| [Background]Adrenocortical carcinoma (ACC) is a primary malignant tumor in the adrenal cortex with low annual incidence rate which is 0.5-2/100 million,It is rare in clinical and accounted for 0.02% of all the malignant tumors.ACC is know as a tumor with high malignancy and strong neoplasm invasion.The diagnosis of ACC is difficult and a 5-year overall survival is about 40%. Most cases of ACC are unilateral,but 2%-6% cases of ACC are found in the both side of adrenal cortex.It also can be found with Li-Fraumeni syndrome,TypeImultiple adenomatous syndrome,Beckwith-Wiedemann syndrome and Garney syndrome in children.The pathogenesis of adrenocortical carcinoma is not clear. Chromosome 11p,13q andl7p may have effects on chromosome heterozygosity,ACC is testified as a multi-gene-related tumor, multiple signaling pathways involved in the pathogenesis is complex.Recent studies found that some of signal pathways like Wnt,IGF,P53 are connected with tumorigenesis and tumor progression.It seem hard for pathologists to distinguish adrenocortical carcinoma between adrenocortical adenoma in clinical.Based on this situation,Weiss put forward nine histological standards which is diagnosed malignancy if three of them match the criterion.Therapeutic options beside the surgical operation for the ACC patients are scarce,and available chemotherapies of limited effectiveness.Nowadays,some studies had confirmed the over-expressed PCAF and activated β-catenin play a key role in progression of ACC while the molecular mechanism was still not clear.Therefore,It will be important to find effective and stable genomic target for the early diagnose and therapy of adrenocortical carcinoma.PCAF (P300/CBP Associated Factor),a cofactor of activated nucleoprotein,is a important nonhistones regulate factor tightly connected physiological process of tumorigenesis like the process of cell cycle, differentiation,apoptosis and transcription.These compounds on the transcriptional regulation basically has two kinds:one is seem as auxiliary activation factor, the other is used to activate the transcription by making the histones and the histone specific sites of lysine residues (Lys) acetylation. The research of PCAF gene in tumors is still a new field while the expression in different tumors has discrepancy,PCAF showed reduced expression or no expression in the tumors like esophageal squamous cell carcinoma, breast cancer, liver cancer or tumor tissue in a variety of tumor cell lines including:Ovmana, OVI-P, etc, the reasons may due to loss of heterozygosity genome, promoter methylation and mutation. Nevertheless,The expression of PCAF in pediatric central nervous system tumors, Wilms tumor and prostate cancer show highly expressed.Recent studies showed CBP and p300 could acetylate β-catenin and regulate the affinity of β-catenin for Tcf4 and E2F1.This study provides theoretical references for the research of PCAF and Wnt/β-catenin signal pathway.Wnt/β-catenin is a highly conservative pathway in biological evolution.lt regulate large of biological characteristics and life activities. β-catenin as the major regulator play a significant role in transcriptional regulation. Disordered Wnt signal pathway cause abnormal accumulation of β-catenin protein and the behavior of transcelluar acumulation,then specific to activate target genes like cyby bonding to TCF/LEF.By this way,The signal pathway promotes tumorigenesis. Numerous malignancy,like hepatic carcinoma,gastric carcinoma,cervical cancer,had been proved the process of abnormal activation of Wnt/β-catenin signal pathway. Similarly,It also be reported in adrenocortical carcinoma.Epithelial-mesenchymal transition(epithelialmesenchymal transition,EMT) provides the basis for the invasion and metastasis of tumor cells of epithelial origin. A variety of signal transduction pathways involved in the EMT of tumor cells,include Mark,Notch and Wnt signal pathways.While the Wnt/β-catenin pathway is abnormally activated,inducing the process of EMT,the adhesion between tumor cells turn weakened,finally enhancing the ability of tumor cell invasion and metastasis.Many of malignancy and blood cancers are connected with EMT.However,Whether the EMT process exist in adrenocortical carcinoma,few study had reported.Based on above background,we try to figure out the development mechanism of PCAF gene and Wnt/(3-catenin signal pathway in adrenocortical carcinoma though clinical specimens and cellular level.Then we can lay the foundation of early diagnosis, effective treatment and prevention medicine in ACC.(Objective]By collecting the clinical pathological section of adrenal cortical carcinoma,adrenocortical adenoma and normal adrenal tissue,we make an expeiment to detect PCAF gene,proteins related to EMT and the key protein of Wnt/β-catenin signal pathway.Using the technology of siRNA deduce the expression of PCAF gene,then we make a invesgation of mechanism and influence on Wnt/β-catenin signal pathway in ACC cell.[Method]1).collect 15 cases of adrenal cortical carcinoma paraffin biopsy,20 cases of adrenocortical adenoma and 13 normal adrenal tissue in the General Hospital of Guangzhou Military Region from 2003-2014 year.The PCAF protein、EMT related protein E-Cadherin、Vimentin and β-catenin was detected in adrenal cortical carcinoma specimens by immunohistochemical staining.2).Three of the PCAF siRNA were designed and synthesized.According to technology of siRNA,adrenal cortical carcinoma SW13 cells were transfected by using transfection reagents like Lipo 2000.Observed transfection efficiency by theInversed fluorescent microscope.Real-time quantitative PCR(qPCR) was used to detected the inhibition of PCAF expression and screened effective siRNA sequences by qPCR.3).The most efficient PCAF-siRNA was transiently transfected into SW13 cells.SW13 cells were divided into three group:experiment group(PCAF-siRNA transfection group),negative control group (NC-siRNA transfection group)and normal group.After transfection 2 days,SW13 cells were collected and detected the expression of β-catenin protein by immunocytochemistry.The mean density of immunocytochemistry straining was analyzed by IPP software. β-catenin protein of ecach group was detected by qPCR. Total proteins and nuclear protein were extracted then the expression of P-catenin was detected by western blot.Dual luciferase reporter assay were performed to text the activity of Wnt/p-catenin signal pathway.4).SiRNA were used to transiently transfected into three group SW13 cells(PCAF-siRNA group、NC-siRNA group、normal group).Various target genes like c-myc、Cyclin D1、CD44、VEGF、MMP-7、PPAR-y in Wnt/β-catenin signal pathway in SW13 cells were detected by qPCR and western blot.[Results]1).Compared with groups of adrenocortical adenoma and normal adrenal tissue,PCAF was over expressed in adrenocortical carcinoma group,the difference was statistically significant(P=0.018,P=0.01),14 cases in the adrenocortical carcinoma group β-catenin was expressed in nucleus and more expressed in ACC group than adrenocortical adenoma and normal adrenal tissue. Compared with groups of adrenocortical adenoma and normal adrenal tissue,The protein of E-Cadherin in ACC group was decreased but the protein of Vimentin was over expressed,and the difference was statistically significant(P<0.05).The Spearman rank test indicated that the positive expression of PCAF was positively correlated with β-catenin nuclear expresion(r=0.375,P<0.05) and Vimentin(r=-0.432,P<0.05),but negatively associated with E-caherin protein expression (r=0.420,P<0.05).2).The SW13 cells were transfected by using transfection reagents and Cy3-siRNA with 50nM final density.Observed transfection efficiency was about 90% by the Inversed fluorescent microscope after 24h later.Finally,The most efficiency condition of transfection was 50nM final density,added in 3μl per orifice with 5h transfection.The expression of PCAF in each candidate siRNA group was detected by qPCR and the most efficient siRNA was PCAF siRNA 002 group.Further more,The propagated gene of PCAF was successfully observed in the right position by agarose gel electrophoresis.3).Protein of β-catenin detected by immunocytochemistry showed that compared to control group,the expression of β-catenin obviously decreased and lower expressed in nuclear in the PCAF-siRNA group.The activity of Wnt/β-catenin pathway detected by luciferase turned highly weaker in experiment group (9.53±1.92)than control group(15.41±1.24),the difference was statistically significant(P<0.05);The expression of β-catenin in PCAF-siRNA group was less than the other two groups,The same result was also found in the Western blot test,The expression of β-catenin in PCAF-siRNA group was less he other two groups not only in total protein but also in the nuclear protein.4). Various target genes like c-myc、Cyclin Dl、CD4、VEGF、MMP-7、PPAR-y in Wnt/β-catenin signal pathway in SW13 cells were detected by qPCR and Western blot. Compared to NC-siRNA group and normal group c-my、Cyclin D1、CD44、 VEGF、MMP-7、PPAR-γ was decreased in PCAF-siRNA while the diffrence about Survivin、TCF-1 genes was no statistically significant(p>0.05),Except c-myc protein,the same result by western blot test was same as the outcome of qPCR.(Conclusion]1). PCAF is highly expressed in adrenal cortical carcinoma, may involved in the EMT progress in ACC.The phenomenon of β-catenin gathering in nuclear was more obvious in ACC group than ADA and normal group,Further more,the result provided a basis theory for the future study about the Wnt/β-catenin signal pathway in vivo.2).The expression of β-catenin obviously decreased in nuclear of SW13 cells by down-regulation of PCAF gene and the activity of Wnt/β-catenin pathway detected by luciferase turned highly weaker.3).Down-regulation of PCAF gene decreased the various target genes like Cyclin D1、CD44、VEGF、MMP-7、PPAR-γ in Wnt/β-catenin signal pathway.The gene of PCAF may play a key role for tumorigenesis in adrenocortical carcinoma. |
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