MiR-96-5p inhibition induces cell apoptosis in gastric adenocarcinoma

BACKGROUND Gastric adenocarcinoma(GAC)mortality rates have remained relatively changed over the past 30 years,and it continues to be one of the leading causes of cancerrelated death.AIM To search for novel miRNAs related to GAC prognosis and further investigate the effect of miR-96-5p on MGC-803 cells.METHODS The miRNA expression profile data of GAC based on The Cancer Genome Atlas were obtained and used to screen differently expressed miRNAs(DEMs)and DEMs related to GAC prognosis.Then,the expression of DEMs related to GAC prognosis was identified in GAC tumor samples and adjacent normal samples by qRT-PCR.The target gene,ZDHHC5,of miR-96-5p was predicted using TargetScan,miRTarBase,and miRDB databases and confirmed by luciferase reporter assay.Furthermore,MGC-803 cells were transfected with inhibitor NC,miR-96-5p inhibitor,si-ZDHHC5,or miR-96-5p inhibitor+si-ZDHHC5,and then cell apoptosis was detected by flow cytometry.The expression of ZDHHC5,Bcl-2,and COX-2 was detected using western blotting.RESULTS A total of 299 DEMs and 35 DEMs related to GAC prognosis were screened based on The Cancer Genome Atlas.Then compared with adjacent normal samples,the levels of miR-96-5p,miR-222-5p,and miR-652-5p were remarkably increased,while miR-125-5p,miR-145-3p,and miR-379-3p levels were reduced in GAC tumor samples(P<0.01),which were consistent with bioinformatics analysis.Furthermore,ZDHHC5 was defined as a direct target gene of miR-96-5p.miR-96-5p inhibition increased the number of apoptotic cells as well as promoted the expression of ZDHHC5,Bcl-2,and COX-2 in MGC-803 cells(P<0.01).After ZDHHC5 inhibition,the number of apoptotic cells and the expression of ZDHHC5,Bcl-2,and COX-2 were reduced.The addition of an miR-96-5p inhibitor partly reversed these effects(P<0.01).CONCLUSION Our findings identified six miRNAs related to GAC prognosis and suggested that downregulated miR-96-5p might induce cell apoptosis via upregulating ZDHHC5 expression in MGC-803 cells.

Adjuvant chemotherapy,p53,carcinoembryonic antigen expression and prognosis after D2 gastrectomy for gastric adenocarcinoma

AIM: To investigate adjuvant chemotherapy, p53 and carcinoembryonic antigen (CEA) expression and prognosis after D2 gastrectomy for stage II/III gastric adenocarcinoma.

Effect of cis-9,trans-11-conjugated linoleic acid on cell cycle of gastric adenocarcinoma cell line(SGC-7901)

AIM: To determine the effect of cis -9, trans -11-conjugated linoleic acid (c9, t11-CLA) on the cell cycle of gastric cancer cells (SGC-7901) and its possible mechanism in inhibition cancer growth. METHODS: Using cell culture and immunocytochemical techniques, we examined the cell growth, DNA synthesis, expression of PCNA, cyclin A, B(1), D(1), p16(ink4a) and p21(cip/waf1) of SGC-7901 cells which were treated with various c9, t11-CLA concentrations (25, 50, 100 and 200 micromol.L(-1))of c 9, t 11-CLA for 24 and 48h, with a negative control (0.1% ethane). RESULTS: The cell growth and DNA synthesis of SGC-7901 cells were inhibited by c9, t11-CLA.SGC-7901 cells. Eight day after treatment with various concentrations of c9, t11-CLA mentioned above, the inhibition rates were 5.92%, 20.15%, 75.61% and 82.44%, respectively and inhibitory effect of c9, t11-CLA on DNA synthesis (except for 25 micromol.L, 24h) showed significantly less (3)H-TdR incorporation than that in the negative controls (P【0.05 and P【0.01). Immunocytochemical staining demonstrated that SGC-7901 cells preincubated in media supplemented with different c9, t11-CLA concentrations at various times significantly decreased the expressions of PCNA (the expression rates were 7.2-3.0%, 24h and 9.1-0.9% at 48h, respectively), Cyclin A (11.0-2.3%, 24h and 8.5-0.5%,48h), B(1) (4.8-1.8% at 24h and 5.5-0.6% at 48h)and D(1) (3.6-1.4% at 24h and 3.7%-0 at 48h) as compared with those in the negative controls(the expressions of PCNA, Cyclin A, B(1) and D(1) were 6.5% at 24h and 9.0% at 48h, 4.2% at 24h and 5.1% at 48h, 9.5% at 24h and 6.0% at 48h,respectively)(P【0.01), whereas the expressions of P16(ink4a) and P21(cip/waf1), cyclin-dependent kinases inhibitors(CDKI), were increased. CONCLUSION: The cell growth and proliferation of SGC-7901 cell is inhibited by c9, t11-CLA via blocking the cell cycle, with reduced expressions of cyclin A,B(1) and D(1) and enhanced expressions of CDKI(P16(ink4a) and p21(cip/waf1)).

Expression of p16 gene and Rb protein in gastric carcinoma and their clinicopathological significance

AIM:To analyze the correlation between the protein expression of p16 and Rb genes in gastric carcinoma (GC), to investigate the role of p16 gene in invasion and lymph node metastasis of GC, and to examine the deletion and mutation in exon 2 of p16 gene in GC. METHODS: The protein expression of p16 and Rb genes was examined by streptavidin-peroxidase conjugated method (S-P) in normal gastric mucosa, dysplastic gastric mucosa and GC. The deletion and mutation of p16 gene were examined by polymerase chain reaction (PCR) and polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) respectively in normal gastric mucosa and GC. RESULTS: The positive rates of P16 and Rb protein expression respectively were 96% (77/80) and 99% (79/80) in normal gastric mucosa, 92% (45/50) and 80% (40/50) in dysplastic gastric mucosa, 48% (58/122) and 60% (73/122) in GC. The positive rates of P16 and Rb protein expression in GC were significantly lower than that in normal gastric mucosa and dysplastic gastric mucosa (P<0.05). The positive rate of P16 protein expression in mucoid carcinoma (10%, 1/10) was significantly lower than that in poorly differentiated carcinoma (51%, 21/41), undifferentiated carcinoma (58%, 15/26) and signet ring cell carcinoma (62%, 10/16) (P<0.05). The positive rates of P16 protein in 30 cases of paired primary and lymph node metastatic GC were 47% (14/30) and 17% (5/30) respectively, being significantly lower in the later than in the former (P<0.05). There was no mutation in exon 2 of p16 gene in the 25 freshly resected primary GCs. But five cases in the 25 freshly resected primary GCs displayed deletion in exon 2 of p16 gene. The positive rate of both P16 and Rb proteins was 16% (14/90), and the negative rate of both P16 and Rb proteins was 8% (7/90) in 90 GCs. The rate of positive P16 protein with negative Rb protein was 33% (30/90). The rate of negative P16 protein with positive Rb protein was 43% (39/90). There was reverse correlation between P16 and Rb expression in 90 GCs CONCLUSION: The loss protein expression of p16 and Rb genes is related to GC. The loss expression of P16 protein is related to the histopathologic subtypes and lymph node metastasis of GC. Expression of P16 and Rb proteins in GC is reversely correlated. The deletion but not mutation in exon 2 of p16 gene may be involved in GC.

CSN6 promotes tumorigenesis of gastric cancer by ubiquitin-independent proteasomal degradation of p16INK4a

Objective: CSN6 is a vital subunit of the constitutive photomorphogenesis 9(COP9) signalosome(CSN), which is responsible for development disorders and promotes ubiquitin-26 S proteasome-dependent degradation in vitro and vivo.Its role in the tumor development of gastric cancer remains unclear.In this study, we investigated the role of CSN6 in gastric cancer progression.Methods: Human gastric cancer samples were collected and immunohistochemistry was performed to identify the role of CSN6 in gastric cancer.The cell proliferation was measured by CCK-8 and the EdU incorporation method.Immunofluorescence localization and a co-immunoprecipitation study were used to show the interaction between the protein CSN6 and p16.Ubiquitination assay was performed to validate whether ubiquitination is involved in CSN6-mediated p16 degradation.BALB/c nude mice were used to produce a tumor model in order to test the effect of CSN6 on cancer growth in vivo.Results: CSN6 expression was dramatically increased in gastric cancer tissues compared with paired adjacent non-tumor tissues and CSN6 was correlated with worse overall and disease-specific survival.Additionally, we also found that CSN6 downregulated p16 protein expression, thereby promoting gastric cancer cell growth and proliferation.Moreover, CSN6 interacted with p16 and a proteasome activator REGγ(PA28γ), thereby facilitating ubiquitin-independent degradation of p16.Conclusions: CSN6 promoted the loss of p16-mediated tumor progression and played an important role in regulating ubiquitin-independent proteasomal degradation of p16.

Role of Helicobacter Pylori Infection in Pathogenesis of Gastric Adenocarcinoma

To study the effect of Helicobacter Pylori ( Hp ) on the process of gastric carcinogenesis, 35 cases of chronic gastritis, 20 cases of gastric adenocarcinoma were studied by use of transmission electron microscopy, immunohistochemical and molecular biological technique. The results showed that 24 of 35 cases of chronic gastritis were positive for Hp, 11/20 cases of gastric adenocarcinoma were Hp positive. PCNA positive cell labeling index (LI) in Hp associated chronic gastritis (LI= 20.6±4.7) was higher than that in Hp negative chronic gastritis (LI=11.3±5.2) ( P <0.05). HSP70 expression of gastric adenocarcinoma tissues in Hp infected patients were lower than that of non Hp infected gastric cancer. p53 gene mutation was found in gastric adenocarcinoma with positive Hp . It was suggested that Hp may enhance gastric cell proliferation, decrease the expression of HSP70 which induces p53 mutation.

EXPRESSION OF p16,CYCLIN D1 AND RB PROTEIN IN GASTRIC CARCINOMA AND PREMALIGNANT LESIONS

Objective: To investigate the expression of p16, cyclin D1 and Rb protein in gastric carcinoma and premalignant lesions including dysplastic gastric mucosa and intestinal metaplasia gastric mucosa. Methods: Using SP immunohistochemical methods, the expression of pl6, cyclin D1 and Rb proteins was detected in 10 specimens of normal gastric mucosa, 15 specimens of dysplastic gastric mucosa, 15 specimens of intestinal metaplasia gastric mucosa, 30 specimens of gastric carcinoma. The clinical characteristics of the 30 patients with gastric carcinoma were analysed to explore the relationship between the parameter detected and biological action of gastric cancer. Results: Expression of p16 protein was detected in 90% of normal gastric mucosa, 86.67% of dysplastic gastric mucosa, 86.67% of intestinal metaplasia gastric mucosa, 36.67% of gastric carcinoma. The positive rate of p16 protein expression in gastric carcinoma is significantly lower than that in normal gastric mucosa and gastric premalignant lesions mucosa (P<0.01). Expression of cyclin D1 protein was detected in 10% of normal gastric mucosa, 20% of dysplastic gastric mucosa, 20% of intestinal metaplasia gastric mucosa, 53.33% of gastric carcinoma. The positive rate of cyclin D1, protein expression in gastric carcinoma is significantly higher than that in normal gastric mucosa and gastric premalignant lesions mucosa (P<0.05). Expression of Rb protein was detected in 90% of normal gastric mucosa, 80% of dysplastic gastric mucosa, 80% of intestinal metaplasia gastric mucosa, 50% of gastric carcinoma. The positive rate of Rb protein expression in gastric carcinoma is significantly lower than that in normal gastric mucosa (P<0.05). The expression of p16, cyclin D1 gene were associated with the degree of differentiation of gastric carcinoma, lymphnodes metastasis and distant metastasis. Conclusion: p16, Cyclin D1 and Rb gene play important role in gastric carcinoma genesis. The expression of p16, cyclin D1 and Rb gene have some value to the diagnosis at earlier stage of gastric cancer. Detection of expression of p16, cyclin D1 gene would be helpful to judge the prognosis of gastric cancer.

EXPRESSION OF P16 AND CYCLIN D1 IN THE COURSE OFCARCINOGENESIS OF THE STOMACH

Objective: To determine p16 and cyclin D1 expression in the specimen of gastric carcinoma, atypic hyperplasia, atrophic gastritis, superficial gastritis and normal gastric mucosa. Methods: Using immunohistochemical method (ABC), the samples of 58 adenocarcinomas, 22 atypic hyperplasias, 28 atrophic gastritis, 27 superficial gastritis and 15 gastric epitheliums were analyzed. Results: Positive immunostaining rate for p16 protein was the highest in normal gastric mucosa and decreased with the lesions progressing from superficial gastritis to atrophic gastritis to atypital hyperplasia and to adenocarcinoma (85%, 78.6%, 31.8%, 48.3% respectively); Positive immunostaining of cyclin D1 can observed in atrophic gastritis. With the lesions progressing from atrophic gastritis to atypical hyperplasia to adenocarcinoma, its expression rate increased (17.9%, 36.4%, 53.4% respectively), and there was a significant difference between adenocarcinoma and atrophic gastritis group (P<0.05). An interesting observation was that inverse expression between p16 and cyclin D1, was shown in most of gastric cancer detected. Conclusion: It is indicated that p16 and cyclin D1 play an important role in the gastric carcinogenesis, the inverse expression between p16 and cyclin D1 suggested that there is a suppression trend in them.

The SMAD2/miR-4256/HDAC5/p16^(INK4a) signaling axis contributes to gastric cancer progression

The dysregulation of exosomal microRNAs(miRNAs)plays a crucial role in the development and progression of cancer.This study investigated the role of a newly identified serum exosomal miRNA miR-4256 in gastric cancer(GC)and the underlying mechanisms.The differentially expressed miRNAs were firstly identified in serum exosomes of GC patients and healthy individuals using next-generation sequencing and bioinformatics.Next,the expression of serum exosomal miR-4256 was analyzed in GC cells and GC tissues,and the role of miR-4256 in GC was investigated by in vitro and in vivo experiments.Then,the effect of miR-4256 on its downstream target genes HDAC5/p16^(INK4a) was studied in GC cells,and the underlying mechanisms were evaluated using dual luciferase reporter assay and Chromatin Immunoprecipitation(ChIP).Additionally,the role of the miR-4256/HDAC5/p16^(INK4a) axis in GC was studied using in vitro and in vivo experiments.Finally,the upstream regulators SMAD2/p300 that regulate miR-4256 expression and their role in GC were explored using in vitro experiments.miR-4256 was the most significantly upregulated miRNA and was overexpressed in GC cell lines and GC tissues;in vitro and in vivo results showed that miR-4256 promoted GC growth and progression.Mechanistically,miR-4256 enhanced HDAC5 expression by targeting the promoter of the HDAC5 gene in GC cells,and then restrained the expression of p16^(INK4a) through the epigenetic modulation of HDAC5 at the p16INK4a promoter.Furthermore,miR-4256 overexpression was positively regulated by the SMAD2/p300 complex in GC cells.Our data indicate that miR-4256 functions as an oncogene in GC via the SMAD2/miR-4256/HDAC5/p16^(INK4a) axis,which participates in GC progression and provides novel therapeutic and prognostic biomarkers for GC.

p16 promoter hypermethylation:A useful serum marker for early detection of gastric cancer

AIM： TO determine p15 promoter hypermethylation in gastric tumoral tissue and serum samples, its impact on p16-protein expression, and correlation with clinical and histological features. METHODS： Samples were obtained from 52 histologically confirmed cases of gastric adenocarcinoma. Gastric tissue and serum of 50 age- and sex-matched individuals with normal gastroscopy and biopsy were obtained as control samples. Methylation-specific polymerase chain reaction （MSP） was used to evaluate methylation status of p16 promoter, p16-protein expression was analyzed by immunohistochemical staining on paraffin-embedded sections. RESULTS： Methylation was detected in 44.2% （23/52） of tumoral tissues. 60.9% of them were also methylated in serum, i.e., 26.9% of all patients （14/52）. Methylation was not detected in tissue and sera of control samples. p16-protein expression was decreased in 61.5% of cases （32/52）, and was significantly associated with promoter hypermethylation （P 〈 0.001）. Methylation was significantly more frequent in higher pathological grades （P 〈 0.05）. Methylation was not associated with other clinicopathological features and environmental factors including Hpylori infection and smoking. CONCLUSION： p16 promoter hypermethylation is an important event in gastric carcinogenesis. It is the principle mechanism of p16 gene silencing. It is related to malignant tumor behavior. Detection of DNA methylation in serum may be a biomarker for early detection of gastric cancer.

胃癌及癌前病变中p16基因启动子异常甲基化研究

目的探讨p16基因异常甲基化在胃癌发生中的作用。方法应用甲基化特异性PCR技术检测胃腺癌及癌前病变组织中p16基因甲基化状态,并应用免疫组化染色检测p16蛋白的表达。结果慢性萎缩性胃炎、重度异型增生及胃腺癌组织p16基因启动子甲基化阳性率分别为11.1%、40%和43.3%。胃腺癌组与浅表性胃炎组、萎缩性胃炎组及轻-中度异型增生组间p16基因甲基化阳性率差异有显著性(P<0.05),而与重度异型增生组间差异无显著性(P>0.05)。p16蛋白阳性率在胃腺癌组、重度异型增生、轻-中度异型增生、萎缩性胃炎组和浅表性胃炎组分别为36.7%、40%、85.7%、83.3%和100%;在19例p16基因甲基化阳性病例中,18例p16蛋白缺失。结论 p16基因启动子异常甲基化可能是其在胃癌蛋白表达缺失的主要原因。p16基因启动子异常甲基化可能是胃癌发生的早期事件,在胃癌的发生起重要作用。

食管贲门双源癌患者癌组织中mdm2,CyclinD1和p16蛋白的表达

目的:探讨食管贲门双源癌组织中mdm2、CyclinD1和p16蛋白的表达。方法:采用免疫组化ABC法,对河南食管癌高发区30例双源癌患者食管鳞癌(SCC)和贲门腺癌(GCA)组织中mdm2、CyclinD1和p16蛋白的表达进行检测。结果:30例双源癌患者SCC组织中mdm2、CyclinD1和p16蛋白阳性率分别为57%(17/30)、45%(13/29)和24%(7/29),GCA组织中分别为73%(22/30)、33%(10/30)和17%(5/30),SCC和GCA组织中3种蛋白的阳性率差异均无统计学意义(P均>0.05)。SCC和GCA组织中mdm2、CyclinD1和p16蛋白表达的一致性差异无统计学意义(P均>0.05)。结论:食管和贲门双源癌存在较高的mdm2、CyclinD1和p16蛋白表达一致性改变,提示河南食管癌高发区食管贲门双源癌可能具有相似的发病因素和分子机制。

p16、p53、PR、Vim在原发宫颈腺癌中的表达及意义

目的探讨p16、p53、PR、Vim蛋白在原发性宫颈腺癌中的表达及临床病理意义。方法收集江西省妇幼保健院存档的子宫颈腺癌标本120例,取正常宫颈组织30例作为对照组,采用PV9000两步法检测p16、p53、PR、Vim蛋白的表达。SPF10 PCR技术进行DNA扩增,检测宫颈腺癌的HPV DNA。对所有石蜡切片进行病理阅片和诊断,分析子宫颈腺癌标本的p16、p53、PR、Vim蛋白表达情况。结果在30例正常宫颈组织中,p16、p53、PR、Vim的阳性表达率分别为3.3%、0%、43.3%、0%。在120例宫颈腺癌中,p16、p53、PR、Vim的阳性表达率分别为86.67%、56.67%、11.67%、15.00%。四组实验结果,差异均有统计学意义(P<0.05)。120例子宫颈腺癌标本中,HPV阳性率为77.5%。p16、p53、PR、Vim在93例HPV阳性的宫颈腺癌中的阳性表达率分别为94.62%、49.46%、11.83%、16.13%。p16蛋白表达与HPV感染有相关性(P<0.001),p53蛋白表达与HPV感染有相关性(P=0.003),PR蛋白表达与HPV感染无相关性(P=1.000),Vim蛋白表达与HPV感染无相关性(P=0.760)。结论p16、p53、PR、Vim对鉴别腺体良恶性病变具有重要意义,为正确估计宫颈腺癌的预后、指导临床治疗提供了重要依据。

不同类型的胃炎和胃癌组织中p21、p16蛋白的表达及临床意义

目的探讨不同类型胃炎与胃癌的相关性。方法采用免疫组化法检测p21、p16在疣状胃炎、非萎缩性胃炎、萎缩性胃炎及胃癌各40例组织中的表达,并对各组值进行比较,得出结论。结果 p21在非萎缩性胃炎、萎缩性胃炎、疣状胃炎及胃癌组织的表达分别为0、22.5%、35.0%、60.0%,疣状胃炎的p21阳性率显著高于非萎缩性胃炎,低于胃癌,各组间比较差异有统计学意义(P<0.05),但与萎缩性胃炎比较差异无统计学意义;p16在非萎缩性胃炎、萎缩性胃炎、疣状胃炎及胃癌组织的表达分别为90.0%、50.0%、45.0%、17.5%,疣状胃炎组p16阳性率显著低于非萎缩性胃炎,明显高于胃癌组,各组间比较差异有统计学意义(P<0.01),但与萎缩性胃炎比较差异无统计学意义。结论 Ras基因激活后形成的p21蛋白及抑癌基因p16失活可能参与疣状胃炎的癌变过程。

低分化胃腺癌组织AUF1与p16表达的负相关

RNA结合蛋白AUF1(AU-富含元件结合/降解因子)通过结合并促进抑癌基因p16 mRNA降解来抑制p16表达.然而,AUF1-p16调控过程在肿瘤发生发展过程中的意义有待探讨.本研究用Western印迹与RT-PCR技术分别检测临床50例患者低分化胃腺癌组织和癌旁组织细胞中AUF1和p16蛋白、p16 mRNA的表达情况,并分析其关联性;用RNA Pull-down技术检测其AUF1与p16mRNA的结合情况.结果显示,低分化胃腺癌组织AUF1蛋白表达水平明显增高,且与p16蛋白和p16 mRNA相对表达水平呈负相关;RNA pull-down分析结果显示,癌组织AUF1与p16-3'UTR的结合活性明显强于癌旁组织.提示AUF1-p16调控过程可能是低分化胃腺癌组织p16水平降低的重要机制.

抑癌基因p16与胃腺癌患者XELOX方案化疗预后的关系

目的:探讨病灶组织抑癌基因p16的表达与胃腺癌患者化疗后2年和5年生存率之间的关系.方法:选择2004-03/2007-02来我院接受治疗的胃腺癌晚期患者47例作为观察组,均接受XELOX(奥沙利铂+希罗达)方案化疗.选择25例健康志愿者作为对照组.采用免疫组织化学检测观察组和对照组患者胃黏膜组织上皮组织内p16表达情况,并比较观察组病灶组织p16低表达和正常表达组2年和5年生存率.结果:观察组p16阳性率为17.4%±3.6%,显著低于对照组(P<0.05).p16正常表达组有19例(40.43%),低表达组有28例(59.57%).p16低表达组16例患者经化疗后2年生存6例(31.58%),5年生存4例(21.05%),均显著低于p16正常表达组(P<0.05).结论:p16过低表达胃腺癌患者化疗预后较差,且p16基因表达可以作为预测胃腺癌XELOX方案化疗预后.

胃腺癌p16与Rb蛋白的表达及相关性研究

目的探讨p16、Rb蛋白与胃腺癌发生发展的关系,分析p16与Rb蛋白的表达及其相关性。方法应用免疫组化SP法检测102例胃腺癌中p16与Rb蛋白的表达情况。结果在31例Rb蛋白阳性标本中,24例显示p16蛋白表达缺失或低表达,在23例Rb蛋白阴性标本中有19例显示p16蛋白阳性或强阳性。结论p16蛋白表达缺失及Rb蛋白表达阳性在胃腺癌发生和发展中发挥重要作用。p16与Rb蛋白阳性表达的相互抑制,是胃腺癌的特征之一。

Implication of HPV16 Infection and P21 Gene Mutation in the Carcinogenesis and Prognosis of Gastric Cancer

Objective： To investigate whether there is a synergistic carcinogenesis between the infection of human papilloma virus （HPV） and the P21 gene mutation in gastric cancer tissue and their relationship with prognosis of the patients with gastric cancer. Methods： By using PCR technique, HPV16 infection in 46 gastric cancer tissue samples was measured and by using immunohistochemical S-P method, the P21 gene mutation in gastric cancer was detected. All patients were regularly followed up for 3 years by writing letter or clinics, to detect the infection of HPV16 by PCR and the p21 gene mutation by immunohistochemical method in 46 gastric cancer tissue specimens. Results： The positive rate of HPV16 was 41.3% and the gene mutation rate of p21 was 52.17% respectively. The recurrence or remote metastasis was observed in 21 of 46 patients. The recurrence rate was 73.68% in the patients positive for HPV16 and 66.6% in those positive for p21 gene mutation. In 8 cases positive for both HPV16 and P21, 6 had recurrence or remote metastasis. Conclusion： The HPV16 infection may be one factor causing gastric cancer and it has a synergistic carcinogenesis with the p21gene mutation. The latter may be one of the prognostic indices in gastric cancer.

Expression,deleton and mnutation of ρ16 gene in human gastric cancer

AIM To investigate the relationship between the expression of p16 gene and the gastric carcinogenesis,depth of invasion and lymph node metastases, and to evaluate the deletion and mutation of exon 2 in p16 gene in gastric carcinoma.METHODS The expression of P16 protein was examined by streptavidin-peroxidase conjugated method (S-P); the deletion and mutation of p16 gene were respectively examined by polymerase chain reaction (PCR) and polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP) in gastric carcinoma.RESULTS Expression of P16 protein was detected in 96.25% (77/80) of the normal gastric mucosa, in 92.00% (45/50) of the dysplastic gastric mucosa and in 47.54% (58/122) of the gastric carcinoma. The positive rate of P16 protein expression in gastric carcinoma was significantly lower than that in normal gastric mucosa and dysplastic gastric mucosa (P＜0.05). The positive rate of P16 protein expression in mucoid carcinoma 10.00% (1/ 10) was significantly lower than that in poorly differentiated carcinoma 51.22% ( 21/ 41 ),undifferentiated carcinoma 57.69% (15/26) and signet ring cell carcinoma 62.50% (10/ 16) (P＜0.05). The positive rate of p16 protein in 30 cases paired primary and lymph node metastatic gastric carcinoma: There was 46.67% (14/30) in primary gastric carcinoma, 16.67% (5/30) in lymph node metastatic gastric carcinoma. The positive rate of lymph node metastatic carcinoma was significantly lower than that of primary carcinoma (P＜0.05). There was of p16 gene mutation in exon 2, but 5 cases displayed deletion of p16 gene in exon 2 in the 25 primary gastric carcinomas.CONCLUSIONS The expression loss of P16 protein related to the gastric carcinogenesis, gastric carcinoma histopathological subtypes and lymph metastasis. The mutation of p16 gene in exon 2 may not be involved in gastric carcinogenesis. But the deletion of p16 gene in exon 2 may be involved in gastric carcinogenesis.

Re-expression of methylation-induced tumor suppressor gene silencing is associated with the state of histone modification in gastric cancer cell lines

AIM： To identify the relationship between DNA hyper- methylation and histone modification at a hyperme- thylated, silenced tumor suppressor gene promoter in human gastric cancer cell lines and to elucidate whether alteration of DNA methylation could affect histone modification. METHODS： We used chromatin immunoprecipitation （CHIP） assay to assess the status of histone acetylation and methylation in promoter regions of the p16 and rnutL homolog 1 （MLH1） genes in 2 gastric cancer cell lines, SGC-7901 and MGC-803. We used methylation- specific PCR （MSP） to evaluate the effect of 5-Aza-2＇- deoxycytidine （5-Aza-dC）, trichostatin A （TSA） or their combination treatment on DNA methylation status. We used RT-PCR to determine whether alterations of histone modification status after 5-Aza-dC and TSA treatment are reflected in gene expression. RESULTS： For thep16 and MLH1 genes in two cell lines, silenced loci associated with DNA hypermethylation were characterized by histone H3-K9 hypoacetylation and hypermethylation and histone H3-K4 hypomethylation. Treatment with TSA resulted in moderately increased histone H3-K9 acetylation at the silenced loci with no effect on histone H3-K9 methylation and minimal effects on gene expression. In contrast, treatment with 5-Aza- dC rapidly reduced histone H3-K9 methylation at the silenced loci and resulted in reactivation of the two genes. Combined treatment with 5-Aza-dC and TSA was synergistic in reactivating gene expression at the loci showing DNA hypermethylation. Similarly, histone H3-K4 methylation was not affected alter TSA treatment, andincreased moderately at the silenced loci after 5-Aza-dC treatment. CONCLUSION： Hypermethylation of DNA in promoter CpG islands is related to transcriptional silencing of tumor suppressor genes. Histone H3-K9 methylation in different regions of the promoters studied correlates with DNA methylation status of each gene in gastric cancer cells. However, histone H3-K9 acetylation and H3-K4 methylation inversely correlate with DNA methylation status of each gene in gastric cancer cells. Alteration of DNA methylation affects histone modification.