3 expression and clinicopathological features of patients,such as age,gender,TNM stage,lymph node metastasis.3.Construction of esophageal cancer cell line with overexpression/inhibition of RCN3.The proliferation ability of ESCC cells after RCN3 overexpression/inhibition was tested using the CCK-8 assay,plate colony formation assay,nude mouse tumorigenesis test in vivo and vitro.The metastatic ability of ESCC cells after RCN3 overexpression/inhibition was tested using the transwell assay,wound healing test and tail vein metastasis assay.Matrigel tube formation experiments and cell adhesion experiments were used to detect the effects of RCN3 overexpression/inhibition on tube formation and cell adhesion.4.The interaction proteins of RCN3 were predicted by bioinformatics analysis.The effects of RCN3 on the expression levels of MMP-2 and MMP-9 in ESCC cells were verified by qRT-PCR and western blot.The relationship between RCN3 and IP3R/Ca2+/CaMK Ⅱ were verified by GEPIA,co-immunoprecipitation,immunofluorescence co-localization,western blot and Flou AM assays.5.Using low-dose cisplatin to induce drug resistance in ESCC cells.The expression level of RCN3 protein in chemo-resistant ESCC cells was detected by western blot.Apoptosis,mitochondrial membrane potential and ROS of esophageal cancer cells were detected by flow cytometry and ROS fluorescence probe after overexpression/inhibition of RCN3.The effects of RCN3 on the sensitivity of esophageal cancer cells to cisplatin were detected by siRNA interference technology and calcium chelator.6.RCN3 transcription factors were predicted by bioinformatics analysis.The transcriptional regulation effect of HIF-1α on RCN3 was detected by CHIP and siRNA interference.The effects of HIF-la and RCN3 on the sensitivity of cisplatin chemotherapy were detected by CCK8 drug sensitivity test.Results1.RCN3 highly expressed in esophageal cancer.The expression level of RCN3 was significantly related to TNM stage,primary tumor size,and lymph node metastasis.So it can be used as one of the factors to evaluate the tumor progression.2.RCN3 promoted the proliferation,invasion,metastasis,tube formation and cellular adhesion,of ESCC cells in vitro and vivo.3.RCN3 promoted the expression of MMP-2 and MMP-9 by regulating the IP3R1/Ca2+/CaMK Ⅱ/c-Jun signaling pathway,so as to promote progress in esophageal cancer.4.RCN3 induced cisplatin resistance in esophageal cancer by regulated IP3R1/Ca2+to maintain intracellular Ca2+ homeostasis and reduced oxidative stress in ESCC cells.5.HIF-1α regulated the expression of RCN3.ConclusionRCN3 regulates Ca2+homeostasis by targeting IP3R1 to promote the progression and chemoresistance of esophageal squamous cell carcinoma.