Study On The Mechanism Of MiR-874 Regulating Gastric Cancer Function By Targeting SPAG9

Background:Gastric cancer(GC)is one of the most common malignant tumors in the world.GC has a high incidence in China.Compared with developed countries such as Japan,our country has a large number of GC patients in the progressive or late stages.So the early diagnosis rate is low,while the mortality rate is high.GC is derived from dysplasia of gastric mucosal gland epithelial cells,which is affected by factors such as environment,diet,race,and genetic factors.Studies have shown that different types of genes in gastric mucosal cells are involved in the process of GC development,such as dysplasia,precancerous lesions under the stimulation of environmental pollution,poor diet and Helicobacter pylori infection,which are manifested as abnormal gene expression profiles.miRNA is a kind of endogenous single-stranded small molecule RNA of about 19-25 nucleotides in eukaryotes.It can negatively regulate target genes by binding to the 3’UTR of target gene mRNA,but has no function of coding protein.Studies have found that,compared with normal tissues,miRNAs in many tumor tissues are abnormally expressed.miRNAs can affect the formation,progression,metastasis,and prognosis of various human tumors by participating in the expression and regulation of tumor-promoting or tumor-suppressing genes.miR-874 is located on chromosome 5q31.2,which is reduced in multiple human malignancies.It is involved in tumorigenesis and development as a tumor suppressor miRNA.The previous research of this study conducted a gene chip of the miRNA expression spectrum of gastric adenocarcinoma tissue was studied,and found that miR-874 was significantly low-expressed in gastric adenocarcinoma tissues.However,the regulatory function and specific mechanism of miR-874 in the occurrence and development of GC have not been fully elucidated.Further in-depth studies are needed to evaluate the significance of miRNAs in the research on the mechanism and diagnosis and treatment value of GC.Objective:To study the differences between the expression of miR-874 in GC tissues or cell lines and in normal gastric mucosa or cells.To investigate the effect of miR-874 on malignant biological behaviors of GC cells.To predict and identify the functional target genes of miR-874 in GC,and to preliminarily explore the regulatory effect and mechanism of miR-874 on GC.Methods:(1)Real-time quantitative PCR(qRT-PCR)was used to detect the expression of miR-874 in GC tissues and paired adjacent normal tissues,GC cell lines(MKN-74,BGC-823,AGS and MKN-45)and GES-1.The correlation between the expression of miR-874 and the clinicopathological characteristics and prognosis of GC patients was analyzed.(2)Transient transfection was used to up-regulate and down-regulate the expression of miR-874 in GC cell lines,which was verified by qRT-PCR.The effects of miR-874 on the proliferation,apoptosis,migration and invasion of GC cells were detected by CCK-8 assay,plate clone formation assay,flow cytometry,Transwell assay as well as expression and functional rescue experiments.(3)A subcutaneous transplanted tumor model of human gastric cancer in nude mice was constructed.The effect of miR-874 on the growth of transplanted tumor was observed by measuring and counting the tumor volume and weight.TUNEL staining was used to detect the effect of miR-874 on the apoptosis of transplanted tumor tissue.(4)The potential target genes of miR-874 were first predicted using bioinformatics software,and then verified by experiments,including dual-luciferase reporter gene assay,qRT-PCR and Western blot detection.(5)The effects of miR-874 and target gene SPAG9 on the malignant biological behavior of GC cells were further verified by CCK-8 assay,clone formation assay,Transwell assay,and cell apoptosis assay.Meanwhile,Western blot was used to detect the expressions of effector proteins(JNK,p-JNK,c-Jun and MMP9)in JNK signal transduction pathway,and to explore the mechanism of miR-874 targeting SPAG9 in regulating GC.Results:(1)The results of qRT-PCR showed that the expression levels of miR-874 in 62 GC tissues were significantly lower than those in adjacent normal tissues(p<0.05).The expression levels in GC cell lines(MKN-74,BGC-823,AGS and MKN-45)were significantly lower than those in gastric mucosa epithelial cell lines GES-1(p<0.05).The expression of miR-874 was significantly correlated with GC tissue size,differentiation degree,Bormann type,TNM stage and lymphatic metastasis(p<0.05),but not with gender,age,tumor site,tissue type,invasion depth and distant metastasis(p>0.05).Compared with the patients with low miR-874 expression,the OS and DFS of patients with high miR-874 expression were significantly prolonged(P<0.05).(2)The results of CCK-8 assay and clone formation assay both showed that up-regulation of miR-874 could significantly inhibit the proliferation of GC cells(p<0.05),while down-regulation of miR-874 expression could significantly enhance the proliferation of GC cells(p<0.05).Flow cytometry results showed that compared with the control group,miR-874 mimics could significantly promote the apoptosis of GC cells(p<0.05),while miR-874 inhibitor could significantly inhibit the apoptosis of GC cells(p<0.05).The results of Transwell assay showed that compared with the control group,miR-874 mimics could significantly inhibit the migration and invasion of GC cells(p<0.05),while miR-874 inhibitor could significantly promote the migration and invasion of GC cells(p<0.05).(3)The results of the nude mouse tumorigenesis test showed that compared with the control group,the transplanted tumors in the miR-874 overexpression group grew slowly,the tumor volume and weight decreased,and the cell apoptosis was enhanced,while the growth of transplanted tumors in miR-874 low expression group was accelerated,the tumor volume and weight increased,and the cell apoptosis was weakened.The differences were statistically significant(p<0.05).(4)Bioinformatics software predicts SPAG9 as a predicted target gene of miR-874.The results of the dual luciferase reporter gene assay showed that miR-874 significantly inhibited the luciferase activity of the wild-type pGL3-SPAG9-WT(p<0.05),but had no significant effect on the luciferase activity of the mutant pGL3-SPAG9-Mut.qRT-PCR and Western blot results showed that miR-874 could negatively regulate the expression of SPAG9 mRNA and protein in AGS cells.(5)Functional experiments showed that SPAG9 can promote the proliferation,migration and invasion of GC cells and inhibit cell apoptosis,contrary to the effect of miR-874 on GC cells.The rescue experiment found that the inhibitory effect of miR-874 on GC could be partially offset by SPAG9.miR-874 regulated the expression of effector proteins(JNK,p-JNK,c-Jun and MMP9)of the JNK signaling pathway by targeting SPAG9.Conclusion:(1)The low expression of miR-874 in GC tissues and cell lines is significantly correlated with tumor size,degree of differentiation,Bormann classification,TNM stage,lymphatic metastasis and tumor prognosis.(2)Over-expression of miR-874 can inhibit the proliferation,migration and invasion of GC cells,inhibit the growth of graft tumor,and promote cell apoptosis.On the contrary,low expression of miR-874 can promote the proliferation,migration and invasion of GC cells,promote the growth of graft tumor,and inhibit cell apoptosis.It is suggested that miR-874 plays a tumor suppressive function in GC.(3)SPAG9 is a target gene regulated by miR-874 in GC.miR-874 inhibits JNK signaling pathway effector proteins(JNK,p-JNK,c-Jun and MMP9)by negatively regulating SPAG9 expression in GC cells,thereby inhibiting cell proliferation,migration and invasion,promoting cell apoptosis,and exerting tumor suppressor effect.