Role Of Androgen On Proliferation And Differentiation Of Small Intestine Stem Cells By Acting On Stromal Cell And Its Underlying Mechanisms

BackgroundColorectal cancer(CRC)is one of the most commonly cancers worldwide.In the past three decades,molecular genetic studies have revealed that the pathogenesis of sporadic and hereditary colorectal cancer is due to the existence of certain genetic changes,including the loss of some functional tumor suppressors and the activation of oncogenes caused by mutations.Colorectal cancer is one of the leading causes of cancer-related death and development.Relevant literature indicates that specific genetic changes in certain sequences are the basis for the development of adenomas to colorectal cancer.In most cases,activation of the Wnt signaling pathway will cause APC inactivating mutation,which eventually initiates the formation of polyps.However it was thought to be caused by the inactivating mutation of P53 and the activation of the transforming growth factor-?(TGF-?)signaling pathway latterly.Studies have shown that men are going to develop colon adenomas and cancers at an earlier age.However,the molecular mechanism of this gender difference remains unclear.Recent articles have shown that the development of colon cancer may be related to gender,suggesting that it may be related to sex hormones.Estrogen inhibits the development of colon cancer,yet androgen have the opposite effect,which can promote the development of colon adenoma.Dihydrotestosterone(DHT),a major active form of androgen in the body,has been reported that it can promote the adenoma development in AOM-induced colon cancer mice.Moreover,a small amount of DHT receptor(Androgen receptor,AR)can be detected in intestinal epithelial mesenchyme stromal cells.However,there is no data indicating that DHT can affect the proliferation and differentiation of intestinal epithelium,and the related mechanisms are still unclear.ObjectivesTo explore the role of androgen in the regulation of proliferation and differentiation of intestinal epithelial stem cells by mesenchymal cells,revealing the role of androgen in the maintenance of intestinal homeostasis and its mechanism.Material and Methods1.Cell culture1.1 Stromal cell cultureStromal cells extracted from the small intestine were cultured in RPMI1640 medium containing 10%FBS,1%PS and 1%Glutamax,and Stromal cells were mixed with androgen for 4h,8h,12h,24h,and Stromal cells were cultured for 4-5 days.It can be passed down.1.2 Co-culture of crypt and Stromal cellsCrypt is co-cultured with stromal cells.The crypt cells are extracted first,and the remaining intestines are digested for 3 hours to obtain stromal cells.The two cells are mixed firstly,and then mixed them with the pre-melted Matrigel,and seeded in a preheated 48-well plate.Divided them into 4 groups,Crypt group,Crypt+DHT group,Crypt+Stromal group,crypt+Stromal+DHT group,with DMEM F-12 medium containing N2,B27,Glutamax,NAC,PS,EGF,Nogging,R-Sonding.They were cultured for 4h,8h,24h,and 7day,respectively,and photographed every day.2.Animal model2.1 Male castration model(ORX model)Dividing 6-8-week-old male C57 mice into Control group,ORX group,ORX +T group randomly.After anesthetizing mice with 2%pentobarbital,ORX,ORX+T Groups underwent testicular castration surgery and the control group underwent sham surgery.The ORX+T group was intraperitoneally injected with dihydrotestosterone at 0.53 mg/kg/day.The mice in the Control group and the ORX group were intraperitoneally injected with normal saline for 21 days.All mice were housed under the same conditions,and different groups were housed in separate cages.2.2 Female castration model(OVX model)Dividing 6-8-week-old female C57 mice into Control group,OVX group,OVX+ T group randomly.After anesthetizing mice with 2%pentobarbital,OVX and OVX+T underwent ovarian castration surgery and the Control group underwent sham surgery.The OVX+T group was intraperitoneally injected with dihydrotestosterone at 0.53 mg/kg/day.The mice in the Control group and the OVX group were intraperitoneally injected with normal saline for 21 days.All mice were housed under the same conditions,and different groups were housed in separate cages.2.3 Androgen receptor(AR)inhibition modelDividing 6-8-week-old male C57 mice into Control group and Flutamine group.After anesthetizing mice with 2%pentobarbital,the Flutamine group was intraperitoneally injected with 10 mg/kg 2-hydroxyflutamide,Control group intraperitoneal injection of normal saline for14 days.All mice were housed under the same conditions,and different groups were housed in separate cages.2.4 BMP pathway inhibition modelDividing 6-8-week-old male C57 mice into Control group,ORX group,ORX +inhibitor group randomly.After anesthetizing mice with 2%pentobarbital,The ORX and ORX+ inhibitor groups underwent testicular castration surgery,and the Control group underwent sham surgery.ORX + inhibitor group intraperitoneal injection 3mg/kg of BMP inhibitor,LDN193189.The control group mice and the ORX group were intraperitoneally injected with normal saline.All mice were housed under the same conditions,and different groups were housed in separate cages.3.Protein extractionMixed the small intestine tissues with the lysate.After thorough homogenization,the cells were centrifuged at 12,000 rpm for 30 min at 4° C,and collected the supernatant.The protein extract was measured by a kit,then added the loading buffer,mixed,and denatured in a metal bath at 100° C for 7-8 mim.4.Western blotA certain amount of the protein was subjected to SDS-PAGE gel electrophoresis,transfer,blocking,and development.5.Total RNA extraction5.1 TissueMixed the small intestine tissues with RNA fixer firstly,secondly put the tissue at 4 ° C overnight,and then extracting RNA by a kit.After measuring the RNA concentration,RNA was reverse-transcribed to obtain cDNA.5.2 Cel sThe stromal cells were cultured at different times,and removed the medium,using trypsinization and centrifuged to obtain precipitate.The cells co-cultured with Crypt and Stromal were extracted with cell recovery solution at dififerent time points,and the precipitate was collected by centrifugation.Added the lysate to the precipitate and lysed them on ice for 30 minutes,and RNA was extracted using an RNA extraction kit.After measuring the RNA concentration,RNA was reverse-transcribed to obtain cDNA.6.Real-time fluorescence quantitative PCR(qPCR)Real-time quantitative PCR was used to analyze the expression levels of Muc2,Mmp7,Cha,Klf4 and other mRNA expression levels of Wnt,BMP,Notch pathway in stromal cells and crypt+stromal cells.7.Staining analysisThe mice were euthanized to obtain intestinal tissue,and then the tissues were fixed,dehydrated,embedded,and sectioned for Brdu,ChA,Lysozyme staining analysis and Periodic Acid-Schiff(PAS)staining.8.Statistical analysisThe data obtained from at least three or more independent experiments.GraphPad was used to statistical analysis through t test or one-way ANOVA.P<0.05 indicates that the results have statistically significant.Results1.The histochemical results showed that the expression of three intestinal secretory cells were increased,and the addition of androgen dihydrotestosterone(DHT)could reverse this phenomenon.The results of AR model and female castration model further confirmed that androgen may inhibit intestinal secretory cells.2.qPCR detected the expression of Klf4,Elf3,Sox9,Ngn3,Atohl and Hesl in male mice were increased after castration.After adding androgen,this phenomenon was reversed.It indicates that Androgen may inhibit the expression of determinants related to secretory cell differentiation,which eventually causes the abnormal expression of intestinal secretory cells.3.After male castration,the number of Brdu+ cells decreased,the expression of active ?-catenin protein was down-regulated,and the expression of Bmp4 and Bmp type I receptor A(BmpRIA)was detected to be up-regulated.This phenomenon was reversed after adding androgen.4.There were almost no AR-positive cells in the intestinal epithelial crypt,however a certain number of AR were expressed in the mesenchyme stromal cell.There was no difference in the survival rate of the 4 groups.Androgen had no effect on the crypts when cultured separately.The increase of crypts' proliferation and expansion can only be observed when cytoplasmic cells are co-cultured.5.The expression of Wnt4,Wnt5a,Wnt5b and Wnt2b was not significantly changed in stromal cell when cultured separately.However,the expression of Wnt antagonistic signals Dkk2,Dkk3 and Sfrpl was significantly decreased after DHT treatment,at same time,Bmp4,Tgfbl decreased obviously.We also observed that,in the co-cultured cells,the expression of downstream genes in Tgf-? signaling pathway and BMP target genes was reduced after DHT treatment.6.In the BMP pathway inhibition model,the expression of Msxl and Idl was up-regulated after male castration.it was found that Bmp signaling pathway inhibitors and androgens have similar effects in the proliferation and differentiation of intestinal stem cells in male ovariectomized mice.Accordingly,the expression of three intestinal secretory cells increases after castration,and the BMP pathway inhibitor LDN193189 can reverse this phenomenon.ConclusionsWe found that intestinal Stromal cells express AR and activate AR to promote intestinal crypt stem cell proliferation and inhibit the differentiation of secretory cells.This change is achieved by affecting the BMP signaling pathway.Androgen stimulates AR to participate in the production of BMP inhibitors,which leads to inhibition of the BMP signaling pathway and thereby promoting intestinal stem cell proliferation.Our findings provide a reasonable explanation for the high incidence of colorectal cancer in men.