| Background InformationLiver cancer is the six most common cancer in the world,its cancer-related death rate ranks third and every year there are more than 700000 person diagnosed with liver cancer,the incidence rate was 16 per 100000.The five-year survival rate is 5-9%,the reason for this is the late stage when diagnosed with liver cancer.About 70-90%liver cancer is correlated with HBV infected or chronic liver disease.Nearly 80%liver cancer happens in Southeast Asia or Subsahara Africa,this was due to the wide spreading of HBV infection and aflatoxin B pollution.In North America,Europe and Japan,HCV infection is the main risk for liver cancer,next is drinking.The generation of liver cancer is a complex process,with many mutations and pathway deregulation.The most common mutanted gene is TP53,with a rate of 20-40%according to clinical stage.CTNNB1,encoding ? catenin,mutanted a rate of 25%,mainly in HCV related liver cancer.The gene amplification of 1q,6p,8q,17q and 20q and deletion at 4q,8p,1l q,13q,16q and 17p were most common in liver cancer.They had impacted a variety of important oncogene and tumor suppressor gene.The EGFR and RAS pathway in activated in more than 50%liver cancer,and mTOR signaling pathway is activated due the inactivation of PTEN.MicroRNA is short non-coding RNA with a length of 15-25 nucleotide.They match with the 3' UTR or 5' UTR of the targeted gene.When microRNA binding to mRNAs,they will lead to the translation suppression or degradation of mRNA,enabling a quick and sensitive gene expression regulation.MicroRNA has proved to influence more than 30%human gene and more than 60%Mrna have predicted binding sites for microRNA at 3' UTR.One microRNA can regulate the expression of many genes and one gene can be regulated by many microRNAs,and the expression of microRNA could be tissue or organ specific.More and more evidence proved that microRNA could be act as oncogene or tumor-suppressor gene,directly or indirectly regulate cancer related pathway.MicroRNA is enrolled in many biological process,it plays an important role in maintaining normal liver function.Some of them are downregulated in liver cancer,indicating they may be tumor-suppressor gene in liver and re-expression of them may stop the cell cycle,increase cell apoptosis,decrease tumor blood vessel formation and tumor migration.MicroRNA and its targeted sites are sequence dependent so change in the nucleotide of the microRNA or its targeted sites can have a profound influence on their function.Sequence analysis revealed that microRNA and its targeted sites are highly conserved and the SNPs for microRNA are rare.This indicated that in the genetic selection,SNP in microRNA or their targeted sites may have function.MicroRNA related SNP can impact tumor cell by two mechanisms.First,mutation leads to the inactivation of microRNA thereafter inactivation of tumor-suppressor gene.Second,mutation leads to the upregulation of oncogenic microRNA.In the biogenesis process of microRNA,they will be edited sequentially by Drosha and Dicer.This process mainly rely on the correct folding of pre-microRNA,and the sequence mutation in the pri-microRNA,pre-microRNA or mature microRNA can either increase or decrease the function of microRNA.SNP can influence microRNA indirectly,for example they are in the promoter region and influenced the translation process or they are in the targeted sites of mRNA.The SNP for microRNA could correlated with the cancer risk and prognosis.For example the SNP rs3783553 in 3' UTR of IL1A can create a new binding site for miR-122 and miR-378.They suppressed the expression of IL1A,decreased anti-tumor immune response and increase liver cancer risk.The insert-deletion of?TrCP in the 3' UTR could break the binding sites for miR-920 and increase the expression of ?TrCP,leading to the nuclear translocation of NF-kB and survival of tumor cells.One SNP at 3' UTR of SET8 is correlated with increasing of liver cancer patient survival.IHC staining revealed that this SNP could downregulate the expression of SET8 and create a new binding sites for miR-502 to suppress the activation of p53 pathway by SET8.ObjectiveThe main objective of this study includes:(1)study the expression of miR-146a in liver cancer tissue,adjacent normal tissue or liver cancer cell lines,are they deregulated?(2)study the function of miR-146a in liver cancer cell lines,such as cell proliferation,colony formation assay,cell migration and tumor xenograft formation.(3)The gene distribution of miR-146a rs2910164 SNP in liver cancer patients and normal control,analyzed the liver cancer risk of this SNP.MiR-146 family contains miR-146a and miR-146b.When THP1 was stimulated by LPS,they were activated and their expression increased depending on NF-kB.MiR-146a regulated NF-kB by targeting IRAK1 and influenced the invasion and metastasis of breast cancer and pancreatic cancer.One SNP rs2910164 in pre-miR-146a was the risk factor of papillary thyroid carcinoma,prostate cancer and esophagus cancer.In a recent study,this SNP was correlated with the decreasing death rate and relapse in bladder cancer.Function study revealed that miR-146a rs2910164 SNP could change the editing process of Drosha to downregulate the expression of miR-146a.The low expression of miR-146 was relate with the androgen independent prostate cancer and rs2910164 SNP is related to hormone resistance prostate cancer,indicating an important role of miR-146a in prostate cancer.MiR-146a was deregulated in liver cancer,acting as a potential tumor-suppressor gene.In this article,we first evaluated the expression of miR-146a in liver cancer tissue,adjacent normal tissue or liver cancer cell lines.Then we used low-miR-146a expression liver cancer cell line as experimental model to study the function of miR-146a in liver cancer.By re-expression miR-146a in liver cancer cell lines,we evaluated the cell proliferation,invasion,colony formation and tumor xenograft formation.The correlation of rs2910164 SNP and liver cancer risk was studied in 188 liver cancer patients and 187 healthy control.Through these studies,we wanted to know if miR-146a was a tumor-suppressor gene in liver cancer and rs2910164 SNP is correlated with liver cancer risk or not.This study would provide new molecular mechanism for liver cancer progression and theoretical basis for liver cancer treatment.Methods(1)Tissue expression of miR-146a:collected the tumor tissue and adjacent normal tissue,extracted total RNA,detected the expression of miR-146a by qRT-PCR.Regroup the patients by cancer clinic stage,analyzed that if the expression of miR-146a was correlated with clinical stage.(2)MiR-146a expression in liver cancer cell line:choose a serious of liver cancer cell lines,extracted total RNA after culture,then evaluated the expression of miR-146a by qRT-PCR,used normal cell lines as control.Choose low-miR-146a cell line as model,constructing the miR-146a expression vector,study the function of miR-146a by re-expression of them in liver cancer cell lines.(3)Cell viability assay:HepG2 and SMMC-7721(transfected cells with miR-146a or empty vector)cells were seeded in 96-well-plate at 2000/well,using CellTiter-Glo(?)Luminescent Cell Viability Assay(Promega#G7252)kit to determine the cell number at different time point.(4)Soft agar assay:HepG2 and SMMC-7721(transfected cells with miR-146a or empty vector)cells were seeded in 6-well-plate at 4000/well in 0.4%top agar above the 0.6%bottom agar,culture for 3 weeks at 37?,evaluate the colony formation rate by crystal violet staining.(5)Cell invasion assay:HepG2 and SMMC-7721(transfected cells with miR-146a or empty vector)cells were starved overnight.Then 100000 cells were seeded into transwell cell room with FBS-free medium adding BSA,under the cell room was 500ul DMEM medium with FBS.Cultured at 37? for 24h,then fixed with 4%PFA.(6)Tumor formation in nude mice:Transplant HepG2 and SMMC-7721(infected with miR-146a or PLKO.1)to the armpits of 6-week old female nude mice.Measure the tumor volume at different time points,the tumor volume is calculate by V=L*W2.(7)Western blot:HepG2 and SMMC-7721(transfected cells with miR-146a or empty vector)cells were seeded in 6-well-plate at 5×105/well.Collected protein lysates at 72h.Evaluated the pAkt,p-P38MAPK,PCNA and NF-kB p65 by western blot.(8)Genotyping of miR-146a rs2910164 SNP:Collected the blood of 188 liver cancer patients and 187 healthy controls,using QIAamp DNA Mini kit to extract DNA,then amplified miR-146a by PCR and analyzed the sequence by Sanger sequencing.Evaluated the SNP with liver cancer risk.ResultsThis study evaluated the expression level of miR-146a in liver cancer patients,adjacent normal tissue and liver cancer cell line.We upregulated the expression of miR-146a in HepG2 and SMMC-7721,then evaluated the cell proliferation,invasion,colony formation,tumor xenograft formation and related pathway protein expression,finally we evaluated the liver cancer risk of miR-146a rs2910164 SNP.The results were list below.(1)The expression level of miR-146a in liver cancer tissue were significantly lower than adjacent normal tissue.The normalized expression level of miR-146a was 3.72±2.5 in liver cancer tissue and 9.55±5.3 in adjacent normal tissue.The liver cancer patients were divided according to the clinical stages,as a results,expression of miR-146a was correlated with clinical stages.The patients with higher clinical stages(?a or ?b)had lower level of miR-146a and the patients with lower clinical stages(?a,Ib,?a and ?b)had higher level of miR-146a.(2)MiR-146a expression level was higher in normal liver cell line L02 and liver cancer cell line LM3,and lower in liver cancer cell line HepG2,SMMC-7721 and QGY-7701.This indicated that the expression level of HepG2,SMMC-7721 and QGY-7701 were lower than normal liver cell line L02.(3)We used HepG2 and SMMC-7721 as experimental model and constructed the expression vector of miR-146a.We transfected HepG2 and SMMC-7721 with miR-146a or PLKO.1 empty vector,then the expression level of miR-146a was verified by qRT-PCR.After re-expression miR-146a,comparing with the parental cells or cells infected with PLKO.1,the cell proliferation,invasion,colony formation and tumor xenograft formation was suppressed in HepG2 and SMMC-7721.The protein expression of pAkt,p65,PCNA was downregulated,indicating the enrollment of PIK3/Akt and NF-kB pathway.(4)The genotype of miR-146a rs2910164 SNP between liver cancer patients and healthy controls were statistically significant.GC(OR=1.99,95%CI 1.27-3.11,P=0.003)orCC(OR=3.3,95%CI 1.72-6.32,P=0.0003)genotype have higher liver cancer risk than GG genotype.There were significant differences in HBV infection,family history of cancer and drinking status between the two groups.The rs2910164 SNP had no correlation with tumor clinical stage,tumor volume,cell differentiation,metastatis,blood vessel and lymphatic infiltration.ConclusionThe expression of miR-146a in liver cancer tissue was significantly lower than adjacent normal tissue,and the expression level of miR-146a was correlated with clinical stage,indicating miR-146a expression level could be prognostic marker for liver cancer patients.MiR-146a acted as a tumor suppressor in liver cancer.Re-expression of them in liver cancer cell line HepG2 and SMMC-7721 could suppress cell proliferation,invasion,colony formation and xenograft formation.This may be the result of downregulate of p-P38MAPK,pAkt,p65 and PCNA.MiR-146a rs2910164 SNP GC and CC genotype had a higher liver cancer risk than GG genotype,but had no correlation with tumor clinical stage,volume,cell differentiation,metastatis,blood vessel and and lymphatic infiltration. |
PDF Full Text Request