The Expression Of LAT1Signal In Gastric Carcinoma And Related Molecular Mechanisms

Part ⅠThe relationship between the expression of LAT1,CD98hc andclinical pathological characteristics of gastric carcinomaObjective To investigate the expression of L-type amino acidtransporter-1(LAT1), CD98hc in gastric carcinoma and elucidate therelationship between their overexpression and clinical pathologicalcharacteristics.Methods The expression of LAT1,CD98hc and Ki67in45cases ofgastric cancer,45cases of adjacent normal gastric tissue were detected byusing semiquantitative reverse transcription polymerase chain reaction(RT-PCR) and immunohistochemistry.Results RT-PCR analysis showed that the expression of LAT1,CD98hc mRNA in gastric carcinoma tissues were significantly higher thanin adjacent normal gastric tissues(P﹤0.05). Immunohistochemistryshowed that the positive rate of LAT1accounted for64.4%(29/45) in gastric carcinoma, and6.67%in adjacent normal gastric tissues (P﹤0.01).The positive expression rates of CD98hc was60.0%in gastric carcinomatissues, and22.2%in adjacent normal gastric tissues (P﹤0.01). Theexpression of LAT1, CD98hc in poorly differentiated gastric carcinoma,stage Ⅲ～Ⅳwere significantly higher than in well differentiated gastriccarcinoma, stage Ⅰ～Ⅱ(P﹤0.05). In gastric cancer with lymph metastasis,tumor size≥5cm their expression were significantly higher than that ingastric cancer without lymph metastasis, tumor size＜5cm(P<0.05). Thehigher expression of LAT1, CD98hc and Ki67in gastric carcinoma werewith a positive correlation among them (P﹤0.01).Conclusions LAT1, CD98hc specifically highly expressed ingastric carcinoma. The abnormal expression may play an important role inthe carcinogenesis and metastasis of gastric carcinoma. Part ⅡThe expression of LAT1and CD98hc in gastric cancer SGC-7901cells and the effect of LAT1antagonist on the cell biologicalbehaviorsObjective To investigate the expression of LAT1and CD98hc inSGC-7901cells and the effects of cell proliferation, cell cycle andmigration.Methods RT-PCR, IF and Western Blot were used to detect theexpression LAT1and CD98hc in gastric cancer cells SGC-7901. SGC-7901cells were cultured for48h with1μM,10μM,100μM BCH, SGC-7901cells were cultured in transwell chamer for24h with1μM,10μM,100μMBCH. MTS assay was used to examine cell proliferation. Flow cytometrywas used to analyses cell cycle distribution. The number of cells whichmigrate through micropores and stay on the outer bottom side of insertsystems were observed and counted.Results LAT1and CD98hc were expressed in SGC-7901cells.Compared with blank group, The proliferation and migration capability ofSGC-7901were obviously decreased by the higher concentration of BCH.Flow cytometry analysis showed that the number of cells in S phasedecreased while the number of cells in G0/G1phase increased after BCHtreatment(p<0.05). Conclusion Higher expression of LAT1and CD98hc in SGC-7901cells may promote cell proliferation, migration and cell cycle. Part ⅢThe effect of RNA interference targeting LAT1on theproliferation, migration and invasion of SGC7901cellsObjective To investigate the effects of downregulation of LAT1expression on the proliferation, migration, and invasion of SGC7901cells.Methods Two sets of shRNA targeting LAT1and one scrambleshRNA were synthesized and inserted into the plasmid-pGPU6/GFP/Neo.Then, the constructs containing the shRNAs were transfected to SGC7901cells and the knockdown efficiency were determined by detecting themRNA and protein expression levels LAT1and CD98hc, which forms theheterodimer with LAT1, using RT-PCR and Western blot assay. The cellswith higher knockdown efficiency were subjected to a subsequent selectionusing G418to establish stable cell lines with constitutive lower expressionof LAT1compared to cells transfected with constructs containing scrambleshRNA. MTT assay, flowcytometry, and transwell assay were conducted todetermine the potency of proliferation, cell cycle, migration, and invasionof SGC7901cells.Results The corrected sequences of LAT-shRNA construct weredetermined by Restriction endonuclease analysis and DNA sequencing. ThemRNA and protein levels of SGC7901cells were decreased aftertransfection with LAT1-shRNA1and LAT1-shRNA2for48h compared with control constructs transfection, which contained the scramble-shRNA.LAT-shRNA2constructs had higher knockdown efficiency thanLAT-shRNA1constructs. However, the expression of CD98hc was notaffected. Downregulation of LAT1inhibited the proliferation, migration,and invasion of SGC7901cells(P＜0.05)。Flowcytometry assay showedcell cycle arrest in G0/G1phase.Conclusion Downregulation of LAT1inhibits the proliferation,migration,and invasion of SGC7901cells. LAT1plays an important role inthe proliferation, migration,and invasion of gastric cancer cells.