Investigating Differential Expression Of AGR2 Gene In Barrett's Esophagus And Its Biological Functions And Mechanism In Esophageal Adenocarcinoma

PartⅠGeneration of a retroviral vector containing shRNAtargeting AGR2 gene and establishment of AGR2-expression-silencedstable esophageal adenocarcinoma SEG1 cell lineObjective To generate a retroviral vector containing shRNA targeting AGR2 gene,deliver shRNA into SEG1 cells by the retrovirus derived from this retroviral vector andestablish an AGR2-expression-silenced stable esophageal adenocarcinoma SEG1 cell lineby shRNA-mediated RNA interference (RNAi).Methods The database of RNAi in Cold Spring Harbor, New York, USA wasemployed to design three shRNAs specifically targeting AGR2 gene. The designed shRNAswere subcloned into the recombinant mouse stem cell retroviral vector (LMP). Thepackaging cell line that was transfected with the retroviral vectors containing shRNAs wasused to produce the corresponding retrovirus. The retrovirus was used to infect SEG1 cellsthat highly express AGR2 gene by the method of Low Speed Centrifugation (See PartⅡfordetails). The puromycin at final concentrate of 2μg/ml was used to select SEG1 cells thatwere successfully infected by the retrovirus. The SEG1 cells that were alive under thepuromycin selection were expanded in order to establish the stable cell line. To verify thesilent effects of RNAi on AGR2 gene, the Real-Time PCR and the Western-Blot were usedto detect AGR2 transcripts and AGR2 protein. Also, the immunocytochemistry wasemployed to detect AGR2 protein in the stable SEG1 cells.Results The retroviral vectors that contained shRNAs specifically targeting AGR2gene were successfully generated, named as LMPAG1,LMPAG2 and LMPAG3. The emptyvector containing no shRNA was called as LMPER and used as the control. The retrovirus containing these vectors was used to infect SEG1 cells. The three stable SEG1 cell linestanscribing each of three shRNAs were successfully generated. All three shRNAs targetingAGR2 gene (namely AG1, AG2 and AG3) inhibited AGR2 expression at various efficiency.AG1 shRNA reduced AGR2 mRNA transcripts by 85%, AG2 by 60% and AG3 by 70%. Atthe protein level, AG1 was the one of three shRNAs that inhibited AGR2 expression most.AG1 decreased AGR2 protein by 90%. The stable SEG1 cell line transcribing AG1 shRNAwas named as LMPAG1 SEG1 and will be used in the rest of this research.Conclusion The retroviral vector was an efficient vehicle for delivery of shRNA.The RNAi mediated by shRNA inhibited AGR2 expression at both mRNA level and proteinlevel with high efficiency. The stable SEG1 cell line that expresses AGR2 at very low levelwas established. PartⅡEstablishment of the method of low speed centrifugation toincrease both titer and infection efficiency of retroviral vectorObjective To establish a simple, practical and efficient approach to increase bothtiter and infection efficiency of retroviral vector.Methods The packaging cells were used to generate recombinant retroviruscontaining green fluorescence protein (GFP) gene. We compared Low Speed Centrifugation(LSC) with the routine methods in terms of their effects on recombinant retrovirus titer byusing thc serial dilution to detect titer of retrovirus. Moreover, we compared the effects ofLSC on infection efficiency of retrovirus with those of Lipofectamine pretreatment andPolybrene pretreatment. The flow cytometry was employed to determine the infectionefficiency.Results The recombinant retrovirus was successfully generated. LSC dramaticallyincreased the titer of recombinant retrovirus by 210 folds (P＜0.01) and the infectionefficiency of retrovirus by 16 times (P＜0.01). The cell infection efficiency resulted fromLSC treatment was higher than that resulted from Lipofectarnine pretreatment but showedno statistical significance (P＞0.1). Compared to Polybrene pretreatment, LSC increased cellinfection efficiency up to 7 folds (P＜0.01) and the infection efficiency of 1 volume ofrecombinant retrovirus treated by LSC equals to that of 9 volume of recombinant retroviruspretreated by Polybrene.Conclusion We successfully established the method of Low Speed Centrifugationthat was more efficient than the other methods. Low Speed Centrifugation dramatically andrapidly increased the titer and cell infection efficiency of retrovirus. PartⅢEffects of AGR2 gene silencing on growth and invasivebehavior of SEG1 cellsObjective To study the effects of silencing of AGR2 gene on growth and invasivebehavior of SEG1 cells.Methods The LMPAG1_SEG1 established in PartⅠwith low AGR2 gene expressionat both mRNA and protein level was employed as the experimental group. LMPER_SEG1infected with the retrovirus contatining the empty vector and the wild type SEG1(WT_SEG1) cells were both used as the controls. The soft agar assay was utilized toevaluate the ability of the anchorage-independent growth of LMPAG1_SEG1 andLMPER_SEG1 cells. The Transwell chamber assay was employed to evaluate the invasivecapability of WT_SEG1 cells under either of the conditioned medium from LMPAG1_SEG1 and that from LMPER_SEG1. The nude mice were used to test the capability ofLMPAG1 SEG1 and LMPER SEG1 cells to form tumors in vivo.Results Compared to LMPER_SEG1 and WT_SEG1, the ability of anchorage-independentgrowth of LMPAG1_SEG1 cells was significantly impaired (P＜0.05). Thenumber of WT_SEG1 cells that invaded in the Transwell assay under chemoattractioninduced by LMPAG1_SEG1 conditioned medium was 37% of the number of cells thatinvaded under chemoattraction induced by LMPER_SEG1 conditioned medium. The sizeof the tumors formed by LMPAG1_SEG1 cells in the nude mice was much smaller thanthat of the tumors formed by LMPER_SEG1 cells at all the time points (P＜0.03). Thegrowth rate of the tumors formed by LMPAG1_SEG1 cells was significantly slower thanthat of the tumors formed by LMPER_SEG1 cells.Conclusion AGR2 gene maintained the malignant phenotype of the SEG1 cells,induced WT_SEG1 invasion and promoted the growth of tumor formed by SEG1 cells invivo. From the Transwell chamber assay, it was speculated that AGR2 may exert itsfunctions in paracrine and/or autocrine manners. PartⅣEffects of AGR2 overexpression on biologicalcharacteristics of the benign cell line NIH3T3Objective To study effects of the AGR2 overexpression on biological characteristicsof the NIH3T3 benign cell line.Methods NIH3T3 cells were cotransfected with the plasmid AGR2-pCMV-SPORT6.0containing the full-length AGR2 gene sequence (Openbiosystems Inc., USA)and the plasmid pCDNA3.1(+)GFP carrying a G418 selective marker and GFP marker. TheG418 was used to select the NIH3T3 cells that stably expressed the both plasmids. TheNIH3T3 cells stably expressing both AGR2-pCMV-SPORT6.0 and pCDNA3.1(+)GFPwere named as NIH3T3:AGR2 after G418 selection. Likewise, the NIH3T3 cells stablyexpressing empty pCMV-SPORT6.0 and pCDNA3.1(+)GFP was named as NIH3T3:vector,which was taken as the control. The foci formation assay was used to evaluate thecontact-inhibition growth (also called density-dependent growth) of NIH3T3:AGR2 andNIH3T3:vector. The colony formation assay was employed to test the population-dependentgrowth and the in vitro proliferative capability of NIH3T3:AGR2 and NIH3T3:vector. Thesoft agar was utilized to detect changes on anchorage-independent growth of NIH3T3:AGR2 and NIH3T3:vector. The capability of NIH3T3:AGR2 and NIH3T3:vector to formtumors in vivo was tested and compared in xenograft experiments in nude mice.Results The stable NIH3T3 cell line expressing AGR2 at high level was generated.The number of foci in foci formation assay, the number of colonies in colony formationassay and the number of colonies in the soft agar assay formed by NIH3T3:AGR2, was allsignificantly more than that of foci and colonies formed by NIH3T3:vector cells.NIH3T3:AGR2 was able to form tumors in nude mice, whereas NIH3T3:vector could notform tumors in vivo. The tumors formed by NIH3T3:AGR2 grew at fast rate.Conclusion The overexpression of AGR2 gene transformed NIH3T3 cells, whichconsequently displayed malignant phenotype. The transformed NIH3T3 with the highexpression of AGR2 gene gained the ability of anchorage-independent growth, lostcontact-inhibition-growth and exhibited enhanced proliferation. The overexpression ofAGR2 gene enabled NIH3T3:AGR2 to form tumors in vivo that grew at fast rate. PartⅤExpression pattern of AGR2 gene in small intestinalepithelium in wild type mice, the exploration of its molecularmechanism and the proposed working modelof AGR2 gene in cell fate decisionObjective To characterize the expression pattern of AGR2 gene in small intestinalepithelium of wild type mice, to explore the molecular mechanism of AGR2 gene and topropose the working model ofAGR2 gene in cell fate decision.Methods The immunohistochemistry was employed to detect AGR2 expressiongene in small intestinal epithelium of wild type mice. The Real-Time PCR was used todetect changes on GFI1 mRNA level in both LMPER_SEG1 cells and LMPAG1_SEG1cells in order to determine if GFI1 expression was affected by AGR2 expression. Based onthe results obtained from this study, the possible working model of AGR2 gene in cell fatedecision was proposed.Results AGR2 was found expressed in the cells with the staining of Alcian Blue thatis the specific staining marker for goblet cells. The AGR2 expression was also foundcoexpressed with Chromogranin A that is the specific marker of enteroendocrine cells. ThePaneth cells were stained with AGR2 expression as well. Some of secretory cells thatexpressed AGR2 were found to express Ki-67--the marker for active proliferation.Moreover, AGR2 expression was coexpressed with Musashi-1 that is considered as thespecific marker for intestinal stem cells and early progenitor cells. The mRNA level ofGFI1 gene remained nearly the same in LMPER_SEG1 cells and LMPAG1_SEG1 cells.Conclusion AGR2 gene was expressed in the three types of small intestinalsecretory lineages and in putative stem cells of intestinal epithelium, whereas no AGR2expression was seen in absorptive cells. The mRNA level of GFI1 gene was not affected byAGR2 expression. AGR2 may play an important role in development of the intestinalsecretory cells and their cell fate decision.