| Background and objective:The latest epidemiological data indicated that gastric cancer remained the fifth most frequently diagnosed cancer and the third leading cause of cancer-related death.The incidence of gastric cancer was high in our country,and more than half of the new cases of gastric cancer were diagnosed in China.The prognosis of advanced gastric cancer was poor and the efficient treatment strategies of advanced gastric cancer was lacking.Therefore,it is of great importance to elucidate the mechanism of gastric carcinogenesis and to develop an effective strategy for primary prevention and treatment of gastric cancer.Helicobacter pylori(Hp)is defined as a type-I carcinogen and the major etiological factor for gastric cancer.Hp infection causes chronic active gastritis in all infected subjects,which can progress to gastric atrophy,intestinal metaplasia,dysplasia and ultimately gastric cancer in a subset of individuals.Hp acted as an“initiation factor”during the typical pathological evolution for gastric cancers.Hp infection-initiated gastric inflammation is believed to play an important role in Hp-induced carcinogenesis.Various virulence factors of Hp interact with the receptors or targets of the host,leading to the activation of inflammatory signaling pathways(e.g.,the NF-?B signaling pathway)and the subsequent release of proinflammatory cytokines,which is a major step in the initiation and development of gastric cancer.We also found that the NF-?B signaling pathway is positively associated with Hp infection through Gene Set Enrichment Analysis of differentially expressed genes between Hp-positive and Hp-negative gastric cancer from the TCGA database.Receptor of activated protein kinase C 1(RACK1),a cytosolic scaffold protein,has been reported to have a suppressive effect on gastric cancer by negatively regulating the WNT and NF-?B signaling pathways.Additionally,we analyzed The Cancer Genome Atlas(TCGA)and found that RACK1 expression was decreased in gastric cancer tissues in comparison with the corresponding normal tissues.However,the mechanism underlying RACK1 expression regulation remains unclear.It has been documented that numerous regulators are involved in modulating the NF-?B signaling pathway.RACK1 acted as a novel negative regulator of the NF-?B signaling pathway,and subsequently inhibited cytokine induction and inflammatory reactions.Moreover,recent study identified RACK1 as a negative regulator involved in Hp-induced NF-?B pathway activation by analyzing the nuclear translocation of P65 in AGS cells interfering with 646 kinases,although the underlying mechanisms were unclear.Integrins(ITG)are transmembrane receptors consisting of?and?-subunits that play essential roles in various physiological functions and pathological progression(e.g.,immune response,cell cycle progression,cell death,invasion,metastasis and angiogenesis).Moreover,interactions of ITG with Hp virulence factors activate downstream signaling pathways with oncogenic activity,which contributes to Hp pathogenesis.RACK1 interacts with different beta-ITG and participates in regulating ITG functions.However,the mechanistic linkage among Hp infection,RACK1expression,beta-ITG and the NF-?B signaling pathway has not been reported.Therefore,this project aimed to explore the role of RACK1,ITG-?1 and NF-?B signaling pathway in Hp carcinogenesis via in vivo and in vitro studies,which provided the theoretical basis for better understanding the mechanisms of gastric carcinogenesis and for the development of a novel therapeutic target for primary prevention and treatment of gastric cancer.Materials and methods:RACK1 expression in gastric cancer and its relationship with the prognosis of gastric cancer1.The gastric cancer information from TCGA database was extracted and RACK1 expression in gastric cancer and adjacent normal tissues was analyzed.The different pathology stage of gastric cancer and adjacent normal tissues were collected and the RACK1 expression were detected by Western blot.2.The expression of RACK1 in 90 pairs of human gastric cancer samples in a tissue array was detected by immunohistochemistry.The relationship between RACK1 and the clinical pathological features was analyzed.Kaplan-Meier plotter database was utilized to analyze the association between RACK1 expression and overall survival in gastric cancer patients.3.The RACK1 mRNA expression was detected using several gastric epithelial cells with different differentiation stages by qRT-PCR.The impact of Hp infection on the RACK1 expression and NF-?B signaling pathway in vitro and in vivo1.The signaling pathways associated with Hp infection were analyzed using bioinformatics.2.After co-culture of GES-1 with Hp,the protein expression of RACK1 and some key molecules in the NF-?B signaling pathway were detected by Western blot,and the mRNA expression levels of several NF-?B target genes were detected by qRT-PCR.3.After challenging Mongolian gerbils with Hp,the colonization of Hp in gastric mucosa was confirmed by Giemsa staining and PCR,and the pathology of gastric mucosa induced by Hp infection was evaluated by H&E staining.The expression of RACK1 and some key molecules in the NF-?B signaling pathway were detected by immunohistochemistry.The mechanism of RACK1 regulating NF-?B signaling pathway induced by Hp infection1.After co-culture of GES-1 stably overexpressing RACK1 with Hp,the mRNA expression level of some NF-?B target genes was detected by qRT-PCR and the activity of NF-?B signaling pathway was determined by NF-?B luciferase reporter assay.2.The involvement of ITG-?1 in biological processes,and the expression of ITG-?1 in gastric cancer and adjacent normal tissues was analyzed using bioinformatic tools,while the relationship between ITG-?1 and overall survival in gastric cancer patients was analyzed using GEPIA and Kaplan-Meier plotter database.3.The expression of ITG-?1 was detected by Western blot after GES-1 was co-cultured with Hp.4.AGS with low RACK1 expression was co-cultured with Hp,followed by detection of the expression of ITG-?1 and some key molecules in the NF-?B signaling pathway by Western blot.5.AGS with low ITG-?1 expression was co-cultured with Hp.Some NF-?B target genes were detected by qRT-PCR and the activity of NF-?B signaling pathway was determined by NF-?B luciferase reporter assay.6.The expression of RACK1 and ITG-?1 were intervened simutanously.Whether the regulation of Hp infection induced NF-?B signaling pathway by RACK1is dependent on the expression of ITG-?1 was investigated.The expression of RACK1,ITG-?1 and some key molecules in the NF-?B signaling pathway in different pathology stages of gastric mucosa and its relationships with Hp infection1.The expression of RACK1 and ITG-?1 in Hp positive and negative gastric cancer samples were analyzed using bioinformatic approaches.2.Biopsies of chronic gastritis,intestinal metaplasia and dysplasia were collected.The expression of RACK1,ITG-?1 and some key molecules in the NF-?B signaling pathway for each stage and its relationship with Hp infection were detected by immunohistochemistry.Results:RACK1 expression in gastric cancer and its relationship with the prognosis of gastric cancerTo explore the role of RACK1 in gastric carcinogenesis,we analyzed the TCGA database and found that RACK1 expression was markedly lower in gastric cancer tissues than in the adjacent normal tissues.We further examined RACK1 expression in 23 pairs of human gastric cancer samples using Western blot and in 90 pairs of human gastric cancer samples in a tissue array using immunohistochemistry.Western blot data showed that RACK1 was downregulated in 87.0%(20/23)of gastric cancer patients compared with that in matched normal tissues.Consistent with this result,the microarray data showed that gastric cancer tissues had significantly lower levels of RACK1 expression than the adjacent normal tissues.The Kaplan-Meier plotter database was utilized to analyze the association between RACK1 expression and overall survival in 876 gastric cancer patients.We observed that lower expression levels of RACK1 were significantly associated with poor prognosis.Next,we analyzed the expression level of RACK1 in different gastric cell lines,showing that RACK1 was highly expressed in nonmalignant and moderately differentiated gastric cell lines(GES-1 and SGC-7901)in comparison with poorly and undifferentiated gastric cell lines(AGS and HGC-27).The impact of Hp infection on the RACK1 expression and NF-?B signaling pathway in vitro and in vivoBecause of the decreased RACK1 expression noted in gastric cancer tissues,we sought to investigate how Hp infection modulates RACK1 expression and the activation of the canonical NF-?B signaling pathway.GES-1 cells were cocultured with different MOIs of wild-type Hp strains 43504,7.13 and isogenic cagA~-Hp strain7.13.Western blot data revealed that Hp infection(MOI=100 or 200)downregulated RACK1 and promoted the induction of p-P65(Ser 536)independent of CagA.To further validate this result,GES-1 cells were cocultured with wild-type Hp strain43504(MOI=100)or treated with TNF-?(1 ng/ml or 10 ng/ml)for 0,15,30,45,60and 75 minutes.Both Hp and TNF-?stimulation promoted the production of p-P65(Ser 536)and the degradation of I?B?,while decreased RACK1 expression was observed at 75 minutes in only the Hp group.In addition,expression of NF-?B signaling pathway target genes(TNF-?,A20 and I?B?)was increased by Hp infection,further indicating the activation of the canonical NF-?B signaling pathway.To further validate our in vitro results,Mongolian gerbils were challenged with Brucella broth,wild-type Hp strain 7.13 or isogenic cagA~-Hp strain 7.13 for 3 months.Giemsa staining and PCR confirmed that Hp colonized the gerbils challenged with strain 7.13 and its cagA~-mutant.H&E staining results showed sparse neutrophils in the squamous junctions and squamous epithelial hyperplasia in a subset of animals infected with cagA~+or cagA~-Hp strain 7.13.Immunohistochemistry was performed to evaluate the expression of RACK1,I?B?and nuclear P65.The data showed that Hp infection downregulated the expression of RACK1 and I?B?and subsequently promoted the translocation of P65 from the cytoplasm to the nucleus.Levels of RACK1 were positively correlated with levels of I?B?but negatively correlated with levels of nuclear P65.The mechanism of RACK1 regulating NF-?B signaling pathway induced by Hp infectionTo determine whether RACK1 plays a role in regulating the activity of the NF-?B signaling pathway,GES-1 cells with stable RACK1 overexpression were established and then cocultured with Hp strain 43504 for 75 minutes.Compared to the GES-1 control group,the RACK1 overexpression group had significantly inhibited NF-?B-mediated transcription activity as detected using an NF-?B luciferase reporter assay and decreased expression of selected NF-?B target genes.In our previous study,we identified that the ITG-mediated signaling pathway was positively associated with Hp using TCGA data,and ITG?-1 was involved in multiple biological processes.We showed that the expression of ITG?-1 was higher in gastric cancer tissue than in adjacent normal tissue and gastric cancer patients with high expression of ITG?-1 had a poor prognosis.Additionally,Hp increased ITG?-1protein levels in vitro.Knocking down endogenous RACK1 upregulated the expression of ITG?-1 and increased the formation of p-P65(Ser 536).In contrast,knocking down endogenous ITG?-1 significantly suppressed NF-?B-mediated transcriptional activity and target gene expression.To further establish the role of ITG?-1 in the RACK1-mediated suppression of NF-?B signaling activation,AGS cells overexpressing RACK1 were transfected with an ITG?-1 plasmid and subsequently cocultured with Hp strain 43504.The suppressive effect of RACK1 on the NF-?B signaling pathway was rescued by ITG?-1 overexpression according to NF-?B reporter luciferase assay and qRT-PCR results.The expression of RACK1,ITG-?1 and some key molecules in the NF-?B signaling pathway in different pathology stages of gastric mucosa and its relationships with Hp infectionBy analyzing the different gene expression levels in Hp-positive and Hp-negative gastric cancer from the TCGA database,we found that there were no significant differences in RACK1 and ITG?-1 expression levels between the two groups.Consistent with this result,the RNA-sequencing data showed that the expression of RACK1 and ITG?-1 showed no differences among Hp-positive and Hp-negative gastric cancer.Subsequently,immunohistochemistry was performed to evaluate the expression of RACK1,ITG?-1 and p-P65(Ser536)in gastric tissues from various histopathologic stages of gastric carcinogenesis(chronic gastritis,intestinal metaplasia and dysplasia).RACK1 expression was lower in the Hp-positive groups than in the negative groups with intestinal metaplasia and dysplasia,whereas ITG?-1 was increased in in the Hp-positive groups than in the negative groups with intestinal metaplasia and dysplasia.Additionally,an increased level of p-P65(Ser536),indicating NF-?B activation,was observed in the Hp-positive group with chronic gastritis but not in the negative group.Taken together,our data indicated that Hp downregulated the expression of RACK1 and upregulated the expression of ITG?-1and p-P65(Ser 536)in vivo.Conclusions1.Hp infection could downregulate RACK1 expression and promote the activation of canonical NF-?B signaling pathway,which is independent of virulence factor CagA.2.RACK1 negatively regulate NF-?B signaling pathway through modulating ITG?-1 expression,which plays an essential role in Hp carcinogenesis. |
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