MiR-548b-3p Inhibits Malignant Biological Behavior Of Hepatocellular Carcinoma Through Targeting CIP2A

Objective:The mortality rate induced by primary hepatocellular carcimona is always high worldwide.China is a hepatocellular carcimona?HCC?area with high incidence,and 50 percent of new hepatocellular carcimona patients were diagnosed in China annually^[1-3].The occurrence and development of hepatocellular carcimona is a multi-factor and multi-step complex process.To explore the relationship between gene expression and hepatocellular carcimona is still a hot issue,which contributes to seek effective diagnostic markers and therapeutic targets of HCC research.CIP2A?cancerous inhibitor of protein phosphatase 2A?was initially recognized as an oncoprotein,which was an important endogenous inhibitor of PP2A.CIP2A interacted directly with the oncogenic transcriptionp factor MYC,inhibited PP2A activity toward MYC serine 62?S62?,and thereby prevented MYC roteolytic degradation.An increased expression of CIP2A has been detected in multiple malignancies,such as prostate cancer,breast cancer,lung cancer,gastric cancer and head and neck squamous cell carcinomas^[4-7].MicroRNAs are a class of non-encoded single-stranded small RNAs encoded by endogenous genes,whose length are about20-25 nucleotides.The function of miRNAs are to induce mRNA degradation or inhibit mRNA translation through binding to 3'UTR^[8].miRNAs are involved in regulating various physiological processes,such as early cell development,cell differentiation,cell proliferation,apoptosis and metabolism^[9].Recently,a large number of studies have confirmed that the abnormal expression of miRNAs is closely related to the occurrence and development of a variety of tumors,including hepatocellular carcinoma.miRNAs regulate the processes of tumor cell differentiation,proliferation,apoptosis,migration and invasion^[10].The possible upstream microRNAs of CIP2A was predicted through software.Its expression in hepatocellular carcinoma tissues and biological role were further explored.This sudy was aim to clarify the regulatory network of CIP2A and its upstream miRNA in hepatocellular carcinoma,which may provide new ideas for clinical diagnosis and treatment for hepatocellular carcinoma.Methods:1 Samples80 cases of hepatocellular carcinoma tissue and 10 cases of normal liver tissue were taken from patients who dianosed as hepatocellular carcinoma between September2015 and September 2017 in the first affiliated hospital of China medical university.2 ImmunohistochemistrySamples were buried with paraffin and the slices were prepared.Slices were dewaxed,hydrated,antigen repaired,non-specific closure and antibody incubated.Slices were incubated with primary antibody?CIP2A?at 4?for the night.Secondary antibody was incubated at 37?for 30 minutes.After the antibodies incubation,the DAB kit was used for dyeing,and hematoxylin stain solution was used for redyeing.3 Cell cultureHL7702?L-02?BEL-7402 and HUH7 cell lines were purchased from Typical Culture Preservation Committee of Chinese Academy of Sciences.SK-HEP-1 cell line was purchased from American Typical Culture Center.The medium were DMED and RPMI-1640 medium containing 10%fetal bovine serum.The culture conditions are37?,5%CO2 and saturated humidity.4 Cell transfection and interferenceCIP2A plasmid and empty plasmid were purchased from Origene.CIP2A siRNA and non-targeting siRNA were purchased from Dharmacon.miR-548b-3p mimic,mimic control,miR-548b-3p inhibitor and inhibitor control were purchased from Ribobio.Lipofectamine 2000 and Dharmafect1 were purchased from Thermo and they are used according to the manufactures?instruction.5 Western blotTotal protein was extracted using RIPR cell lysate.The antibodies used in this experiment were one CIP2A,c-myc,bcl-2,cleaved-PARP,actin and horseradish peroxidase coupled secondary antibodies.Samples were visualized according to ECL luminescence method.6 Real-time fluorescence quantitative PCRTotal RNA of samples were extracted using Trizol reagents.Reverse transcription were performed using Primescrip RT Master mix and Primescrip RT Reagent Kit.Real-time fluorescence quantitative PCR was performed using SYBR Green Master Mix Kit.7500 real-time PCR System was used for PCR reaction.The amplification multiples of the genes are calculated according to 2^-??CT.Actin and U6 were used as reference genes.7 Proliferation assayFor MTT assay,cell suspension was incubated in 96-well plate.Then cells were cultured for 24,47,72,96 hours respectively at 37?.MTT solution was added,and the cells were cultured for another 4 hours.Remove the medium and add DMSO.Adjust the wave length and OD of cells was detected.For colony formation,cell suspension was incubated in 6-cm culture dish for 14 days.The cells were dyed with Giamsa solution.The number of colony was counted under a microscope.8 Flow cytometryFor cell cycle,cells were collected and rinse tenderly.A pre-cooled 75%alcohol was added for cell fixation.Remove the ethanol solution and tenderly rinse the cells.PI dyes were added,and the cells were incubated avoiding light.Cell cycle were analyzed by flow cytometry.For cell apoptosis,cells were collected and rinse tenderly.Annexin?-FITC and PI solution were added,and the cell mixture was incubated avoiding light.Cell apoptosis level was detected by flow cytometry.For mitochondrial membrane potential,cells were collected and rinse tenderly.Add JC-1 fluorescent dyes to the cell mixture,and the cells were incubated for 45 minutes.Cells were rinse tenderly with PBS buffer.The mitochondrial membrane potential of cells was analyzed by flow cytometry.9 Fluorescence enzyme Reporting Gene experimentFluorescein enzyme reporting gene plasmid was constructed and it was transfected with miR-548b-3p mimic into hepatocellular carcinoma cells.The fluorescence intensity was detected to analysis whether CIP2A was the direct target gene of mir-548b-3p.10 Statistic analysisSPSS 16.0 software was used for Statistical analysis.The relationship between CIP2A expression and the clinicopathological factors was analyzed using chi-square test.The survival analysis was analyzed using Kaplan-meier method.The difference between treatment group and control group was tested by student?s t test.The data obtained were represented by the mean±standard deviation.p<0.05 indicates that there is a statistically significant.Results:1 CIP2A was upregulted in hepatocellular carcinoma tissueThe expression of CIP2A in hepatocellular carcinoma tissue was increased by 40%?32/80?.CIP2A high level was related with a short lifespan in hepatocellular carcinoma patients.2 CIP2A promoted cell proliferationThe proliferation rate and colony number of hepatoma cancer cells were increased after CIP2A overexpression.On the contrary,the proliferation rate and colony number of hepatoma cancer cells were decreased after CIP2A depletion.3 CIP2A regulated expression of c-myc,bcl-2 and cleaved-PARPCIP2A overexpression upregulated c-myc,bcl-2 and downregulated cleaved-PARP expression.CIP2A depletion showed the opposite results.4 miR-548b-3p was downregulted in hepatocellular carcinoma tissueReal-time fluorescence quantitative PCR results showed that miR-548b-3p was downregulated in hepatocellular carcinoma tissues.Bioinformatics analysis showed the same results.5 miR-548b-3p inhibited proliferation of hepatocellular carcinoma cellsAfter transfection with miR-548b-3p mimic,the proliferation rate and colony number of hepatocellular carcinoma cells were decreased.After transfection of miR-548b-3p inhibitor,the proliferation rate and colony number of hepatocellular carcinoma cells were increased.6 miR-548b-3p inhibited cell cycle transformationTransfection of miR-548b-3p mimic inhibited cell cycle transformation in hepatocellular carcinoma cells.And transfection of miR-548b-3p inhibitor promoted cell cycle transformation.7 miR-548b-3p enhanced chemotherapy sensitivity in hepatocellular carcinoma cells After transfection of miR-548b-3p mimic,the apoptosis level induced by CDDP was increased.While the apoptosis level induced by CDDP was decreased after transfection of miR-548b-3p inhibitor.8 miR-548b-3p regulated mitochondrial membrane potential in hepatocellular carcinoma cellsThe mitochondrial membrane potential was decreased after mir-548b-3p overexpression,while was increased after miR-548b-3p downregulation.9 miR-548b-3p regulated the expression of c-myc,bcl-2 and cleaved-PARPIn hepatocellular carcinoma cells,miR-548b-3p overexpression reduced CIP2A,c-myc,bcl-2,while increased cleaved-PARP expression.miR-548b-3p deficiency increases CIP2A,c-myc,bcl-2,while decreased cleaved-PARP expression.10 CIP2A is a direct target gene for mir-548b-3p in hepatocellular carcinoma cellsThe fluorescence intensity was decreased after co-transfection of wild-type CIP2A gene and miR-548b-3p mimic,while was almostly the same after co-transfection of mutant CIP2A gene and miR-548b-3p mimic in hepatocellular carcinoma cells.11 miR-548b-3p regulated proliferation of hepatocellular carcinoma cells through CIP2AThe activity of hepatocellular carcinoma cells was decreased after transfection of miR-548b-3p mimic,and the activity was decreased further after co-transfection of mir-548b-3p mimic and CIP2A siRNA.The activity of hepatocellular carcinoma cells was increased after transfection of miR-548b-3p inhibitor,while was decreased after co-transfection of miR-548b-3p inhibitor and CIP2A siRNA.Conclusion:CIP2A was increased,while miR-548b-3p was decreased in hepatocellular carcinoma tissue.miR-548b-3p regulated proliferation,periodic transformation,chemotherapy resistance and other biological behaviors of hepatocellular carcinoma cells through targeting CIP2A.