Research On Germline Mutation Of STK11 Gene, STK11 And TGFβ1 Protein Expression In Peutz-Jeghers Syndrome

BACKGROUNDPeutz-Jeghers syndrome(PJS) is a rare autosomal dominant hereditary disease characterized by gastrointestinal hamartomatous polyps and mucocutaneous melanin deposition.Hemminki and Jenne found STK11(serine threonine kinase 11,also known as LKB1) gene germline mutation in PJS patients and families,proved that STK11 gene is the pathogenic gene of PJS.Germline mutation is related to genetic,which is at the level of blood DNA.The main mutation forms were point mutation and small fragments deletion.Predicted effects are occur premature termination signal,resulting in truncated protein and inactivation of protein kinase. STK11 protein play an important role in chromatin remodeling,cell cycle,cell polarity and regulation of energy metabolism.And play a regulatory role in many signaling pathways such as TGFβ/Smad,NF-κB,PTEN/ phosphatidylinosito13' kinase,G protein-coupled signaling pathway.STK11 protein inhibit cell proliferation and promote apoptosis,down-regulated of protein expression will lead to other tumor-associated gene mutations in somatic,eventually lead to malignant tumors.TGFβis a disulfide-linked dimer peptide.Its family members in mammals have very high stability in vivo.TGFβcontrols the growth of epithelial cells,indirectly repairs DNA damage,stimulates apoptosis,regulates the function of stem cell.TGFβhas the dual characteristics of promote and inhibit tumor.TGFβ/Smad pathway is the main way of TGFβsignal transduction.TGFβand its receptor composed receptor complex,activate Smad proteins,then transducte signal to the nucleus,performance the function as transcription factors.To different cells,TGFβhas different effects:for fibroblasts,the main effect is to stimulate cell division;for the majority of epithelial cells,its effect is growth inhibition.In recent years,many literatures reported TGFβsignaling pathway disorder is closely related to tumorigenesis,development and metastasis.Human TGFβfamily including TGFβ1,2,3,which located at 19q13, 1q41,14q24.Any component in the pathway may cause signal transduction disorder, then cells escape TGFβ-mediated growth inhibitory effect,leading to tumorigenesis. In cells and tissues,TGFβ1 is the most abundant member of the TGFβ.Its promoter regulation is complex,so TGFβ1 disorder diseases(including tumorigenesis) are most common.TGFβ1 inhibited cell growth with the negative regulation of biological effect,its expression or activation deficiencies lead to cell malignant proliferation.Ki67(Proliferating cell nuclear antigen) is confirmed as a nuclear proliferation marker.It initial expressed in the G1 phase of cell cycle,then gradually increased in S and G2 phase,in M phase reached the peak and quickly disappeared in the division phase,no expression in GO phase.Half-life of Ki67 is short,rapid degradation out of the cell cycle.Ki67 expression can objective reflect tumor proliferative activity.A recent research found TGFβsignaling defect in the stromal of gastrointestinal polyps by PJS animal model(STK11 gene allele deletion mouse),which caused the adjacent mesenchymal epithelial cell proliferation activity.This feature is not found in wild-type mice(non-STK11 deletions).Researchers proposed "STK11 deletion→TGFβsignal down-regulated→excessive glandular epithelium proliferation→PJ polyps formation" hypothesis,speculated the formation of PJ polyps and TGFβsignal abnormal are closely related.This hypothesis provide a new way to explore the formation mechanism of PJ polyps.Objeetives: 1,Detection STK11 gene germline mutation in PJS family,analysis of possible pathogenic mutation and mutation forms,explore the Characteristics of STK11 gene mutation in Chinese PJS families.2,Research on STK11 and TGFβ1 protein expression in PJ polyp tissue, combined with research the proliferation of PJ polyps,preliminary authenticate "STK11 deletion→TGFβsignal down-regulated→excessive glandular epithelium proliferation→PJ polyps formation" hypothesis from tissue protein level.Materials and Methods:1,PJS families(20 people,including 16 patients and 4 family health members) are experimental group.Randomly selected 20 health people who unrelated to PJS families as the control group,peripheral blood were taken from two groups,extract genomic DNA.Design 9 pairs of primers according to all the exons of STK11 gene.Purified PCR amplified products,then detect STK11 germline mutation by DNA direct sequencing method.Initial screening by online BLAST in NCBI,positive samples compared with family health people and control group,analysis the characteristics of STK11 germline mutation and mutation forms in PJS families.2,23 paraffin specimens of PJ polyps(from 11 patients) as the experimental group,10 normal gastrointestinal mucosa specimens as control group. Immunohistochemical(IHC) method detected STK11,TGFβ1 and Ki67 protein expression then calculated the positive rate,detected these three proteins at the same time in order to reduce errors,calculated the positive rate.The positive staining rates were expressed by labeling index(LI).We detected TGFβ1 and Ki67 protein expression of colon adenoma(12 cases) and colon adenocarcinoma(11 cases)also by IHC.3,Using SPSS13.0 statistical software to deal with experimental data.If raw data consistent with parameters test conditions,t test were used to compared the mean of two independent samples;analysis of variance(F test)were used to compared many groups independent samples;bivariate correlation analysis using Pearson correlation analysis.If raw data do not consistent with parameters test,Mannn-Whitney U test were used to compare two independent samples differences;Kruskal-Wallis H test were used to compare many independent samples differences;bivariate correlation analysis using Spearman rank correlation analysis,α=0.05 as a tests standard,P＜0.05 for significant difference or there is a significant correlation.Results:1,In experimental group,we found 15 samples from 20 cases existence STK11 gene mutation,the mutation form is point mutation.The predicted effect is caused abnormal protein coding.No mutation was found in control group.Point mutations were found in exon 4,6 and 8.In family A,3 patients have codon 354(exon 8) heterozygous mutation(GAA→CAA),there is no such mutation in family health person.Meanwhile,no such mutation was found in control group.In addition,some missense mutations were found in several patients in codon 154,155,176 and 270.2,Both epithelium and stromal cells of normal gastrointestinal mucosa expressed STK11 protein:cytoplasmic and nuclear staining.STK11 expression types of PJ polyps were as followed:①reduced expression in epithelial cells,completely loss expression in stromal cells;②completely loss expression in epithelial cells and stromal cells;③similar to normal mucosa expression.Both epithelium and stromal cells of normal gastrointestinal mucosa expressed TGFβ1 protein:cytoplasmic staining,no nuclear staining.Expression of TGFβ1 in PJ polyps:stromal staining, little epithelial cells staining,no nuclear staining.Expression of TGFβ1 in colon adenoma and adenocarcinoma:cytoplasmic,membrane and nucleus staining.Ki67 is specific nuclear staining,Ki67 expressed in PJ polyps and normal tissues are lower than colon adenoma and adenocarcinoma.3,Mann-Whitney U test showed LI average rank of STK11 protein staining in PJ polyps group was 13.5,the normal group was 25.05,u=34.500,P=0.002＜0.05, two groups have significant differences,PJ polyps expressed STK11 protein lower than normal.That of TGFβ1 protein staining in PJ polyps group was 14.61,the normal group was 22.5,u=60.000,P=0.031＜0.05,two groups also have significant differences,PJ polyps expressed TGFβ1 protein lower than normal tissue.Using Kruskal-Wallis H test comparing PJ polyps,normal gastrointestinal tissues,colon adenoma and colon adenocarcinoma of-Ki67 protein expression,the staining LI of average rank were:18.43,15.75,37.79,51.0 as followed.χ~2=39.711,P=0.000＜0.05, four groups were significantly different,according to the average rank,the highest expression of Ki67 is colon adenocarcinoma,followed by colon adenoma group,PJS group,normal group.4,Using Spearman rank correlation analyzed PJ polyps STK11 and TGFβ1 relevance of staining LI,r_s=0.863,P=0.000＜0.05,two have significant positive correlation.TGFβ1 and Ki67 of PJP rank correlation analysis results:r_s=-0.422,P= 0.045＜0.05,two have significantly negatively correlation.TGFβ1,Ki67 in normal gastrointestinal tissues,colon adenoma and colon adenocarcinoma correlation analysis of the LI with Pearson correlation analysis,r=0.216,0.349,0.095,P= 0.549,0.266,0.782,P＞0.05,no significant correlation.Conclusion1,A number of STK11 gene germline mutations in PJS pedigrees,confirmed STK11 gene mutation is closely related with this syndrome.A missense mutations: GAA→CAA had been found in codon 354,this mutation is related with PJS in family A.The missense mutation in codon 154,155,176(exon 4),and codon 270 (exon 6) may also be correspond with pathogenic of those PJS families.2,PJ polyps reduced express STK11 protein suggest that abnormal function of this protein probably play a key role in the formation of PJ polyps.Reduced express TGFβ1 protein suggest the formation of PJ polyps may be related to the up-regulation TGFβsignals.3,In PJ polyps,STK11 protein inactivation may lead to low expression of TGFβ1 protein;low expression of TGFβ1 protein may enhance the activity of epithelial tissue proliferation(Ki67 expression higher).This correlation did not found in normal tissue,colon adenoma and adenocarcinoma,suggested this correlation is probably the characteristics of PJ polyps,the trend of the IHC results, supported the "STK11 deletion→TGFβsignal down-regulated→excessive glandular epithelium proliferation→PJ polyps formation" hypothesis in some extent.