The Regulation Mechanism Of LSD1 Engages In Gastric Cancer Metastasis By Estrogen And Androgen Receptor Signals

Histone lysine specific demethylase LSD1 is the first lysine specific demethylase identified by professor Shi Yang group in 2004 and proved that it was high expressed in many cancer cells.Estrogen Receptor α and Androgen Receptor were researched as hormone receptors related to theirs target genes transcription research mainly focused on target organs,such as breast and prostate cancer and so on.It has been indicated that E2 can activate ERα serve as the ligand’s of ERα,DHT can activate AR serve as the ligand’s of AR receptor and activate the downstream target genes.In the prophase research experimental results indicated an unexpected discovery that LSD1 in gastric cancer cell line MGC 803 for the metastasis of females and males rats produced a different effect.Therefore,the purpose of this paper is to study whether LSD1 mediated ERα and AR signal in gastric cancer metastasis.(1)The correlation expression of LSD1 and ERα with AR in gastric cancer tissuesThere have been many studies reported that LSD1 has a high expression in various cancers including gastric cancer,prostate cancer,breast cancer,lung cancer and other malignant tumors,at the same time,high expression of LSD1 in the cancer patients shows the low cure after the effect,and the incidence of gastric cancer in men and women and in different gender.Through the above phenomenon and the conclusion,we select 146 cases of different gender patients with tumor and cancer adjacent tissues for our immunohistochemical analysis and Pearson analysis test.we found that in gastric cancer tissues,the LSD1 and AR proteins expression level in cancerous tissue are generally higher than that of tissue adjacent to carcinoma,but for ERα,the expression of ERα level is higher in the tissue adjacent to carcinoma.and for LSD1 and ERα,there may be a negative regulation in gastric cancer cells.(2)Explore the impact of E2 and DHT to the expression of LSD1 in two kinds of gastric cancer cellsIn the topic of prophase work,there may be a negative regulation between LSD1 and ERα and that LSD1 can serve as the members of activated complex associated with ERα and AR and it can regulate the downstream target genes of transcription activation.E2 as ERα receptor ligand can activate ERα,DHT as AR receptor ligand and activate the downstream target genes.Therefore,this section is intended to study the influence of E2,DHT to LSD1 and explore wthether there has a relationship between the ERα with LSD1.By treating different concentrations of E2 in MGC 803 cells,after 48 hours using Western blot to detect the LSD1 expression capacity in MGC 803 cells,we can find that E2 can decrease the expression of LSD1,but when treating MPP,the inhibitor of ERα,MPP can inhibit this effect,indicating that E2 regulated the expression of ERα through LSD1.the same result was also validated in SGC 7901 cell lines.By treating different concentrations of DHT in MGC 803 cells,using Western blot to detect the LSD1 expression capacity in MGC 803 cells,we can find that DHT can decrease the expression of LSD1,but when treating BIC,the inhibitor of AR,BIC can inhibit this effect,indicating that DHT regulated the expression of AR through LSD1.We can find the same result in SGC 7901 cell lines.(3)E2 and DHT regulate the gastric cell in proliferation,invasion and metastasis through the corresponding hormone receptors.We use the MGC 803 LSD1 knockdown cells,MGC 803 LSD1 overexpression cells and MGC 803 cells to detect the proliferation of different cell lines with different concentrations of E2 and DHT,we can found that when treating with E2 and DHT,it can not be influenced of proliferation in different cell lines,even LSD1 was knockdown or overexpression.The same results were detected in the SGC 7901 cells,the MKN 45 cells and the MKN 7 cells.We use the wound healing assay to detect the invasion of treating E2 or DHT in different cells.As we can find,E2 can inhibit the invasion in MGC 803 cells,SGC 7901 cells,MKN 45 cells and MKN 7 cells comparing with control cells,DHT can induce the invasion in these cells comparing with control cells.In these four gastric cancer cells when treating in a certain concentration of E2 and we used the transwell experiment to detect the migration of cells.The results demonstrated that E2 can inhibit the migration of cells,but after treating the ERα inhibitor MPP after E2 for transwell experimental,we can find that MPP can inhibit the effect of E2 of decreasing the migration of cells,but when treating with DHT,there is a opposite result in these cells.The levels of E-cadherin and N-cadherin in MGC-803 cells were detected after treating E2 by immunofluorescence assay,and we found that E2 can inhibit the expression of N-Cadherin and increase the expression of E-Cadherin.We use the EMT proteins markers to detect the expression of EMT antibodies.The levels of proteins associated with epithelial cell and proteins associated with mesenchymal cell in MGC 803 cells,SGC 7901 cells,MKN 45 cells and MKN 7cells were detected by Western Blot assay,we found that E2 can inhibit the expression of EMT,DHT can increase expression of EMT.(4)the regulation mechanism of E2 regulate the ERα and LSD1In many articles that LSD1 can be used as members of ERα and AR activated complexs and it can regulate the transcription activation of downstream target genes of the ERα and AR.In adding the certain concentration of E2 in MGC 803 cells by the immune co-precipitation(IP)assay and immunofluorescence(IF)assay,we found that E2 can increase the combination of LSD1 and ERα,the same results were detected in SGC 7901 cells MKN 45 cells and MKN 7 cells.In MGC 803 cells we used liposome 2000(Lippfectamine ? 2000,Lipo2000)and liposome 3000(Lippfectamine ? 3000,Lipo3000)to build LSD1 siRNA and Firefly Luciferase plasmid ERE-Luciferase and Renilla Luciferase plasmid SV-40 treated into cells,treating with different concentrations of E2 to measure the expression of ERE transcription.we found that E2 increase the activation ERE transcription but the ERE transcription activity was suppressesed after LSD1 was knockdown.The same results were decected in SGC 7901,MKN 45 and MKN7 cells.We select MGC 803 cells and MGC 803 LSD1 KD cells to extract total RNA for RT-qPCR assay,the result indicated that E2 activated the mRNA levels of transcription for the genes activation of transcription complexs of ERα and LSD1,but after LSD1 was knockdown the mRNA levels were inhibited.It demonstrate that LSD1 influenced the mRNA levels of transcription for the genes activation of transcription complex of ERα and LSD1.In the MGC 803 cells,LSD1 IP complex were used for the influence of substrate enzyme activity.The result shows that the increase of histone demethylation H3K9me2 protein expression,indicating that the complexs of LSD1 and ERα significantly inhibited the activity of LSD1,after treating with E2,E2 can increase the combination of LSD1 and ERα,and increase the inhibition of the activity of LSD1 decreased by complex of LSD1 and ERα.