| 1Background and ObjectiveGastric cancer is the fourth most common human cancer in the world, themortality rate of gastric cancer was the second in the world, only lower to lungcancer. There was also a high incidence of gastric cancer in China, and it is stillone of the deadliest malignant tumor. Therefore, a deep understanding of themolecular mechanisms of gastric cancer is very important in guiding therapy.Helicobacter pylori（H.pylori） is I type of carcinogen for gastric cancer, theoccurrence and development of gastric cancer was a interaction result ofmultiple genes. The molecular mechanisms of H.pylori lead to gastric cancer isthe focus of the studies. Researchs had indicating that apoptosis-related genesmay play a key role in the development of H.pylori infected gastric cancer.Our’s preliminary results suggested that after infected gastric epithelial cells,H.pylorican change the expression of FAF1mRNA. If or not H.pylori infectioncan influence the FAF1expression and it’s corresponding signaling pathways inthe occurrence and development of gastric cancer is required to validate in thistopic.By further elucidate the pathogenesis of gastric cancer, the new drug oftarget genes and tumor chemopreventive agents will be found, and provide anew direction for the treatment of cancer.2Method2.1The expression of FAF1mRNA in gastric epithelial cells and varying degrees of differention of gastric cancer cells were detected by Real time PCR.Human gastric cancer cells(HGC-27、SGC-7901) and human gastric epithelialcell (GES-1)Co-culture with different degree of H.pylori standard strainNCTC11637for24h, cells were divided into three group by multiplicity ofinfection(MOI): control group （without co-culture with H.pylori）,1:100co-culture group,1:200co-culture group. Thiazolyl blue (MTT) assay was usedto determine the proliferation and flow cytometry was used to determine theapoptosis of these cells, real-time fluorescent quantitative reverse transcriptasepolymerase chain reaction technology was used to detect FAF1mRNA relativeexpression.2.2Lentiviral vector was selected to express FAF1gene, FAF1full-lengthcDNA was amplified and cloned to AgeI digestED lentiviral vectors, and DNAsequencing of the recombinant plasmid. FAF1recombinated expressionplasmids and lentiviral packaging plasmids pHelper1.0, pHelper2.0andLipofectamine2000co-transfected cells293T,48hours later, the recombinatedexpression FAF1virus particles named Lenti-FAF1in the cells supernatant werecollected and concentrated. The titer was detected by quantitative PCR. Thesame method to construct negative control Lentiviral Particles named Lenti-NC,and lentiviral titer was detected by quantitative PCR.2.3HGC-27cells in logarithmic growth phase were transfected with lentivirus,they are separate into three group: Lenti-FAF1/HGC-27group (HGC-27cellstransfected with Lenti-FAF1) and Lenti-FAF1/HGC-27group (HGC-27cellstransfected with Lenti-NC lentiviral) and HGC-27group (non-transfectedHGC-27cells), cell cycle and apoptosis were detected by flow cytometry, cellproliferation was detected by MTT, tumorigenicity ability were detectey bycolony formation assay. Cell suspension of Lenti-FAF1/HGC-27group， Lenti-NC/HGC-27group and HGC-27group were conventional digested, sixnude mice were randomly selected,0.2ml cells of1×107were injected on theright armpit of each nude mices. general condition diet, weight and movement ofthese nude mices were observed daily. subcutaneous tumor growth was observedevery3-4d. The tumor long and short diameter were measured by vernier caliperfor the growth curve and calculation of tumor volume. the mice were killed bycervical dislocation for28d after injection, the tumor morphological changeswere observed by HE staining, FAF1protein expression were detected bywestern blot and immunohistochemistry SP method.2.4The expression of IL-8and TNF-α of Lenti-FAF1/HGC-27group,Lenti-FAF1/HGC-27group, HGC-27group, Lenti-FAF1/HGC-27group+H.pylori, Lenti-NC/HGC-27group+H.pylori and HGC-27group+H.pylori weredetected by ELISA. The expression levels of p65、 FAF1and Iκκβ inLenti-FAF1/HGC-27group,Lenti-NC/HGC-27group, HGC-27group,Lenti-FAF1/HGC-27group+H.pylori were detected by real time PCR and western blot.3Results3.1The influence of H.pylori infection on FAF1expression in gastric epithelialcells:3.1.1The expression level of FAF1mRNA in cells: compared with GES-1FAF1mRNA expression(1.04±0.23),The level of FAF1mRNA in HGC-27(0.36±0.19)and in SGC-7901(0.52±0.17) was significantly decreased（P<0.05）, and it waslower in HGC-27than in SGC-7901（P <0.05）.3.1.2After the cells co-cultured with H.pylori for24h, dead cells were increased,the "hummingbird" phenotype was seen, cell proliferation and apoptosis wereinhibited for H.pylori infection.3.1.3The expression level of FAF1mRNA in gastric epithelial cells after co-culture with H.pylori: compare with respective control group, FAF1mRNAexpression of1:100and1:200co-culture group in HGC-27cells wassignificantly lower (0.67±0.20vs0.99±0.27P<0.01;0.44±0.30vs0.99±0.27, P<0.001); and that of1:100and1:200co-culture group in SGC-7901cellswas significantly lower (0.72±0.29vs1.05±0.42,0.47±0.31vs1.05±0.42,P<0.05). The expression of co-culture group1:100in GES-1cells wassignificantly increased (1.61±0.77vs0.99±0.41, P<0.013),1:200co-culturegroup was significantly lower (0.36±0.22vs0.99±0.41, P <0.01). Comparedwith the respective1:100co-culture group, FAF1mRNA in HGC-27andSGC-7901cells in1:200co-cultured group had no significantly difference(P>0.05), the FAF1mRNA expression in GES-1cells in1:200co-culture groupwas significantly decreased (P<0.001).3.2The recombinated FAF1expression lentivirus vector was constructedand identificated, it supply a necessary method for the next study.3.2.1The full length of FAF1amplify product was1996bp, the length ofdigestedproduct of recombinated lentivirus plasmid was724bp and wasconfirmed by sequence. The FAF1-GV218plasmid and negative controlplasmid trans-fected293T for48h, the green fluorescence was seen influorescent micro-scope, lentivirus package was successfully.3.2.2The titer of recombinated FAF1expression lentivirus(Lenti-FAF1) is2×108TU/mL and negative control lentivirus (Lenti-NC) is3×109TU/mL.3.3The impact of Lentivirus-mediated FAF1high expression in human gastriccancer cell HGC-27in vivo and in vitro:3.3.1The change of cell morphology: after transfected with Lenti-FAF1for48h,the cell morphology were changed from clostridial form to irregular form inLenti-FAF1/HGC-27group, and there were not obviously form changes in Lenti-NC/HGC-27group.3.3.2FAF1expression in transfected gastric caner cells: compared withLenti-FAF1/HGC-27group(2.436±0.538), the expression levels of FAF1mRNAin HGC-27group（1.086±0.226）and Lenti-NC/HGC-27group(1.182±0.381)was significantly decreased(P<0.001）, the expression levels of FAF1mRNA inHGC-27group had no difference with Lenti-NC/HGC-27（P>0.05）.Comparedwith Lenti-FAF1/HGC-27group(1.515±0.377),The expression of FAF1proteinin HGC-27（0±0）and Lenti-NC/HGC-27group（0±0）was significantlydecreased(P<0.001）.3.3.3The influence in cell proliferation, apoptosis, colony formation rate aftertransfected with Lentivirus: Lenti-FAF1was constructed successful. From d2incell growth,the cell proliferation activity in Lenti-FAF1/HGC-27group waslower than in HGC-27group and in Lenti-NC/HGC-27group (P<0.05). Thecells in G2/M phase in Lenti-FAF1/HGC-27group was significantly lower thanin HGC-27group （29.78%±3.91%vs19.33%±3.82%， P<0.05） andLenti-NC/HGC-27group（29.78%±3.91%vs20.93%±2.46%，P<0.05). The cellsapoptosis rate in Lenti-FAF1/HGC-27group was significantly higher than thatin HGC-27group （84.66±5.92%vs4.60±3.80%， P<0.001） andLenti-NC/HGC-27group（84.66±5.92%vs7.32±3.82%，P<0.001）. The cellscolony formation rate in Lenti-FAF1/HGC-27group was significantly decreasedthan in HGC-27group（45.75%±5.73%vs62.5%±11.25%，P<0.05）andLenti-NC/HGC-27group（45.75%±5.73%vs56.75%±8.57%，P<0.05）.3.3.4The influence of tumorigenicity in vivo: the growth of tumor xenograftswas slowly and the volume tumor xenografts were significantly smaller inLenti-FAF1/HGC-27group than in HGC-27group and Lenti-NC/HGC-27group cells from d8after injection (P<0.05). HE staining showed atypia and mitotic was obviously showed in HGC-27group and Lenti-NC/HGC-27group;whereas lower atypia and rarely mitotic showed in Lenti-FAF1/HGC-27group.The FAF1positive cells expression rate and strength was significantly increasedin Lenti-FAF1/HGC-27group than in HGC-27group and Lenti-NC/HGC-27group by immuneHistochemistry (P<0.05).The expression levels of FAF1protein in Lenti-FAF1/HGC-27group (0.87±0.34) was higher than inHGC-27group (non expression) and Lenti-NC/HGC-27group (non expression)(P <0.05).3.4The influence of H.pylori in FAF1expression in gastric cancer cells and itsrelated pathway:3.4.1Iκκβ gene: compared with Lenti-FAF1/HGC-27group, the Iκκβ mRNAexpression in Lenti-FAF1/HGC-27group and HGC-27group（0.95±0.10vs0.50±0.06，P<0.05）, Lenti-NC/HGC-27group（0.87±0.07vs0.50±0.06，P<0.05） and Lenti-FAF1/HGC-27group+H.pylori（0.75±0.12vs0.50±0.06，P<0.05） were significantly increased; compared with Lenti-FAF1/HGC-27group+H.pylori, the Iκκβ mRNA in HGC-27group and Lenti-NC/HGC-27group were significantly increased（P<0.05）. Compared with Lenti-FAF1/HGC-27group, the Iκκβ protein expression in HGC-27group（0.11±0.04vs0.03±0.02，P<0.05）, Lenti-NC/HGC-27group（0.10±0.08vs0.03±0.02，P<0.05）and Lenti-FAF1/HGC-27group+H.pylori（0.08±0.03vs0.03±0.02，P<0.05）. Compared with Lenti-FAF1/HGC-27group+H.pylori, the Iκκβ proteinexpression in HGC-27group and Lenti-NC/HGC-27group were significantlyincreased（P<0.05）.3.4.2p65gene:Compared with Lenti-FAF1/HGC-27group, the p65mRNAexpression in Lenti-FAF1/HGC-27group and HGC-27group（0.96±0.05vs0.32±0.10，P<0.05）, Lenti-NC/HGC-27group（0.90±0.03vs0.32±0.10， P<0.05） and Lenti-FAF1/HGC-27group+H.pylori（0.50±0.10vs0.32±0.10，P<0.05） were significantly increased; compared with Lenti-FAF1/HGC-27group+H.pylori, the p65mRNA in HGC-27groupand Lenti-NC/HGC-27groupwere significantly increased（P<0.05）. Compared with Lenti-FAF1/HGC-27group, the p65protein expression in HGC-27group（0.64±0.13vs0.14±0.03，P<0.05）, Lenti-NC/HGC-27group（0.49±0.06vs0.14±0.03，P<0.05） and Lenti-FAF1/HGC-27group+H.pylori（0.28±0.02vs0.14±0.03，P<0.05）were significantly increased. Compared with Lenti-FAF1/HGC-27group+H.pylori, the p65protein expression in HGC-27group andLenti-NC/HGC-27group were significantly increased（P<0.05）.3.4.3FAF1gene: Compared with Lenti-FAF1/HGC-27group, the FAF1mRNAexpression in Lenti-FAF1/HGC-27group and HGC-27group（1.086±0.226vs2.436±0.538，P<0.05）, Lenti-NC/HGC-27group（1.182±0.381vs2.436±0.538，P<0.05） and Lenti-FAF1/HGC-27group+H.pylor（i1.75±0.12vs2.436±0.538，P<0.05）were significantly decreased; compared with Lenti-FAF1/HGC-27group+H.pylori, the FAF1mRNA in HGC-27group and Lenti-NC/HGC-27group were significantly decreased（P<0.05）. Compared with Lenti-FAF1/HGC-27group, the FAF1protein expression in HGC-27group（0±0vs1.515±0.377，P<0.05）, Lenti-NC/HGC-27group（0±0vs1.515±0.377，P<0.05）and Lenti-FAF1/HGC-27group+H.pylori0.76±0.24vs1.515±0.377,P<0.05）were significantly decreased. Compared with Lenti-FAF1/HGC-27group+H.pylori, the FAF1protein expression in HGC-27group andLenti-NC/HGC-27group were significantly decreased（P<0.05）.3.4.4Detection of IL-8and TNF-α expression: after co-culture of Helicobacterpylori, the cell supernatant levels of IL-8and TNF-α were elevated. Comparedwith Lenti-FAF1/HGC-27group+H. pylori TNF-α, IL-8expression levels in Lenti-NC/HGC-27group+H. pylori and HGC-27group+H. pylori wassignificantly increased (P <0.05)4Conclusions4.1The expression level of FAF1gene was lower in gastric cancer cell linesthan that in normal gastric epithelial cells, the expression level of FAF1genemay correlate with differentiation of gastric cancer cells, it suggested aimportant role of FAF1in progress and differentiation of gastric cancer. H.pyloriinfection can down-regulated FAF1expression in gastric epithelial cells. One ofmechanism of cell proliferation and apotosis disorder caused by H.pylori mayachieve by disturbing FAF1gene expression.4.2Lenti-FAF1and Lenti-NC was successfully constructed, it was supply thenecessary means for further experiments.4.3FAF1mRNA and protein expression was significantly up-regulated in gastriccancer cells HGC-27after transfected with Lenti-FAF1. HGC-27cellmorphology changed from clostridial form to irregular form after transfectedwith Lenti-FAF1, it is suggested the start of apoptosis.High expression of FAF1can inhibit gastric cancer cell proliferation and apoptosis and cell colonyformation rate. And also resulting in gastric cancer cell cycle arrest in G2/Mphase. High expression of FAF1can inhibit tumor growth in nude mice, reducingthe atypia and the degree of and malignant of gastric cancer cells, it is suggestedthat gene deletion of FAF1is one of the cause of gastric cancer.4.4High expression of FAF1significantly reduce the expression of NF-κB p65and Iκκβ, H.pylori infection can reduce the expression of FAF1and up-regulatethe expression of p65and Iκκβ. High expression of FAF1can reduce theexpression level of IL-8and TNF-α induced by H.pylori. It is suggest thatFAF1high expression may negatively regulate NF-κB signaling pathway that can inhibit the growth of gastric cancer.InnovationThere are few studies in relationship between FAF1and gastric cancer in ourcountry and abroad, and the exploration of FAF1impact on the development ofgastric cancer and the relationship between H.pylori from the cellular level hasnot been reported. This issue initially clarify the FAF1mRNA expression levelin human gastric cancer cell line HGC-27and SGC-7901, gastric epithelial cellline GES-1, the expression level of FAF1may be associated with the degree ofcell differentiation, H.pylori infection can down-regulation the expression levelof FAF1. The recombinanted FAF1expression vector of lentivirus and the stabletransfected cell line with high expression of FAF1was was successfullyconstructed, and the followed results were fouded: the high expression ofFAF1can reduce cell proliferation,enhance cell apoptosis,reduce cell colonyrate, and arrest cancer cells in G2/M period, it also can reduce thetumorigenicity of cancer cells in vivo. It initially clarified the relationship ofFAF1and H.pylori in gastric cancer and it related pathway,so we shouldprovide theoretical and experimental evidence for gastric cancer therapy intarget gene FAF1. |
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