MiR-409-3p Regulates The Proliferation And Apoptosis Of THP-1 Through Targeting Rab10

Objective: Acute myeloid leukemia(AML)is a malignant hematological tumor with extremely poor prognosis.More and more micro RNAs,as important regulatory factors,are involved in the occurrence and development of tumors.The abnormal expression of micro RNA-409-3p(mi R-409-3p)is associated with a variety of tumors,including gastric cancer,breast cancer,colorectal cancer,ovarian cancer and cervical cancer.Nevertheless,its mechanism of action in AML has not been reported.Therefore,this article explores the biological effects and mechanisms of mi R-409-3p in AML,providing molecular targets for the treatment of AML.Methods:(1)The expression of mi R-409-3p and Rab10 in human myeloid leukemia mononuclear cells(THP-1)and human bone marrow stromal cells(HS-5)was detected by real-time quantitative PCR(RT-q PCR);(2)Design and synthesize mi R-409-3p analogues(agomi R-409-3p and agomi R-409-3p NC)and Rab10 small interference analogues(Si-Rab10 and Si-control),transfect the above compounds into THP-1 cells by Lipofectamine3000 liposome transfection method,and detect the transfection efficiency by RT-q PCR;(3)Cell counting kit(CCK-8)was used to detect the effect of mi R-409-3p overexpression or Rab10 knockdown on THP-1 cell proliferation;(4)Annexin V-FITC/PI method was used to detect the overexpression of mi R-409-3p and the effect of Rab10 knockdown on THP-1 proliferation and apoptosis;(5)The apoptosis-related proteins Bax and Bcl-2 were detected by Western blot after mi R-409-3p overexpression or Rab10 knockdown;(6)Bioinformatics method was used to predict the binding ability of mi R-409-3p and Rab10 m RNA.The targeted binding site between mi R-409-3p and Rab10 3’UTR were further verified by the dual Dual-Luciferase Reporter assay.RT-q PCR and Western blot analysis were used to determine the expression of Rab10 m RNA and protein following mi R-409-3p overexpression;(7)The THP-1 cell with stable mi R-409-3p was constructed and subcutaneously injected into SCID-Beige mice to observe the effect of mi R-409-3p on the transplanted tumor of mice;(8)HE(Hematoxylin-eosin)staining was used to detect the degree of tissue inflammation and necrosis after overexpression of mi R-409-3p;(9)Immunohistochemistry(IHC)was used to detect the changes of Ki-67 in Rab10 after overexpression of mi R-409-3p.Results:(1)RT-q PCR results showed that compared with HS-5,mi R-409-3p in THP-1 was significantly down-regulated,while Rab10 was significantly up-regulated(P<0.05);(2)RT-q PCR confirmed that mi R-409-3p overexpression and Rab knockdown had good transfection effect(P<0.05);(3)The cell function test showed that the overexpression of mi R-409-3p or the knockdown of Rab10 could inhibit the proliferation and apoptosis of leukemia cell line THP-1 compared with the respective control group(P<0.05);(4)Western blot results showed that overexpression of mi R-409-3p or knockdown of Rab10 could up-regulate the expression of apoptosis-related protein Bax,while the expression of Bcl-2 was down-regulated(P<0.05);(5)Bioinformatics prediction showed that there was a possible binding site between mi R-409-3p and Rab10 3’UTR region.RT-q PCR and Western blot results showed that the expression of Rab10 m RNA and protein decreased after mi R-409-3p overexpression was transfected.The double luciferase reporter gene experiment showed that the relative luciferase activity of Rab10-WT+agomi R-409-3p co-transfection group was lower than that of other groups(P<0.05);(6)The tumor-bearing experiment in mice showed that the tumor volume and weight of SCID-Beige mice with stable overexpression of mi R-409-3p were sharply lower than those of the control group(P<0.05);(7)HE results showed that the degree of inflammatory proliferation and necrosis in the overexpression of mi R-409-3p group decreased compared with the control group(P<0.05);(8)The immunohistochemical results showed that the positive degree of Rab10 and Ki-67 in the overexpression mi R-409-3p group was obviously lower than that in the control group(P<0.05).Conclusion: Mi R-409-3p inhibited cell proliferation,induced apoptosis,and promoted the development of AML by downregulating Rab10,providing potential molecular targets for the treatment of AML.