The Correlation Between The Upstream Of P16 Gene And The Nuclear Matrix In Gastric Cancer

Recent reports have shown that free of the G1/S andG2/M checkpoint contro1 contributed to the tumor. Thosece1ls which have genetic defect and se1ectivepro1iferation of malignant ce11 can result intumorigenesis. Gl/S checkpoint c1osely correlates withp16 gene. Protein l6 is an inhibitor of CDK4/6. Itnegative1y regu1ates the ce11 cycle. Gene expression wasregu1ated by transcription factors recognizing andbinding to cis--acting element. Matrix attachedregions (MARs) I a nove1 cis--acting e1ement, had been5yJ,I,I k7 2002 M{d[7{t$)fei2 X PI6 4lkl l:WG#IJ Ij Pj64flllth4R4ffi0jtUR.r4increasing1y thought much of . The nuc1ear matrix p1ays animportant ro1e in repl icat ion, transcript ion andpost--transcript iona1 process ing. The fluc 1ear matrixc1ose1y corre1ated with tumor.The reports on p16 gene concefltrated on the structura1sequences. Up to now, no reports on the corre1ationbetween nuc1ear matrix and p16 gene, the upstream of p16gene have been found. This research aimed to the nuc1earmatrix change in gastric cancer and the corre1at ionbetween the upstream of D16 gene afld the nuc1ear fnatrixin gastri c cancer, and determine if matri x at tached region(MAR) is on the 869bp fragment which is in the upstreamof exon l of p16 gene. The 890bp fragment (including the869bp fragrneflt) was purified from the p16promoter--1uc--pGL2--Bas1c D1asmid, and 1abeIed by randompl1ming wi th di goxi gefli n. The probe sensi t ivity wasdetected by DNA dot blot.22 samp1es, inc1uding tumor tissue, adjacent tumort issue and flormal gastric mucosa were ana1yzed. 10 cases6mNX% 2002 EWnt$Etet Pl6 $N--t:WA'g-'jWf$mat&SmMmXttwere high1y and moderate1y differented gastric cancer, 12cases were poor1y differented gastric cancer; 9 cases werein the I-- lI stage, l3 cases were in the III--IV stagerespective1y. The purified nuc1ei were co11ected bysucrose gradient centrifugat ion, nuc1ear matrix proteinswere prepared by routine methods, and the so1ub1e proteinsin the process of preparation were co11ected, too. Thenuc1ear matrix proteins were separated by 10%po1yacry1amide ge1 e1ectrophoresis (PAGE) containing0. 1% sodium dodecy1 su1fate (SDS) and e1ectrotransferredonto nitroce11u1ose membranes for DNA--binding(Southwestern b1ot). The bands were ana1yzed by Genetoo1ssoftware. Nonparanetric statistics were emp1oyed.The resu1ts were shown:1. The sensitivity and specificity of the probequa1 if i ed.2. The nude nuc1ei were co11ected and purified byc entr ifugat ion.3. the resu1ts of SDS--PAGE:7m)l'lk$2002 MWf4tt$allitX Pl6 $N1@SyIJ4P1Mhlltha4mMtoXttThe mo1ecu1ar weight of so1uble proteins were 1owerthan 43I{Da. The 30, 2SKDa molecu1ar--weight protein bandswere significant1y reduced in the gastric cancer tissue,as compared to that in the norma1 tissue. There was nosignificant difference between we11, moderate1ydifferented and poor1y differented gastric cancer. Andthere was no significant difference between I-- II stage andIII--IVstage gastric cancer.2. the resu1ts of Southwestern b1ot:The major bands were 28, 30, 40, 43, 50KDamo1ecu1ar--weight proteins in normal tissue, whi1e 40, 66KDa mo1ecular--weight proteins in cancer ti ssue. There wasno difference on the 40KDa band. There was significantdiffereflce between florma1 afld cancer tissue on the 66KDaband. There was flo s ignificant difference betweefl high1y,moderate1y differented and poor1y differented gastriccancer. And there was no significant difference betweenI-- II stage and IlI--IVstage gastric cancer.Conc1usions were as fo1lows:82002 Iggf5＃ife:t pie1. The quantity of 30, 28KDa proteins were reduced in nuclear matrix in gastric cancer. There was no significant difference between highly, moderately differented and poorly differented gastric cancer. And there was no significant difference between I- II stage and III-IVstage gastric cancer. This ma...