The Role And Mechanism Of LAT1 In The Occurrence And Development Of Pancreatic Cancer

The relationship between the expression of L-amino acid transporter 1 and the clinical characteristics and prognosis of postoperative patients with pancreatic cancer ObjectiveDomestic and foreign studies have shown that L-type amino acid transporter 1(LAT1)is less expressed in normal tissues,but more expressed in tumor tissues.Many literatures have reported the expression of LAT1 in a variety of solid tumors.It was considered that the expression of LAT1 in tumor tissues is up-regulated and related to tumor differentiation,proliferation,metastasis and prognosis.There are few studies on the expression of LAT1 in pancreatic cancer.The purpose of our study was to investigate the relationship between the expression of LAT1 and the clinical characteristics and prognosis of postoperative patients with pancreatic cancer.Materials and methodsThis study included 32 pancreatic cancer patients hospitalized in Anhui Provincial Hospital from September 2019 to February 2020,with 22 males and 10 females,ranging from 34 to 83 years-old,and the average age at the time of operation was 63.The postoperative pathological sections and adjacent normal tissue sections of the patients were collected,and the clinical stages were defined according to the guidelines of the American Joint Committee on Cancer(AJCC)in 2009.The relevant clinical data for patients were extracted by consulting their electronic medical records,including general condition,pathological type,and serum CEA,CA19-9,and Ki67 expression.The clinical course of the patients was followed up by telephone.This study was approved by the Research Ethics Committee of Anhui Provincial Hospital.Immunohistochemical detection of LAT1 expression:After recovering the antigen,the slide was incubated with the primary antibody and secondary antibody,developed color with DAB,stained with hematoxylin,dehydrated with graded ethanol after bluing,transparent with xylene,and mounted with neutral gum.ResultsThe primary lesions and paracancerous tissues of 32 patients with pancreatic cancer were analyzed by immunohistochemistry.This immunostaining can be detected in pancreatic cancer tissues,mainly located in the cell membrane.All positive cells showed strong membrane immunostaining,while cytoplasmic staining was rare.The expression rate of LAT1 in the pancreatic cancer tissues was 40.6%(13/32),and that in the adjacent tissues was 0%(0/32).The difference was statistically significant(P<0.001).Spearman’s rank test showed that the expression of LAT1 was positively correlated with Ki67 expression(r=0.632,P=0.001)and the degree of differentiation significantly(r=0.390,P=0.027),but negatively correlated with the number of lymph nodes(r=-0.378,P=0.033).According to the LAT1 expression,the DFS and OS rates were analyzed by the Kaplan-Meier test.The results showed a significant difference between the expression of LAT1 and DFS(P=0.043),but no significant difference between the expression of LAT1 and OSAmong the 32 patients,15 died,and 20 relapsed postoperatively.The univariate analysis showed that the CA19-9,CEA,and LAT1 expression levels were important variables affecting the DFS.The multivariate analysis confirmed that the CEA and LAT1 expressions were independent prognostic factors for predicting DFS(PCEA=0.015.PLAT1=0.042).The univariate analysis confirmed that CA19-9 and CEA were important variables affecting the OS,and the multivariate analysis confirmed that CEA and CA19-9 were independent prognostic factors for predicting poor overall survival(PCEA=0.024,PCA19-9)=0.040).ConclusionThe immunostaining of LAT1 was detected in pancreatic cancer tissues mainly located in the cell membrane.The cytoplasm was largely unstained.LAT1 was highly expressed in pancreatic cancer tissues,but not in adjacent normal tissues.LAT1 was associated with the proliferation and differentiation of pancreatic cancer;LAT1 and CEA are markers for predicting DFS of pancreatic cancer.Effect of LAT1 expression on the function of pancreatic cancer cells and its mechanismObjectiveIt was believed that the expression of LAT1 was related to the biological behavior of tumor cells previously,whereas there were few studies concerned on the function and mechanism of LAT1 in pancreatic cancer.To explore the effect of LAT1 on proliferation,invasion,and apoptosis of pancreatic cancer cells,the up-regulated and down-regulated LAT1 stable cell lines were established by lentivirus transfection in vitro.In addition,we investigated whether the PI3K-AKT-mTOR-4EBP1 pathway is involved in the effect of LAT1 on pancreatic cancer.MethodsWe cultured pancreatic cancer cells(Capan-1,SW1990,CFPAC-1,PANC-1,BXPC-3)and normal pancreatic cells(HPDE6-C7).The expression of LAT1mRNA and protein was detected by real-time fluorescence quantitative PCR and Western blot.Pancreatic cancer cell lines with low expression of LAT1 were screened and transfected with lentivirus to construct overexpressed pancreatic cancer cell lines.Western blot was used to verify the expression of LAT1 in pancreatic cancer cell lines transfected with lentivirus.The proliferation,migration,invasion and apoptosis of overexpressed pancreatic cancer cell lines and control pancreatic cancer cell lines were detected by CCK-8,Transwell test and flow cytometry.Pancreatic cancer cell lines overexpressing LAT1 were treated with PI3K inhibitor,and the genes,proteins and phosphorylation related to mTOR pathway were detected by real-time fluorescence quantitative PCR and Western blot to determine whether PI3K-AKTmTOR-4EBP1 pathway was involved in the effect of LAT1 on pancreatic cancer cells.ResultsIn pancreatic cancer cells(Capan-1,SW1990,CFPAC-1,PANC-1,BXPC-3)and normal pancreatic cells,we could detect the expression of LAT1 by RT-qPCR and Western blotting.The expression of LAT1 of SW1990 was the highest,Capan-1 and CFPAC-1 was low,and PANC-1 and BXPC-3 was between the above cells.The expression of HPDE6-C7 LAT1 in normal cells was the lowest.CFPAC-1 cell lines with low expression of LAT1 were selected for lentivirus transfection.CFPAC-1 pancreatic cancer cells were transfected with lentivirus,and the overexpression lentivirus vector was constructed,and the expression of the target protein was verified by Western blot.The results showed that the CFPAC-1-SLC7A5 overexpression target cell line had obvious bands between 48 and 63kDa,while the CFPAC-1-NCcopGFP control cell lines had no obvious bands at the same location.It was suggested that the LAT1 expression level of CFPAC-1-SLC7A5 was significantly up-regulated,which indicated that the lentivirus was successfully transfected and stably expressed.Cell counting kit(CCK8)was used to detect the proliferation ability of pancreatic cancer cells.The relative absorbance of CFPAC-1-SLC7A5 at OD450nm for 24 hours,48 hours and 72 hours was higher than that of CFPAC-1-NC.The difference was statistically significant(t=-15.457,P<0.001;t=-19.173,P<0.001;Z=-2.460,P=0.014).Migration and invasion ability of pancreatic cancer cells were detected by Transwell.The number of transmembrane migration and invasion cells of CFPAC-1SLC7A5 was higher than that of CFPAC-1NC.The difference was statistically significant(t=-18.043,P<0.001;t=-11.2,P<0.001).The apoptosis of pancreatic cancer cells was analyzed by flow cytometry.The results showed that the late apoptosis rate of CFPAC-1-SLC7A5 was lower than that of CFPAC-1NC(t=46.792,P<0.001).In CFPAC-1 NC,CFPAC-1-SLC7A5 and CFPAC-1-SLC7A5 with PI3K inhibitor,PI3K,mTOR,AKT and 4EBP1 mRNA,protein,and protein phosphorylation could be detected by RT-qPCR and Western blot.The results showed that the expression of PI3K,AKT,mTOR,4EBP1 mRNA,protein,and the level of protein phosphorylation in CFPAC-1-SLC7A5 overexpression cell lines were significantly higher than those in the control group.After the addition of PI3K inhibitor,the expression of mRNA and protein and the level of protein phosphorylation were significantly decreased.It was suggested that PI3k-AKTmTOR-4EBP1 pathway was involved in the function of LAT1 in pancreatic cancer cells.Conclusion1)The expression level of LAT1 was different in different pancreatic cancer cell lines.2)The proliferation,migration,and invasion abilities of CFPAC-1-SLC7A5 cell line with high expression of LAT1 were significantly enhanced,and the apoptosis rate was decreased,which indicated that LAT1 plays a potential role in promoting cancer.3)LAT1 plays an important tumor-promoting role in pancreatic cancer mainly by regulating the PI3K-AKT-mTOR-4EBP1 signaling pathway.4)This study further improves the tumor regulatory network in theory,and provides a reference for finding new pancreatic cancer biomarkers and therapeutic targets in terms of clinical application.