Study On Genetic Instability Of The SFRP1 Gene With Hepatocellular Carcinoma In Chinese

BackgroundThe malignant tumors have threatened human health and lives severely.One of the most important reasons is that the microinvasion or micrometastasis exists at the time of surgery and can not be controlled.Therefore it is very important to clarify the mechanism of invasion and metastasis,and quite significant to establish some definite molecular marker and target for the therapy.It has been investigated that the expression of anti-oncoprotein becomes low with the occurrence and metastasis of tumor,then the growth and metastasis of tumor cell are out of control.The main reason that the expression of anti-oncoprotein reduces is anti-oncogenes inactivity.To investigate the mechanism of genetic inactivity,scientists did lots of research on many anti-oncogenes such as P53,P16,et al,and found that the genetic instability,the representative of microsatellite instability and loss of heterozygosity,might be one important factor that leads to genetic mutation,the anti-oncogenes function maladjustment,and the tumor occurrence.Microsatellite instability(MSI) and loss of heterozygosity(LOH),the alteration in length and strength of short tandem repeat sequences are important molecular characteristics of many human tumors.Many researches indicated that MSI or LOH of anti-oncogenes plays an important role in occurrence of sporadic tumor.MSI was first found in tumors of the hereditary non-polyposis colon carcinoma(HNPCC) syndrome, and then it is found in many other kinds of tumors,such as colon cancer,gastric cancer, carcinoma of endometrium,breast cancer,prostatic cancer and pancreatic cancer,et al. Many studies suggested that the occurrence of MSI increase the frequence of mutation, and moreover,it lead to mutation of genes which has great relationship with tumor which may be an important mechanism of tumor occurrence.According to Knudson's two-hit hypothesis of TSG(tumor suppressor gene) inactivation,the common chromosomal region of LOH is a potential site harbouring TSG,thus LOH is regarded as a valuable molecular genetic marker to find TSGThe sFRP1 gene is found at chromosome 8p12-p1 1.1,composed with about 313 amino acid and 35.3kDa in molecular weight,containing three exons and two introns. sFRP1 encodes a soluble Wnt antagonist,a putative tumor suppressor gene. Wnt/β-cateninn pathway is a conserved molecular system that plays a major role in development and homeostatic tissue self-renewal.In a resting state,β-catenin level remains low due toconstant turnover mostly by the destruction complex APC/Axin/GSK-3β. Phosphorylation by this complex causes P-catenin to be degraded by the proteasome. When the Wnt binds to the cell surface receptor Frz and activates disheveled(Dv1 or Dsh),glycogen synthase kinase 3β(GSK-3β) is dissociated from the destruction complex.As a result,accumulated cytoplasmicβ-catenin translocates to the nucleus and binds to members of the T-cell factor/lymphoi-d enhancer factor(TCF/LEF) protein family;the resulting complexes then activate oncogenic target genes such as c-myc, cyclin D1,which regulate cellular growth and apoptosis.Structurally sFRP1 protein resembles the Frizzled receptors although lacking the latters'transmembrane domains.Thus,sFRP1 may block Wnt signal either by interacting with Wnt proteins to prevent them from binding to Frz proteins or by forming nonfunctional complexes with Frz.The down-regulation of sFRP1 can lead to the persistence of Wnt signal and active target genes,resulting in malignant tumors worsened.Moreover,the down-regulation of sFRP1 has been observed in many tumors. It is frequently inactivated by promoter methylation in many human cancers including breast cancer,ovarian cancer,bladder cancer,mesothelioma,prostate cancer,colorectal cancer,and non-small cell lung cancers.Recently,the frequent down-regulation of the gene was also reported in HCC by Shih.YH,where 43 of 47 HCC(91.5%)exhibited the depressed SFRP1 as compared with non-cancerous livers through quantitative RT-PCR,suggesting it worked as a tumor suppressor gene in HCC.One mechanism leading to the down-regulation of sFRP1 was promoter methylation.Insteresting,the down-regulation of sFRP1 was found in the cases without promoter methylation, suggesting there is that other mechanisms involved in its expression of silence,such as LOH.Most of the studies about sFRP1 gene focused on the promoter methylation,it was uncommon to have the study on the genetic stability about sFRP1 gene.ObjectiveTo investigate MSI and LOH of locus D8S532,D8S1722 of sFRP1 gene,and their effect on the expression of sFRP1,which would reveal the function mechanism of anti-oncogene and provide experimental basis for metastasis mechanism of cancer.Methods(1) Phenol-chloroform Method for extraction DNA from paraffin-embedded hepatocellular carcinoma tissues.(2) Polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) and displaying band by ordinary silver stain to analyze the genetic instability of the locuses.(3) Expression of protein was tetected by Envision immunohistochernistry.(4) Leica-Qwin computer image analysis system was used for camera into the computer,Image-Pro PluS(IPP) Version4.5 professional image analysis software for analyzing expression of protein.(5) SPSS statistic software analyzed the relationship among experimental results.ResultsStudy on genetic instability of sFRP1 gene with the clinical pathological behaviors of HCCs in Chinese①LOH and MSI at locus D8S532 were detected in 11.90%(5/42) and 9.52%(4/42) of 42 HCC tissues,respectively.The frequency of LOH was not correlated with the patient's age and gender,lymphatic and vessel metastasis,tumor TNM stage,tumor differentiation,tumor size,levels of AFP and ALT,or the status of tuberculosis.The frequency of MSI was higher in patients who were 60 years or older(>=60) compared with that in the younger patients below 60 years of age(<60)(23.53%vs.0.00%, P<.05),but it was not correlated with other clinicopathological features.②LOH and MSI at D8S1722 were detected in 14.29%(6/42) and 9.52%(4/42) of 42 HCC tissues, respectively.The frequencies of both LOH and MSI at this locus were not correlated with a patient's age,gender,lymphatic and vessel metastasis,tumor TNM stage,tumor differentiation,tumor size,levels of AFP and ALT,or the status of tuberculosis.Expression of sFRP1 protein and genetic instability of sFRP1 gene,also and clinical pathological behaviors in Chinese with HCCs①Expression of sFRP1 protein was down-regulated to various degrees in 88.10% (37/42) of the HCC tissues compared with the corresponding non-carcinoma liver tissues,and the sFRP1 protein was only detected in 47.62%(20/42) of the tumor tissues, but in 100%of the normal tissues.②The sFRPl positive rate in the highly differentiated HCC cases was higher than that in the moderately and poorly differentiated cases(100.00%vs.23.53%,P < 0.05).The level of sFRP1 protein expression was not correlated with other clinicopathological parameters.③The frequency of LOH at locus D8S532 in sFRP1 positive cases was lower than that in sFRP1 negative cases(0.00%vs.22.73%,P < 0.05).④The frequency of LOH at locus D8S1722 in sFRPl positive cases was not significantly different to that in sFRP1 negative cases(5.00%vs.22.73%,P > 0.05).ConclusionsThis study revealed that down-regulation of sFRP1 protein is an important factor in the progression of HCC.Up-regulation of the protein might improve the prognosis of the disease in Chinese patients.At the same time,LOH of sFRP1 gene may be one of the mechanisms causing the gene silencing and the protein expression deficiency. Therefore,the genetic instability of sFRP1 gene can be considered as a prognostic maker for HCC.