| Background: Hepatocellular carcinoma(HCC)is the third for most cause of cancer related deaths.The HCC development and progression is potentially linked with tumor growth and metastasis.The metastasis of tumor is the capacity and ability of malignant cells to move from originated place to around and sites,the origin place called primary tumor and the moved place called metastasis tumor.The tumor metastasis is one of the main challenges to cause the progression and spread of malignancy to it's around.The metastasis is very complicated that including many steps and factors.Migration,invasion and adhesion are the very important steps and properties of malignancy in which many RNAs,proteins and genes are involved.Micro R-124-3p(mi R-124)dysregulation is observed in HCC.As small non-coding RNAs(mi RNAs)are able to regulate many genes and proteins through direct binding to 3'UTR of protein coding genes.Next gene included in this study is Annexin-A11(ANXA11).ANXA11 is a member of calcium-dependent phospholipids-binding annexin family proteins,and a potential direct target of mi R-124.However the role of mi R-124 in HCC and effect of mi R-124 on ANXA11 in HCC have not been investigated.Objectives:1.To check and measure the expression level of mi R-124 and ANXA11 in HCC(HCC-LM3 and Huh7)cell lines and normal liver(LO2)cell lines.2.To study the effect of mi R-124 on ANXA11 in HCC-LM3 and Huh7 cells.3.To determine the effects of mi R-124 and ANXA11 on cell invasion and migration ability of HCC-LM3 and Huh7 cells.4.To examine the effect of level change of mi R-124 and ANXA11 on cell growth and proliferationMethods: A total 17 pairs of human HCC tissues samples with its adjacent normal were used to measure the mi R-124 expression level.Two human HCC cell lines,HCC-LM3 a high metastasis and Huh7 a low metastasis capacity and one normal liver cell lines LO2 were used in this study.First,all these three cell line were cultured in 10% fetal bovine serum(FBS)in DMEM and placed in humidified atmosphere with 5% CO2 at 37 oC for 24 h.Before any transfection the quantitative real time polymerase chain reaction(q-RT-PCR)was performed to measure the level of mi R-124 and ANXA11 in the two malignant cell lines compared with the normal cell lines.For the measurement protein level of ANXA11,Western Blotting assay was performed.For further investigation the malignant cells Huh7 and HCC-LM3 were transfected with mi R-124 mimic/NC to over-express mi R-124 or transfected with siANXA11/NC to knockdown ANXA11.To determine the over-expression of mi R-124,and the knockdown of ANXA11 on the cell proliferation,the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)method assay and soft after colony forming assay were performed.Transwell chamber assay was performed to investigate the expression level changes of mi R-124 and ANXA11 on the invasion and migration capacities of Huh7 and HCC-LM3 cells.Results: The mi R124 level was down-expressed in human HCC tissue samples than their corresponding matched normal liver samples(P=0.045).Compared with LO2,mi R-124 was down-regulated in HCC-LM3 95.8%(P=0.0001)and Huh7 94.9%(P=0.0001)cells.Mi R-124 mimic transfections resulted in the over-expressions of mi R-124 by 30 fold and 174 fold changes in HCC-LM3(P=0.002)and Huh7(P=0.0003)cells,respectively.The MTT assay result showed that mi R-124 significantly inhibited the cell's growth ability of HCC-LM3 at the time of 48 h 33.2%(P=0.0001),72 h 34.7%(P=0.0001),96 h 33.6%(P=0.0001)and 120 h 18.7%(P=0.0004)than mimic-NC.Over-expressed of mi R-124 decreased the Huh7 cells growth at different time intervals 72 h 24.8%(P=0.009),96 h 21.9%(P=0.0023)and 120 h 27.94%(P=0.0001).Mi R-124 mimic diminished the colony formation growths of HCC-LM3 50%(P= 0.0003)and Huh7 41.1%(P=0.004)cells,compared with mimic-NC,respectively.Transfected mi R-124 mimic suppressed the invasion 22.9%(P=0.0021)and migration 32.8%(P=0.0014)of HCC-LM cells.Over-expressed of mi R-124 significantly suppressed the invasion 33.3%(P=0.008)and migration 38.9%(P=0.004)capacity of Huh7 cells.Bioinformatics analysis indicated ANXA11 is a potential direct target of mi R-124.Compared with LO2 cells the m RNA and protein level of ANXA11 was up-regulated in HCC-LM3(P=0.031 and P=0.013)and Huh7(P=0.041 and P=0.032)cells,respectively.Over-expressed of mi R-124 decreased the ANXA11 m RNA level into Huh7 58%(P=0.00062)and HCC-LM3 65.7%(P=0.0001)cells.Furthermore,over-expressed of mi R-124 down-regulated the ANXA11 protein level after 48 h 15%(P=0.042)and 72 h 24%(P=0.026)in HCC-LM3 cells.Transfected of mi R-124 mimic into Huh7 cells,inhibited the expression of ANXA11 in protein level at 48 h 34%(P=0.016)and 72 h 21%(P=0.0162).The Si-ANXA11 transfection was used to knockdown the ANXA11 expression.MTT results indicated,Si-ANXA11 decreased the HCC-LM3 cell's growth after 48 h 18%(P=0.0005),72 h 17.4%(P=0.0001)and 120 h 14.1%(P=0.018),and as well in Huh7 cells at 96 h 20.8%(P=0.004).Furthermore,SiANXA11 resulted the inhibition of cell's colony formation growths of HCC-LM3 21%(P=0.0015)and Huh7 56%(P=0.028).Si-ANXA11 by down-regulating the ANXA11,induced the cell invasion 22.9%(P=0.0021)and migration 32.8%(P=0.00140)of HCC-LM3.In addition,Si-ANXA11 promotes the invasion 61%(P= 0.0068)and migration 65%(P=0.009)of Huh7 cells.Conclusion: The mi R-124 level was down-regulated in human HCC clinical samples than their adjacent normal tissues.Compared with normal liver LO2 cells,mi R-124 was downexpressed and ANXA11 was over-expressed in both HCC(Huh7 and HCC-LM3)cell lines.Mi R-124 over-expression decreased the in vitro proliferation,invasion and migration capacities of Huh7 and HCC-LM3 cells.Mi R-124 over-expression resulted in downexpression of ANXA11 in Huh7 and HCC-LM3 cells.Mi R-124 probably directly functions on ANXA11 in HCC carcinogenesis and progression.Their functions with the underlying mediation mechanisms in HCC deserve further investigation. 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