MiR-30a-Dbf4 Target Regulates Gastric Cancer Cell Proliferation,Migration And Its Sensitivity To 5-Fu

Background:Gastric cancer is the most common malignant tumor of the digestive system.Although research on the diagnosis and treatment of gastric cancer continues to increase,its morbidity and mortality are still high at present.The occurrence of gastric cancer involves many factors.Helicobacter pylori,unhealthy diet,alcohol abuse,gene mutations,etc.are closely related to the occurrence of gastric cancer.Among them,pathogenic Helicobacter pylori infection plays an important part in the pathogenesis of gastric cancer.The diagnosis of gastric cancer generally relies on clinical symptoms,endoscopy,histopathology and imaging examinations.The treatment of gastric cancer includes early surgery,radiotherapy and chemotherapy,and targeted therapy.At present,the exact mechanism of gastric cancer is still not fully understood.Abnormal gene expression and gene mutation are closely related to the occurrence of gastric cancer.Exploring the genetic changes and signal transduction pathways of gastric cancer cells at the molecular level contributes to diagnosis and targeted therapy of gastric cancer.MicroRNA（miRNA）is a type of highly conserved endogenous non-coding small RNA,containing 18～25 nucleotides.In the process of protein synthesis,miRNA is mainly used to specifically bind to the 3’-UTR of target mRNA through RNA polymerase Ⅱ to regulate the transcription of target genes,and to influence protein expression by regulating the translation of target gene mRNA.A large number of studies have shown that miRNA is involved in the occurrence and development of a variety of cancers.Research on the pathogenesis of gastric cancer and formulating effective treatment plans for miRNA are one of the important directions of current gastric cancer research.During the pathogenesis of tumors,different miRNAs play different functions of tumor suppressor genes or oncogenes.Since miRNA is dysregulated in most tumor cells,it is considered to be a biomarker associated with almost all tumors.miR-30a is an important member of the miRNA family and belongs to intron miRNAs,which can participate in the regulation of tumor cell proliferation,invasion and migration by inhibiting the growth of tumor cells.There are few research reports on miR-30a in gastric cancer,and the existing data are not enough to prove the detailed mechanism of its expression and regulation in the proliferation and migration of gastric cancer cells.Dbf4 is the regulatory activation subunit of cell division cycle factor 7（CDC7）and a key checkpoint for the G1/S transition.It plays an important role in initiating DNA replication and damage tolerance.In the past 10 years,the CDC7-Dbf4 complex has become a potential new biological therapeutic target.We aim to figure out whether miR-30a can inhibit the proliferation and migration of gastric cancer cells by targeting Dbf4 mRNA and affect the sensitivity of 5-fluorouracil（5-FU）through the miR-30a-Dbf4 pathway,the effect of lactic acid accumulation on miR-30a in tumor microenvironment and the detailed regulation effect and mechanism of miR-30a on Dbf4.Objective:Gastric cancer shows obvious infiltration,proliferation,and migration.It is necessary to determine the potential pathogenic molecular mechanism of gastric cancer.This study inhibits the proliferation and migration of gastric cancer cells through the miR-30a-Dbf4 pathway and aims to clarify the functional significance and molecular mechanism of miR-30a、Dbf4 in order to develop effective targeted therapy ideas.Method:SiRNA was transfected into human gastric cancer cell lines MGC-803 and AGS,and qRT-PCR was used to detect the relative expression of Dbf4 and miR-30a in peripheral blood and gastric cancer tissues;Western blot analyze Dbf4 expression at the protein level;Tarbase Biology Information database predicts the interaction between miR-30a and Dbf4;dual luciferase reporter gene assesses whether miR-30a directly targets Dbf4;CCK-8 and colony formation analysis are used to detect the effect of Dbf4 on the proliferation of gastric cancer cells;the transfection efficiency was verified by qRT-PCR and Western blot;the effect of Dbf4 on the migration ability of gastric cancer cell lines was further determined by transwell assay and wound healing assay;Rescue experiment was used to determine whether miR-30a plays the role of tumor suppressor gene by regulating Dbf4;Dbf4 was overexpressed in vitro on the basis of MGC-803 and AGS cells by plasmid transfection,and Dbf4 siRNA（siDbf4）was used to inhibit the expression of Dbf4 in MGC-803 and AGS cells.The cells were treated with 5-Fu and its proliferation was observed.Result:1.Compared with adjacent tissue cells,Dbf4 in gastric cancer cells was significantly increased.2.Compared with the control group,the proliferation of gastric cancer cells in the siDbf4 group was reduced（P<0.01）.In gastric cancer cells transfected with siDbf4,the expression of PCNA and cyclin-A also decreased.By Western blot detection,the expression of PCNA and cyclin-A increased in the gastric cancer cells with over-expressed Dbf4.3.Dbf4 weakened the sensitivity of MGC-803 and AGS cells to 5-FU.4.Silencing Dbf4 inhibited the migration of two gastric cancer cells;overexpression of Dbf4 restored the migration ability of MGC-803 and AGS cells.5.The expression of miR-30a in gastric cancer tissues was significantly reduced,and the expression levels of miR-30a and Dbf4 were negatively correlated in gastric cancer tissues（r=-0.4271,P<0.0001）.In gastric cancer cells transfected with miR-30a mimics,the expression of Dbf4 was significantly down-regulated（all P<0.01）.Overexpression of miR-30a significantly reduced the activity of wild-type Dbf4 promoter in gastric cancer cells and inhibited the expression of Dbf4.6.After transfection with miR-30a mimic,MGC-803 and AGS cell lines showed a significant reduction in proliferation.qRT-PCR analysis showed that the expression of Dbf4 in the miR-30a analog group was down-regulated,while the expression of Dbf4 in the Dbf4 overexpression group was up-regulated.CCK-8 and colony formation analysis showed that overexpression of DBR4 eliminated the inhibitory effect of miR-30a overexpression on cell proliferation.When Dbf4 was overexpressed,the inhibitory effect of miR-30a overexpression on cell migration was offset.7.Lactic acid inhibited the expression of miR-30a,while IL-6 showed only a weak effect on the expression of miR-30a.Conclusion:1.Dbf4 is a key tumor-promoting factor in gastric cancer,and the up-regulation of Dbf4 increases the proliferation of gastric cancer cells.2.The accumulation of lactic acid in the tumor microenvironment can induce an increase in the expression of Dbf4,while inhibiting the expression of miR-30a.3.The miR-30a-Dbf4 pathway can inhibit the proliferation and migration of gastric cancer cells and increase the sensitivity to 5-Fu.Dbf4 can be used as a potential target for the treatment of gastric cancer.