| Part one the expression and clinical significance of miR-29s family in hepatocellular carcinomaBackground and objectiveHepatocellular carcinoma(HCC)is one of the most common primary malignancies;it’s the fifth most frequently diagnosed cancer worldwide and the third most frequent cause of cancer death.HCC is difficult to diagnose and confirm at the initial stage,prognosis remains poor for high tumor recurrence and tumor progression.Thus,exploring the reliable biomarkers to predict the prognosis of HCC and improve poor prognosis of patients becomes urgent and meaningful.MicroRNAs(MiRNAs)are non-coding small molecule RNA,miRNAs can regulate genes expression,and increasing researches demonstrated that miRNAs can not only participate in cellular biological process,but have emerged as biomarkers to predict prognosis of tumor patients.MiR-29s family includes miR-29a,miR-29b and miR-29c,lots of studies showed miR-29s family closely relates with cancers,however,whether miR-29s can predict the prognosis of HCC as biomarkers remains unclear,therefore,the present study aims to detect the expression of miR-29s family in HCC tissues and evaluate their reliability of predicting the prognosis of HCC patients.Methods1.Research subjects:122 primary HCC patients who underwent a curative liver resection from 2008 to 2015 were included.After a detailed history and a thorough physical examination,blood was collected for liver function test and tumor markers;Contrast-enhanced computerized tomography(CT)and/or magnetic resonance imaging(MRI)was used to confirm the TNM staging at diagnosis;liver resection was carried out,taking note of the tumor diameter,location,tumor extension and estimated volume of the remaining liver.Liver resection was performed following couinaud’s segments,sectors and hemilivers.2.Specimens storage:specimens were immediately soaked into liquid nitrogen after surgery,then removed to-80 ℃ fridge or stored as paraffin-embedded tissue.3.RNA extraction and miRNA quantification:Total RNA was extracted from tissues by phenol-chloroform method;miRNAs were amplified by miRNA kits and quantified by ABI 7300 system.4.Follow-up:postoperative serum AFP and abdominal ultrasound were carried out monthly.Patients received abdominal contrast-enhanced CT scan or MRI once every 3 months in the first two years after surgery,and once every 6 months thereafter.Overall survival(OS)was defined as the period from the date of surgery until an occurrence of death or last contact.5.COX proportional hazard analysis was performed to explore risk factors that influence prognosis by the result of miRNA and clinicopathologic features of patients.Results1、122 patients with HCC were recruited into the study.The median follow-up was 6.5 years(ranging from 6.2 months to 8.2 years).The ratio of male to female was 2.49:1.The ratio of tumor size<5cm,microvascular invasion and solitary tumor was 63.1%,62.3%and 81.1%,respectively.The low-expression of miR-29a,miR-29b and miR-29c were 42.6%,78.7%and 35.3%,respectively.2、Compared with non-tumor tissues,miR-29a,miR-29b and miR-29c were down-regulated in tumor tissues(P<0.001).3、For all 122 patients,the overall 1,3,5-year OS were 87.5%,53.3%and 44.1%,respectively.Descriptive survival statistics and Kaplan-Meier curves suggested that AFP>400ng/ml,tumor size>5cm,microvascular invasion,tumor multifocality,low-expression of miR-29b and miR-29c had prognostic significance.Low-expression of miR-29a showed no significantly prognostic value.4、AFP>400(ng/ml)was associated with a decreasing 5-year OS rate from 53.4%to 34.3%(P=0.010);tumor size>5cm was associated with a decreasing 5-year OS rate from 53.1%to 28.6%(P<0.001);Microascular invasion(MVI)was associated with a decreasing 5-year OS rate from 57.1%to 34.4%(P=0.009);Multiple tumors was associated with a decreasing 5-year OS rate from 48.7%to 24.8%(P =0.036);Low miR-29b was associated with a decreasing 5-year OS rate from 70.2%to 39.1%(P=0.020);Low miR-29c was associated with a decreasing 5-year OS rate from 53.6%to 23.7%(P=0.012);Survival analysis showed that combined low miR-29b and low miR-29c was associated with a decreasing 5-year OS rate from 51.7%to 30.5%(P=0.006).5、Univariate analysis showed that AFP>400ng/ml,tumor size>5cm,microvascular invasion,tumor mutilfocality,low expression of miR-29b and miR-29c were associated with a worse prognosis in the univariable analysis;combined low miR-29b and low miR-29c.The results demonstrated that combined low miR-29b and miR-29c(HR:2.561,P=0.012)was an independent prognostic factor of patients with HCC.Conclusion1、MiR-29a,miR-29b and miR-29c are down-regulated in HCC.2、AFP>400ng/ml,tumor size>5cm,microvascular invasion,tumor mutilfocality,combined low miR-29b and miR-29c are associated with decreased OS,besides,combined low miR-29b and miR-29c was an independent prognostic factor of patients with HCC.Part two:effect of miR-29s family on the proliferation and apoptosis of hepatocellular carcinoma cellsBackground and objectiveThe underlying mechanisms of HCC remain unclear;researches on molecular mechanisms of HCC are popular.The previous study has demonstrated that miR-29s family was an independent prognostic factor of patients with HCC,thus,we aim to investigate the effect of miR-29s family on the proliferation and apoptosis of HCC in this part.Methods1、Human hepatocytes cell line L02 and HCC cell line SMMC-7721 were cultured in vitro,RT-qPCR was used to detect the expression of miR-29a,miR-29b and miR-29c in L02 and SMMC-7721.2、MiR-29a mimics,miR-29b mimics and miR-29c mimics were transfected into SMMC-7721 cells to overexpression miR-29a,miR-29b and miR-29c,respectively.MTS assay was performed to detect cell viability after transfection;colony formation assay was used to explore the clone-forming ability of SMMC-7721 after transfection;flow cytometry was used to investigate cell apoptosis of SMMC-7721 after transfection.3、Target genes of miR-29s family were predicted by miRDB database,venn diagram was used to observe the difference of prediction of each miRNA,related KEGG pathways of predicted genes was performed by using Metascape software.4、Expression of CDK2、CDK6、AKT2、Bcl-2 and Bax after transfection were detected by western blot.Results1、Compared with L02 cells,miR-29a,miR-29b and miR-29c were down-regulated in SMMC-7721 cells.2、Cell viability was decreased after overexpression of miR-29a,miR-29b and miR-29c in HCC cells;clone-forming ability was decreased after transfection;cell apoptosis was increased after transfection.3、For miR-29a,miR-29b and miR-29c,number of predicted genes by miRDB database 623,the shared genes of 3 miRNAs was 631;the top 5 KEGG pathways,which related with predicted genes,were protein digestion and absorption,small cell lung cancer,TNF signaling pathway,Toxoplasmosis and mTOR signaling pathway,respectively.4、Overexpression of miR-29a,miR-29b and miR-29c inhibited the expression of CDK2、CDK6、AKT2 and Bcl-2 and promote the expression of Bax.Conclusion1、MiR-29s family is down-regulated in HCC cells.2、MiR-29s family inhibits proliferation and promotes apoptosis of HCC cells.3、CDK2,CDK6 and AKT2 are target genes of miR-29s family;miR-29s family might inhibit proliferation and promote apoptosis of HCC cells via inhibiting the expression of those genes. |
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