Molecular Mechanisms Of MiR-301a-3p Mediating Trastuzumab Resistance Of Gastric Cancer During Endoplasmic Reticulum Stress

BackgroundGastric cancer(GC)is a malignant tumor that ranks the 4th in mortality of global cancer deaths.Trastuzumab remarkably prolongs the survival of HER2-positive gastric cancer patients.Endoplasmic reticulum stress(ERS)is associated with the drug resistance of cancer.miRNA is an endogenous gene regulator that plays an important part in the signal regulation pathway of ERS.Our previous research has proven that GC cells in ERS regulate the resistance of GC to trastuzumab by inducing miR-301a-3p expression,but the detailed molecular mechanism remains to be investigated.The purpose of this research is to explore the specific mechanism of ERS regulating trastuzumab resistance of GC via miR-301a-3p.Contents and Methods1.Target gene predictive analysis was obtained by software TargetScanHuman7.1 and miRDB.2.The target gene LRIG1 was verified by Dual-Luciferase Reporter assay.3.The LRIG1 expression level of GC cells in different status with trastuzumab resistance was determined by qRT-PCR and Western Blot.4.The protein expressions of LRIG1,GRP78,p-AKT,AKT,p-ERK,and ERK were detected by Western Blot with overexpression and inhibition of mir-301a-3p in NCI-N87 or MKN45 cells under ERS.5.The protein expressions of LRIG1,GRP78,p-AKT,AKT,p-ERK,and ERK were detected by Western Blot with overexpression and inhibition of mir-301a-3p in NCI-N87 or MKN45 cells with trastuzumab treatment under ERS.6.Phospho-receptor tyrosine kinase array for the detection of altered RTK receptors in NCI-N87 cells under ERS.7.Representative Western Blot images and semi-quantification for IGF-1R and FGFR1 protein level alterations in NCI-N87 or MKN45 cells under ERS.8.qRT-PCR analysis of the mRNA levels of IGF-1R and FGFR1 in miR-301a-3p/RNAi and siLRIG1 cotransfected NCI-N87 or MKN45 cells under ERS.9.Western blot analysis of protein levels of GRP78,LRIG1,p-AKT,AKT,p-ERK,ERK,IGF-1Rand FGFR1 in miR-301a-3p/RNAi and siLRIG1 cotransfected NCI-N87 or MKN45 cells under ERS.10.Western blot analysis of protein levels of GRP78,LRIG1,p-AKT,AKT,p-ERK,ERK,IGF-1R and FGFR1 in miR-301a-3p/RNAi and siLRIG1 cotransfected.NCI-N87 or MKN45 cells with trastuzumab treatment under ERS.Results1.Online target gene prediction suggested that there was a binding site between LRIG1 gene 3 ’-UTR sequence and miR-301a-3p.2.The target gene of LRIG1 was verified,and miR-301a-3p influenced 3 ’-UTR of LRIG1 and regulated LRIG1 directly,according to the results of Dual-Luciferase Reporter assay.3.The expression of LRIG1 protein in MKN45 or NCI-N87 cells was enhanced with interference of traztuzumab resistance,while it was weakened during ERS.4.After miR-3 01 a-3p expression was inhibitedd,the expression of LRIG1 protein in MKN45 or NCI-N87 cells induced by ERS was enhanced,and the expressions of p-AKT and p-ERK protein were weakened.5.After traztuzumab was administrated and miR-301a-3p expression was inhibited,the LRIG1 protein expression of MKN45 or NCI-N87 in ERS was enhanced,and the protein expression of p-ERK and p-AKT was apparently decreased.6.The expressions of IGF-1R and FGFR1 in gastric cancer cells were significantly increased under ERS.7.When cotransfected with miR-301a-3p/RNAi and siLRIG1,the protein levels of p-AKT and p-ERK,and the mRNA and protein levels of IGF-1R and FGFR1 in MKN45 or NCI-N87 cells were up-regulated under ERS.8.When cotransfectied with miR-301a-3p/RNAi and siLRIG1,the protein levels of p-AKT and p-ERK,the protein expressions of IGF-1R and FGFR1 in MKN45 or NCI-N87 cells were up-regulated with trastuzumab treatment under ERS.Conclusions1.ERS-induced miR-301a-3p mediates the resistance of gastric cancer cells to trastuzumab by downregulating the expression of LRIG1 and the activation of AKT and ERK signaling pathways.2.ERS-induced miR-301a-3p mediates the resistance of gastric cancer cells to trastuzumab by downregulating the expression of LRIG1 and upregulating the expression of IGF-1R and FGFR1.