Parafibromin Expression And Its Mechanism In Colorectal Carcinoma

Colorectal cancer is one of the most common cancers in the world,accounting for nearly10%of new cases of all cancer. Pathological and geneticobservations demonstrated that colorectal adenoma precedes the majority ofcolorectal adenocarcinoma and could undergo malignant transformation intoadenocarcinoma. However, the molecular mechanisms underlying thiscolorectal carcinogenesis are still poorly understood.Parafibromin is a protein encoded by the HRPT2(hyperpara-thyroidism2)oncosuppressor gene and its down-regulated expression is involved inpathogenesis of parathyroid, breast and gastric carcinomas. It is intimatelyrelated to malignant pathological behavior of tumors. Parafibromin can formcomplexes with cleavage and polyadenylation specificity factor(CPSF),leavage stimulation factor (CStF) and symplekin.It can also interact withhistone methyltransferase(HMT) and poly adenosine element binding protein1(CPEB1).These compounds can regulate activity of promotor or maturationof mRNA after its binding to nucleotide sequence in order to participate in cellcycle,proliferation,apoptosis,differentiation,migration and transition.First part Expression and clinicopathological meaning of parafibrominin colon cancer tissueObject:To clarify parafibromin location and expression of colorectal carcinomaand its relation with clinicopathological feature, so as to explore the clinicalpathological meaning of parafibromin expression in colorectal carcinoma.Method:1. RT-PCR to test parafibromin mRNA expression in frozen colorectal carcinoma and paracancerous tissue of15cases;2. Western blotting to test parafibromin protein expression in frozencolorectal carcinoma and paracancerous tissue of15cases;3. Construct tissue chip and using immunohistochemistry to testparafibromin expression of colorectal carcinoma (n=306) and paracanceroustissue (n=279), and analyse the clinicopathological meaning of parafibrominexpression in colorectal carcinoma tissue.4. Statistical evaluation was performed using Spearman correlation test toanalyze the rank data. Kaplan-Meier survival plots were generated andcomparisons between survival curves were made with the log-rank statistic.The Cox’s proportional hazards model was employed for multivariate analysis.P<0.05was considered as statistically significant. SPSS13.0software wasemployed to analyze all data.Results:1. mRNA and protein expression in frozen colorectal carcinoma tissueAmong15cases of carcinoma and paracancerous tissue, there are each4cases without objective bands, which suggest positive rate of mRNAexpression is respectively73.3%(11/15).Statistical analysis suggest that thereis no significant difference(P＞0.05) in parafibromin mRNA expressionbetween colorectal carcinoma and paracarcinoma tissue. In the15cases ofparacarcinoma tissue,there are2cases without protein objective bands; in the15cases of carcinoma tissue there are parafibromin protein bands, whichsuggest there is no significant difference with paracarcinoma tissue(P＞0.05).2. Parafibromin protein expression in colorectal carcinoma tissue and itsrelation with clicopathological parameters.Using immunohistochemistry to test parafibromin protein expression incolorectal carcinoma and paracarcinoma tissue, the result suggestparafibromin protein expresses in cell nucleus. Positive rates of parafibrominexpression in colorectal carcinoma and paracarcinoma tissue are respectively53.9%(165/306) and91.4%(255/279).Statistical analysis suggest thatparafibromin expression in colorectal carcinoma tissue is less than in paracarcinoma normal mucosa(P＜0.05).Its expression is unrelated topatient’s gender(P=0.292),age(P=0.758) and vein invasion(P=0.173); isinversely related to tumor diameter(P=0.019),UICC stages(P=0.173) andlymph vessel transition(P=0.009). Kaplan-Meier survival plots weregenerated and suggests patient accumulated survival rate with positiveparafibromin expression is evidently higher than negative ones(P＜0.05).Cox’s proportional hazards model suggests parafibromin expression is aindependently prognostic factor of colorectal carcinoma patients(P＞0.05).Conclusion:1. There is no significant difference in parafibromin mRNA and proteinexpression levels between colorectal carcinoma and paracarcinoma tissue.2. Decrease of parafibromin expression in colorectal carcinoma tissuemay play a role in progression and transition of colorectal carcinoma.3. Decrease of parafibromin expression may impact survival time of thepatients and it can be used as a prognostic biological marker. Second part Parafibromin recombinant transfected cells’ biologicaleffect and its mechanismObject:To study extraneous gene parafibromin’s effect on DLD-1cell line and itsprobable mechanism.Method:1. Transfect parafibromin expression plasmid stably to selected colorectalcarcinoma cell lines, using western and RT-PCR to detect parafibromintranscription and expression levels and then pick out monoclone cell lines withoverexpression of parafibromin.2. Using PI to detect changes of cell cycle between before and aftertransfection, ALP activity to test cell differentiation before and after transfection, scorning test and transwell test to detect cell invasion andtransition degree before and after transfection,in order to study biologicalbehavior change of the cell lines.3. Using qRT-PCR to test mRNA expression levels of cycle and apoptosisfactor.Result:1. Parafibromin mRNA and protein expression after stably parafibromineukaryon plasmid transfectionWe selected DLD-1cell lines and stably transfected parafibromin-expressionrecombinant carrier and then picked out3kinds monoclone cell lines to detectexpression of mRNA and protein. We found that only number1monoclonemRNA and protein expression levels are higher than non-transfected cell lineswithout reaching double of the non-transfected cell lines, the difference isstatistical significantly(P＜0.05),there are no evidently change in othermonoclone cell lines(P＞0.05).2. Biological behavior change of DLD-1cell line after parafibromintransfectionAfter parafibromin transfection,G1(53.5%),S(30.5%) and G2(16.0%)；non-transfected cell line every cell cycle’s proportion is G1(48.5%), S(26.5%)and G2(25.0%),after transfection G1and S stages proportions are evidentlyincreased, while G2stage proportion evidently decreased, the difference isstatistical significantly(P＜0.05);ALP activity is12pg/ug after transfection,ALP activity of non-transfected cell is6.5pg/ug, alkaline phosphatase activityof transfected cell is significantly increased, the difference is statisticallysignificant(P＜0.05).Transwell test didn’t find invasion both in transfectedcells and non-transfected cells, there is no statistical significance(P＞0.05).Inthe scorning test, parafibromin transfected cell transit for68.9um after12h,118.9um after24h, non-transfected cell transit for58.9um after12h,124.6umafter24h,there is no significant difference(P＞0.05).3. Cycle and apoptosis factors mRNA expression levels before and afterparafibromin transfection. In the parafibromin transfected cells,c-myc,cyclinD1,cdc2,AKT1,AKT2and Bad mRNA expression levels decreased, parafibromin,cyclinB1mRNAexpression levels increased, the difference is statistically significant(P＜0.05).Between before and after transfection,there is no significant differencein p21,p27, cyclinE1, AKT3, Bax, β-catenin mRNA expression(P＞0.05).Conclusion:1. After DLD-1cells transfecting by parafibromin recombinant,parafibromin mRNA and protein expression levels increased.2. After transfection,DLD-1cell presented cell cycle stagnation,parafibromin promotes DLD-1cell differentiation, but not the invasion andtransition.3. After transfection, cell cycle protein mRNA expression decreased,supression apoptosis factor mRNA expression decreased, cell adhesion factorshowed no evident difference.