HDAC6/HNF4α Loop Mediated By MiR?1 Promotes Bile Acids?Induced Gastric Intestinal Metaplasia

[Background]Gastric cancer is the third lethal cancer in the world.According to Lauren’s classification,it is mainly divided into intestinal type and diffuse type,and the former is more common.At present,it is generally believed that the development of intestinal gastric cancer follows Correa model:Chronic superficial gastritis-atrophic gastritis-intestinal metaplasia（IM）-dysplasia-gastric cancer.Therefore,gastric cancer may be prevented by blocking the progression of precancerous lesions.Although many studies have found that IM increases the risk of gastric cancer,the initiation and development mechanism of IM is still unclear.It was reported that the degree and range of IM of gastric mucosa were more serious in patients with high concentration bile acid reflux.Hepatocyte nuclear factor-4α（HNF4α）plays an important role in maintaining normal differentiation,development and lipid metabolism of hepatocytes.It is worth noting that the researchers found that the molecule also showed a high expression in intestinal tissue,and participated in the development and function maintenance of the intestine.In our previous study,we found that bile acids（BA）significantly increased the level of HNF4α,and it could promote the expression of downstream intestinal markers through transcriptional activation of caudal-related homeobox transcription factor 2（CDX2）,thus inducing the transformation of gastric cells into intestinal type cells.However,the mechanism of BA-induced activation of HNF4αin gastric cells remains to be further explored.[Objectives]To clarify the activation pathway of HNF4αand further reveal the mechanism ofBA-induced gastric IM.[Methods]1.Using CRISPR/Cas9 technology to construct mice overexpressing HNF4αgene in Lgr5positive gastric stem cells.Hematoxylin-Eosin（HE）staining,electron microscopy and alcian blue-periodic acid-schiff stain（AB-PAS）staining were used to observe the changes of gastric mucosa.Immunohistochemistry（IHC）and immunofluorescence（IF）assays were used to detect the expression of pivotal molecules;2.After stimulation of gastric cells and primary gastric mucosal cells with deoxycholic acid（DCA）,quantitative real-time PCR（q RT-PCR）and western blot were conducted to examine the expression levels of histone deacetylase 6（HDAC6）,HNF4α,miR-1 and intestinal markers MUC2（MUCIN2）,KLF4（Krüppel-like factor 4）and CDX2;3.The expression levels of HDAC6,HNF4αand miR-1 were examined by IHC and in situ hybridization（ISH）assays in normal and gastric intestinal tissues.Ten paired gastric IM specimens were obtained from patients who underwent endoscopy.And total RNA was extracted from these tissues to analyze the expression levels of the above-mentioned molecules;4.The binding sequence between HNF4αand HDAC6 promoter region,as well as miR-1and HDAC6 and HNF4αwere predicted by JASPAR,PROMO and Target scan database,and identified by dual-luciferase reporter gene and chromatin immunoprecipitation（Ch IP）assays;5.The acetylation level of histone in forkhead box protein 3（FOXP3）promoter and CNS region,as well as the combination of HDAC6 and these two regions were detected by Ch IP experiment.[Results]1.Construction of HNF4αtransgenic miceThe gastric mucosa tissues of WT mice and Rosa26^Hnf4αmice were collected for IF staining to detect the expression of Lgr5 and HNF4α.The results showed that the specific expression of HNF4αappeared in the bottom region of gastric gland with Lgr5 positive in Rosa26^Hnf4αmice.Moreover,we also examined the expression of HNF4αin gastric mucosa of WT and Rosa26^Hnf4αmice by IHC staining.The results were consistent with those of IF staining.These results indicated that the mouse model was successfully constructed.The results of electron microscopy showed that the secretion of mucus in the gastric mucosal cells of Rosa26^Hnf4αmice was significantly increased.HE staining showed that there was atrophy in the gastric mucosa of Rosa26^Hnf4αmice.Next,to determine whether the overexpression of HNF4αin gastric mucosal cells can cause IM,the stomach,small intestine and large intestine of WT mice and Rosa26^Hnf4αmice was stained with AB-PAS.It can be seen that the intestinal tissue of the two groups of mice showed blue staining.The surface of gastric mucosa of WT mice was purplish red,and the glands were not stained.However,in Rosa26^Hnf4αmice,similar blue staining appeared at the bottom of the mucosa glands near the gastric antrum.2.HDAC6 increased in IM cellsWe performed RNA-seq to identify differentially expressed genes in gastric cells induced by DCA.The results showed that it could significantly increase HDAC6,and KEEG analysis showed that DCA was closely related to tumorigenesis and intracellular signal transduction.Next,GES-1 and AZ521 cells were treated with DCA at different concentrations（0,25,50,75 and 100μM）,respectively.After 24 hours,HNF4α,MUC2,KLF4 and CDX2 were detected.The results showed that DCA at 100μM could significantly increase these molecules.At the same time,we found that HDAC6 protein and messenger RNA（m RNA）levels were also significantly increased at this concentration.Then,we treated mouse primary gastric mucosal cells with BA and examined the expression levels of these molecules.The results were similar to those in human gastric cell lines.IF results reconfirmed that 100μM DCA could activate HDAC6 and HNF4αin GES-1 cells,and the former was mainly located in the cytoplasm,while the latter was mainly located in the nucleus.IHC staining of HDAC6 and HNF4αin normal gastric mucosa,gastritis and IM showed that the expression level of HDAC6 and HNF4αin IM was significantly higher than that in normal gastric mucosa and gastritis.Correlation analysis showed that HDAC6 and HNF4αwere positively correlated in IM.3.The interaction between HDAC6 and HNF4αHNF4αwas overexpressed or knocked down in GES-1 or AGS cells,respectively.Western blot results showed that HNF4αcould positively regulate HDAC6.And it was found that the decrease of HNF4αcould inhibit the activation of HDAC6 and downstream intestinal markers by BA in GES-1 cells.The results of dual luciferase reporter gene showed that HNF4αcould transcriptionally activate HDAC6 by binding to its promoter.In addition,IHC staining of Rosa26^Hnf4αmice showed that HDAC6 was upregulated in the HNF4αexpression region in gastric mucosa of transgenic mice compared with WT mice.Next,we changed the level of HDAC6 in gastric cells and found that it could promote the expression of downstream intestinal markers.Meanwhile,we observed that HDAC6could also activate HNF4α.Then,combined the results of RNA-seq,literature review and bioinformatics analysis,we speculated that FOXP3 may be an intermediate molecule between HDAC6 and HNF4α.Western blot and q RT-PCR analysis identified that DCA treatment caused the decrease of FOXP3 in GES-1 cells and mouse primary cells.Moreover,HDAC6 overexpression also induced the reduction of it.The results of IHC staining further revealed that FOXP3 is significantly higher in gastritis than that in IM.After detecting the acetylation level of histone,we found that HDAC6 could reduce the acetylation level of H3Ac,H3K9Ac,H3K27Ac and H4Ac in the promoter region and CNS region of FOXP3.Ch IP assay showed that HDAC6 could directly bind to the promoter region and CNS region of FOXP3.Dual luciferase reporter gene and Ch IP assays showed that FOXP3 could inhibit the transcription of HNF4α.4.miR-1 targeted HDAC6 and HNF4αFirst,the gastric cells and primary cells were treated with DCA.Compared with the control group,miR-1 in these cells decreased significantly.Then,the expression level of miR-1 in gastric mucosa was detected by ISH and q RT-PCR assays,it can be seen that miR-1 in IM tissue was significantly lower than that in normal gastric tissue.Next,western blot was used to examine the expression of HDAC6,HNF4αand downstream intestinals in gastric cells transfected with miR-1 agomir or antagomir.The results revealed that miR-1 could negatively regulate these proteins.According to mirwalk2.0（https://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/）,Pic Tar（https://picta r.mdc-berlin.de/）and Target scan（https//www.targetscan.org/vert＿72/）,we constructed wt-3’UTR and mut-3’UTR vectors of HDAC6 and HNF4α,respectively.Finally,the results of dual luciferase reporter gene demonstrated that miR-1 could specifically target the 3’UTR region of HDAC6 and HNF4αto inhibit their activity.And western blot was used to examine the regulation of miR-1 on HDAC6 and HNF4αprotein expression.5.DCA induced IM-like cells in gastric mucosa of Rosa26^Hnf4αmiceAfter 12 months of DCA or PBS treatment,the gastric mucosa tissues of the mice in all four groups were stained with HE.It was found that there were abnormally enlarged glands in the squamocolumnar junction（SCJ）of gastric mucosa in Rosa26^Hnf4αmice and Rosa26^Hnf4αmice treated with DCA.In addition,the antral mucosa of the two groups showed atrophy.The results of AB-PAS staining showed that the expression of HNF4αin gastric stem cells could promote the secretion of intestinal mucus in gastric mucosa.In addition,under the stimulation of DCA,the staining,morphology and size of some gastric mucosal cells of transgenic mice were highly similar to those of intestinal goblet cells.IHC staining showed that the expression of HNF4αin gastric mucosal stem cells could induce the appearance of HDAC6,while the increase of the latter inhibited the expression of FOXP3 in gastric cells,which further confirmed the regulatory relationship among HDAC6,FOXP3 and HNF4αin vivo.[Conclusion]In this study,we found that BA can significantly reduce the expression of miR-1 in gastric cells,which could target both HDAC6 and HNF4α,leading to the increase of them.Then,the enhancement of HDAC6 and HNF4αpromotes the expression of downstream intestinal markers.In addition,there is a mutual regulatory relationship between HNF4αand HDAC6.The former can activate HDAC6 transcriptionally,while the latter can affect the promoter activity of HNF4αby epigenetic regulation of FOXP3,and finally form a closed positive feedback loop.Therefore,BA promotes miR-1-mediated activation of HDAC6/HNF4αloop in gastric cells,and then lead to the increase of downstream intestinal markers and intestinal transformation of cells.Understanding the signal network of HNF4αin gastric cells will help us to further reveal the molecular mechanism of BA-induced gastric IM,and preventing the activation of HDAC6/HNF4αsignal loop may provide a new theoretical basis for intervening the occurrence and development of IM and even intestinal gastric cancer.