| CHAPTER I Splicing form of CD147 gene in tumor tissue of hepatocellular arcinoma and its expression level in m RNA Background and objectiveCD147,also known as matrix metalloproteinase inducer(EMMPRIN),HAb18G/CD147 or basigin(BSG),is a highly glycosylated transmembrane glycoprotein belonging to the immunoglobulin superfamily.The NCBI database discloses that the CD147 gene(BSG)has four different transcript variants expressing different CD147 proteins in different tissues or cells.Studies have shown that CD147 is closely related to the occurrence and development of hepatocellular carcinoma(HCC).However,which isoform of CD147 is expressed in the tumor tissue of liver cancer in Guangxi is not very clear at present.Therefore,to determine the transcript isoform of CD147 in hepatocellular carcinoma and its expression level in m RNA is very important for understanding how CD147 participates in the development of HCC,and providing an important theoretical basis for the application of CD147 in the accurate diagnosis of HCC.MethodsA total of 20 fresh tumor and adjacent normal liver tissues resected from patients in guangxi with HCC were collected.RT-PCR was used to detect the CD147 isoforms of HCC.Real-time fluorescence quantitative PCR was used to detect CD147 expression in m RNA in tumor and normal liver tissues.Results1.CD147 m RNA in HCC is transformed as subtypes BSG-2?BSG-3 and BSG-4.2.The expression level of CD147 in m RNA was higher in the tumor than that in adjacent tissue,but there was no significant statistical difference between them(P>0.05).ConclusionCD147 m RNA in HCC is transformed as subtypes BSG-2?BSG-3 and BSG-4,whose expression level has no significant statistical difference between the tunor tissue and the adjacent tissue(P>0.05).CHAPTER II Association between CD147 gene polymorphisms and the usceptibility of hepatocellular carcinoma Background and objectiveCD147 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily.A number of reports have shown that CD147 plays an important role in the proliferation,differentiation,invasion and metastasis of hepatocellular carcinoma,but studies about the association of CD147 gene polymorphisms with the susceptibility of hepatocellular carcinoma have not been reported.In this study,CD147 was selected as a candidate gene to identify whether the gene is a susceptible gene for hepatocellular carcinma using public databases of genetic polymorphisms,combined with bioinformatics methods.MethodsFirst,we searched public polymorphism databases,single nucleotide polymorphisms(SNPs)of CD147 gene in registered Chinese population(reference sequence is NT011255)using bioinformatics methods.After comprehensively analying the search results,2-3 polymorphisms were selected for further study.We performed a case-control study including 155 patients with hepatocellular carcinoma and 151 healthy controls on random basis.The polymorphisms of rs8259 and rs6757 were detected by gene sequencing.The ?2 test was used to analyze whether the distribution of the SNPs genotype frequency in healthy controls group was consistent with Hardy-Weinberg equilibrium(HWE).Logistic regression was used to adjust for four factors: gender,age,smoking,and dringking.Odds Ratios(OR)and 95% Confidence Intervals(CI)were calculated to analy the association between polymorphisms(rs8259 and rs6757)and the susceptibility of HCC.Result1.The genotypes frequencies of CD147 gene SNPs(rs8259 and rs6757)in control groups were consistent with Hardy-Weinberg equilibrium(all p>0.05),indicating that the samples we selected were in accordance with Mendel inheritance and had group representationmet.2.The rs8259 had AA,AT and TT three genotypes.There was no statisticaly difference in genotypic frequencies distribution between the HCC and control group(all p>0.05).Using the TT genotype as the reference genotype,the AT and AA genotypes had no associstion with the the susceptibility of HCC(all p>0.05).Using the T allele as the reference gene,the A allele was not associated with the risk of HCC(P>0.05).No correlation was found between the rs8259 genotypes and the risk of HCC whether in the dominant model or in the recessive model(all p>0.05).3.The rs6757 had TT,CT and CC genotypes,whose genotypic frequencies distribution had significant difference between the HCC and control group(p<0.05).Using the TT genotype as the reference genotype,the CT genotype was not found to be associated with the risk of HCC(p=0.086).The risk of HCC in patients with CC genotype could be increased to 4.513-fold(95% CI: 1.510-13.489,p=0.007).Using the T allele as the reference gene,the C allele showed 1.826-fold(95% CI: 1.263-2.642,p=0.001).In the dominant model analysis,compared to TT genotype,CT+CC increased the risk of HCC to 1.824-fold(95% CI: 1.122-2.965,p=0.015).In the recessive model analysis,compared with CT and TT genotypes,patients carrying CC genotype showed 3.765-fold(95% CI: 1.286-11.020,p=0.016)increased risk of HCC.ConclusionThe CD147 gene polymorphism rs8259 was not associated with the susceptibility of HCC.The polymorphism rs6757 was associated with the risk of HCC,whose C mutant allele may increase the risk of HCC.The CD147 gene is one of the susceptibility genes of HCC.CHAPTER III The biological effect of the functional SNP rs6757 n CD147 gene.Background and objectiveMicro RNAs,a class of non-coding small RNAs with a length of about 22 nt,are widely found in animal,plant and virus species.As post-transcriptional regulators they can specifically bind to the 3'-untranslated retinion(3'-UTR)of the target mi RNA molecule to regulate the expression level of the target genes.Recently,more and more studies have reported that mi RNAs play fundamental roles in diverse biological processes including physiology in normal cell and pathologyical condition,such as cell proliferation,differentiation and apoptosis,inflammation,tumorigenesis,etc.SNPs in mi RNA target region,also known as mi RSNPs,are known to enhance or weaken mi RNA-m RNA interactions.The purpose of this study is to predict mi RSNPs in the CD147 gene based on the bioinformatic online softwares(Target Scan and mi RBase)and databases enquiries.We aimed to calculate the functional significance of the mi RSNP in regulating CD147 expression level.The double-luciferase reporter assay was used to determine whether the mi RSNPs could influence the CD147 expression by affecting the mi RNA binding ability to the CD147 m RNA,which provides strong theoretical support for the study of the role of CD147 in the pathogenesis of HCC.Method1.Bioinformatic method was used to predict the potential binding sites of mi RNAs in the CD147 gene 3'-UTR based on online softwares,such as Target Scan,Mir SNP,Mir Base,etc.After blasting,1-2 candidate mi RNAs were selected for futher study.2.Construct a luciferase reporter vector carrying different allele of the polymorphic site rs6757.In the following experiment,luciferase reporter constructs were co-transfected into HEK293 T cells,the luciferase activity of each group was detected and analyzed.In addition,luciferase reporter constructs were co-transfected into HEK293 T cells with different concentrations of mimic mi RNA-3976(1.0 n M;2.5 n M;5.0 n M and 10.0 n M).The expression activity of the CD147 gene was calculated by the activity of the luciferase affected by the SNP rs6757 different alleles at different concentrations of mimic mi RNA-3976.Result1.According to the predict results based on Target Scan and Mir Base online softwares,there may be multiple potential binding sites for mi RNAs in the CD147 3'UTR region.After blasting,the polymorphism rs6757: T>C was located in the seed region of the hsa-mi R-3976.So,the hsa-mi R-3976 was selected for the following functional experiments.2.We successful constructed a luciferase reporter gene vector carrying different alleles of the CD147 SNP site rs6757.3.Luciferase reporter vectors carrying the rs6757 T allele were transfected to HEK293 T cells,and luciferase activity assay results were as follows: compared with the blank or NC group,the relative luciferase activity of the mi R-3976 group was significantly decreased(p<0.05)and was restored by mi RNA inhibitors.Luciferase reporter vectors carrying the rs6757 C allele were transfected to HEK293 T cells,luciferase activity assay results showed that there were no significant differences between mi R-3976 group and blank or NC groups(p> 0.05).4.Luciferase reporter vectors carrying the rs6757 different alleles were transfected to HEK293 T cells with different concentrations(1.0n M;2.5n M;5.0n M and 10.0n M)of mimic mi R-3976.The result showed that the mi R-3976 could decrease the expression level of Luciferase reporter vector carrying the rs6757 T allele in a dose-dependent manner.There wsa no obvious change in the expression level of Luciferase reporter vector carrying the rs6757 C allele.ConclusionThese results suggest that the CD147 gene is a target of hsa-mi R-3976.The CD147 3'UTR SNP rs6757: T>C is associated with increased HCC susceptibity through altered mi R-3976-mediated negative regulatory of the CD147 at the post-transcriptional level. |
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