Molecular Mechanism Of DOT1L Regulating Gastric Cancer Cell Proliferation

Objective: To analyze the effect of DOT1 L gene expression difference between gastric cancer(GC)tissues and adjacent tissues on the survival rate of gastric cancer patients by bioinformatics,and to predict the possible proliferation signal pathway of gastric cancer cells,and to observe the changes of proliferation ability of gastric cancer cell lines MGC-803 and SGC-7901 after DOT1 L gene silencing and over-expression objective to investigate whether MAPK signaling pathway regulates the progression of human gastric cancer and its possible mechanism.Methods:(1)Download The gene expression data and clinical data of gastric Cancer from The Cancer Genome Atlas.After analyzing the data of 375 cases of gastric cancer and 32 cases of cancer adjacent,using R language 5678 pairs of differential genes(DEGs)were selected and annotated with go function.Kaplan-Meier was used to analyze the survival prognosis of patients.KEGG pathway in KEGG database was used to analyze the online signal pathway of differential genes,and GSEA software(gene set enrichment)was used Analysis can enrich the genome of gastric cancer patients and predict the possible signal pathway of gastric cancer proliferation.(2)The protein expression levels of DOT1 L gene in gastric cancer cell lines MGC-803,BGC-823,SGC-7901,MKN-45 and AGS were detected by Western blotting.The gastric cancer cell lines with high expression of DOT1 L gene protein were selected as follow-up research objects and set as control group(NC).(3)According to the target gene DOT1 L sh RNA was designed.The viral vector p LKO.1puro-DOT1L-sh RNA was constructed and transfected into gastric cancer cell lines MGC-803 and SGC-7901.(4)Real time PCR was used to detect the silence group expression of DOT1 L m RNA in MGC-803 and SGC-7901 gastric cancer cell lines.(5)The colony forming ability of cancer cells in the silence group and the control group was observed under the microscope by colony cloning experiment.(6)In rescue experiment,the plasmid containing DOT1 L gene was constructed and transfected into gastric cancer cell line of silencing group,which was set as over-expression group(oe-DOT1L).(7)Western blotting was used to detect the expression of DOT1 L protein,Erk-p,p-p38 MAPK and Ki-67 in blank group,silencing group and over-expression group.(8)CCK-8 method was used to detect the changes of cell proliferation in control group,silence group and over-expression group.Results:(1)Compared with the adjacent tissues,the expression level of DOT1 LL gene in gastric cancer tissue was significantly increased(P<0.05).The overall survival rate(OS)of patients in the high expression group of DOT1 L in gastric cancer tissue was significantly reduced by Kaplan-Meier analysis(P<0.05).(2)The results of GSEA enrichment analysis showed that MAPK＿SIGNALING＿Path way signal pathway is enriched significantlyn in DOT1 L gene high expression group(NES:1.59,P-value:0.029),KEGG pathway enrichment analyzing results showed MAPK＿SIGNALING＿Path way signal pathway was significantly correlated in DOT1 L gene high expression(P<0.05).(3)Compared with AGS gastric cancer,the expression level of DOT1 L protein in other four gastric cancer cells was higher,among which MGC-803 and SGC-7901 were the highest.Therefore,MGC-803 and SGC-7901 gastric cancer cells were selected for follow-up experiments to observe the changes of their proliferation ability.(4)The silencing group was successfully constructed by silencing the DOT1 L gene of MGC-803 and SGC-7901 gastric cancer cell line.(5)Compared with the control group,the expression of DOT1 L gene and protein in gastric cell line in silencing group was significantly reduced(P<0.05).(6)Compared with the control group,the proliferation of colony clone in gastric cell line in silencing group was significantly decreased(P<0.05).(7)Compared with the control group,the expression of MAPK signaling pathway regulatory proteins Erk-p and p-p38 MAPK increased,Ki-67 protein expression decreased(P<0.05),OD450 decreased significantly(P<0.05),and the proliferation of cancer cells was significantly reduced.(8)In rescue experiment: compared with the silencing group,the expression levels of Erk-p and p-p38 MAPK,Ki-67 and OD450 in the over-expression group were significantly decreased(P<0.05),and the proliferation ability of cancer cells was significantly restored(P<0.05).Conclusion: DOT1 L maybe an important factor affecting the survival of gastric cancer patients.DOT1 L gene may regulate the proliferation of gastric cancer through MAPK signaling pathway.These suggest that DOT1 L plays an important role in the occurrence and development of human gastric cancer,which provides a new target for clinical work.