Polymorphism of p16INK4a gene and rare mutation of p15INK4b gene exon2 in primary hepatocarcinoma

INTRODUCTION Hepatocellular carcinoma(HCC)is the mostcommon cause of death from cancer in China.Themechanisms of hepatocarcinogenesis are not yetknown clearly,p16INK4a gene,the multiple tumorsuppressor gene 1(MTS1),encodes P16 protein,which acts as an inhibitor by binding directly toCDK4 and CDK6 and preventing its association

The Relationship between Methylation and Expression Defect of Tumor Suppressor Gene p16INK4A in Epithelial Ovarian Cancer

Objective： To evaluate the expression of p16INK4A gene in ovarian cancer and analyze the relation between this alteration and the promoter methylation of p16INK4A DNA. Methods： Seven ovarian cancer cell lines and eighteen ovarian cancer specimens were selected for the study. Genomic DNA and RNA were extracted from fresh tissues and cell lines, DNA was treated with sodium bisulfite and then analyzed with methylation-specific PCR （MSP） to detect p16INK4A methylation. The expression of p16INK4A mRNA was detected by reverse transcription-polymerase chain reaction （RT-PCR）. In addition, the proliferation of methylated cell lines before and after treatment of demethylating agent 5-Aza-2＇-deoxycytidine （5-ADC） was examined with 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay in vivo. Results： Compared with the control, the expression of p16INK4A mRNA decreased significantly or absolutely defaulted in 10 of 18 （55.56%） ovarian cancer specimens and 71.4% （5/7） ovarian cancer cell lines （P〈0.05）, and the expression of p16INK4A protein also decreased （P〈0.05）. The decrease of p16INK4A was due, in part, to p16INK4A methylation, which was found in the first exon of three cell lines and six ovarian cancer specimens and the rate was 42.86% and 33.33% in ovarian cancer cell lines and specimens respectively. All the methylated cells and tissues showed expression defect of p16INK4A, but the treatment of 5-ADC reactivated the expression of p16INK4A in methylated cells and decreased the proliferation of tumor cells in vitro and in vivo. Conclusion： The expression defect of p16INK4A gene possibly has an important role in the development of ovarian cancer, and this alteration is due, in part, to the methylation of the first exon in p16INK4A.

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.

Relationship between alterations of p16^( INK4a) and p14^(ARF) genes of CDKN2A locus and gastric carcinogenesis

Objective To investigate the relationship between alterations of p16 INK4a and p14 ARF genes and gastric carcinogenesis Methods The tumors and neighboring gastric tissues from 48 patients with gastric cancer were studied The homozygous deletion, mutation, methylation of the CpG islands, and mRNA expression of p16 INK4a and p14 ARF genes were assessed by PCR, PCR SSCP, PCR based methylation assay, and RT PCR Results ① The homozygous deletion rate of p16 INK4a and p14 ARF was 35 4% (17/48), and no homozygous deletion was examined in any gastric tissue neighboring the tumor ② There was no point mutation of p16 INK4a and p14 ARF in 31 gastric cancers without homozygous deletion or in the matched gastric tissues adjacent to the tumor ③ Methylation of the CpG islands of p16 INK4a and p14 ARF was detected in 47 9% (23/48) of gastric cancers, while methylation was observed only in 2 of 48 gastric tissues neighboring the cancer with a significant difference ( P <0 01) ④ The loss rate of p16 INK4a mRNA was 47 9% (23/48) in gastric cancer, and the patients of the combined methylation of exons 1α and 2 had a higher loss rate (100%, 6/6) of p16 INK4a mRNA than those of the methylation of the other exons (11 8%, 2/17, P <0 01); the loss rate of p14 ARF mRNA was 45 8%(22/48) in gastric cancer, and patients with the combined methylation of exons 1β and 2 had a higher loss rate (100%, 3/3) of p14 ARF mRNA than those of the methylation of the other exons (15%, 3/20, P <0 05) ⑤ The combined loss of p16 INK4a and p14 ARF mRNAs was examined in 1 (5 6%) of 18 patients of well and moderately differentiated carcinomas, and 11 (36 7%) of 30 patients of poorly and not differentiated carcinomas with a significant difference ( P <0 05) Conclusion p16 INK4a and p14 ARF genes are frequently inactivated by homozygous deletion and methylation of the 5'CpG islands in gastric cancer, which may play an important role in the carcinogenesis of gastric cancer

细胞周期调控因子P16^(INK4)、P57^(kip2)和Ki67在肝癌中的表达及意义

目的探讨细胞周期调控因子P16INK4、P57kip2和Ki67在肝癌中的表达水平及其意义。方法收集正常肝组织标本21例,肝细胞肝癌标本48例及对应的癌旁组织标本48例为研究对象,通过免疫组织化学和Western blot分析P16INK4、P57kip2和Ki67的表达水平。结果 P16INK4及P57kip2蛋白肝癌组织中的表达与癌旁组织及正常肝脏组织比较显著降低,阳性率分别为49.3%和34.2%,Ki67蛋白肝癌组织中的表达水平显著升高,其阳性率为91.7%,与癌旁组织及正常肝脏组织比较差异均有统计学意义(P<0.05),结果与Western blot结果一致;高、低分化组P57kip2、Ki67及P16INK4的表达水平比较差异均有统计学意义(P<0.05)。结论 P16INK4、P57kip2表达降低及Ki67的高表达在肝细胞癌的发生、发展过程中可能有重要的作用。

虎眼万年青总皂苷对诱发大鼠肝癌病变过程中肝组织IGF-2及P16 ink4a影响

目的动态检测虎眼万年青总皂苷对二乙基亚硝胺诱发的大鼠肝癌变过程中肝组织中IGF-2及P16 ink4 a表达的影响。方法二乙基亚硝胺诱发大鼠肝癌变,虎眼万年青总皂苷干预,运用实时定量PCR法检测并分析第14周末预防组、模型组及18周末治疗组癌旁组织、癌组织、模型组癌组织标本中IGF-2及P16 ink4 a的表达。结果肝癌变模型组18周癌组织中IGF-2表达最高,与正常组、14周末预防组、模型组、18周末治疗组癌旁组织及癌组织间比较均有统计学意义(P<0.05)。肝癌变模型组18周癌组织中p16 ink4 a表达最高,与正常组、14周末预防组、模型组、18周末治疗组癌旁组织间比较均有统计学意义(P<0.05)。治疗组癌组织与模型组18周癌组织间无统计学差异(P>0.05)。结论二乙基亚硝胺诱发的肝癌组织中IGF-2及P16 ink4 a表达水平增高,而虎眼万年青总皂苷具有干预肝癌组织中IGF-2、p16 ink4 a表达水平。

p16^(INK4a)、FGF-2和D2-40与宫颈癌浸润转移的关系

目的探讨肿瘤抑制基因p16INK4a、成纤维细胞生长因子-2(FGF-2)和D2-40在CIN3、微小浸润癌、宫颈浸润癌中的表达及其在宫颈癌浸润转移中的作用。方法采用免疫组化学方法,检测35例CIN3、13例微小浸润癌及62例宫颈浸润癌中p16INK4a、FGF-2和D2-40蛋白的表达情况。结果 p16INK4a在微小浸润癌中的过表达率明显高于CIN3(P<0.01);FGF-2在CIN3、微小浸润癌和宫颈浸润鳞癌中的阳性率分别为51.43%、53.85%、76.92%,差异有统计学意义(P<0.05);FGF-2的表达强度与宫颈病变的发展程度呈正相关(P<0.01)。p16INK4a和FGF-2的阳性强度与宫颈癌淋巴结转移呈正相关(P<0.01或P<0.05),FGF-2与宫颈癌浸润深度有关,二者均与宫颈癌的肿瘤大小、组织学分级、临床分期无相关性;D2-40的阳性反应与临床病理因素无相关性。p16INK4a、FGF-2的表达和D2-40阳性反应之间无相关性(P>0.05)。结论 p16INK4a异常表达可能有助于诊断微小浸润癌,其阳性强度与宫颈癌淋巴结转移正相关;检测FGF-2在宫颈活检组织中的表达,对评估宫颈病变的发展以及宫颈癌浸润和转移的情况有重要的临床参考价值。

ASPP2及P16^(INK4a)在食管癌中的表达及其与凋亡的关系

目的探讨ASPP2、P16^(INK4a)在食管癌的表达与患者临床病理特征及凋亡的相关性。方法 SP免疫组化染色法检测112例食管癌及31例正常食管黏膜ASPP2、P16^(INK4a)的表达,分析它们与患者临床病理特征的相关性;在上述蜡块中随机选取37例食管癌及12例正常食管黏膜,采用原位缺口末端标记技术(TUNEL)检测上皮细胞的凋亡情况,分析凋亡阳性率与以上二标记物在食管癌表达的相关性。结果 ASPP2、P16^(INK4a)在食管癌与正常食管黏膜的表达差异有统计学意义,且二者异常表达均与食管癌组织学分级、淋巴结转移相关(P<0.05)。TUNEL检测显示正常食管黏膜与食管癌的凋亡阳性率差异有统计学意义,且其与ASPP2、P16^(INK4a)在食管癌的表达相关(P<0.05)。结论 ASPP2、P16^(INK4a)参与食管癌发生、发展及凋亡等过程,联合检测二者可辅助食管癌诊断并指导临床治疗。

Bcl2转录抑制因子1、E-钙黏蛋白在宫颈鳞状细胞癌不同区域的表达及两者与P16INK4a表达的关系

目的检测宫颈鳞状细胞癌肿瘤出芽区及肿瘤中央区失巢凋亡因子Bcl2转录抑制因子1(Bit1)、上皮-间质转化(EMT)标志物E-钙黏蛋白及P16INK4a的表达情况,探讨Bit1、E-钙黏蛋白在宫颈癌获得高侵袭力过程中的意义及二者与P16INK4a表达的关系。方法收集甘肃省肿瘤医院病理科2014-2018年宫颈鳞状细胞癌石蜡包埋标本77例。采用免疫组织化学法检测宫颈鳞状细胞癌肿瘤出芽区及中央区Bit1、E-钙黏蛋白、P16INK4a的表达情况。以肿瘤中央区及出芽区各蛋白质表达评分的中位数作为分界点,将标本分为高表达组和低表达组,分析在不同P16INK4a表达情况下Bit1及E-钙黏蛋白的表达差异及二者与患者临床病理特征的关系,并进一步分析在肿瘤中央区及出芽区Bit1与E-钙黏蛋白的相关关系。统计学分析采用χ2检验或连续校正χ2检验和Spearman等级相关分析。结果77例宫颈鳞状细胞癌标本中,肿瘤中央区P16INK4a、E-钙黏蛋白、Bit1高表达率分别为32.5%(25/77)、67.5%(52/77)、63.6%(49/77),而在肿瘤出芽区分别为67.5%(52/77)、33.8%(26/77)、37.7%(29/77),差异有统计学意义(χ2=18.935、17.561、10.391,P均<0.01)。无论在肿瘤出芽区还是中央区,P16INK4a高表达组与P16INK4a低表达组Bit1及E-钙黏蛋白的表达差异均无统计学意义(P均>0.05)。肿瘤中央区Bit1低表达与脉管内癌栓及淋巴结转移有关(χ2=5.053、4.400,P均<0.05),肿瘤出芽区E-钙黏蛋白和Bit1低表达均与淋巴结转移有关(χ2=5.580、7.573,P均<0.05)。Spearman等级相关分析显示,在肿瘤中央区及肿瘤出芽区E-钙黏蛋白与Bit1表达均呈正相关(r=0.287,P=0.011;r=0.236,P=0.039)。结论宫颈癌侵袭力的增高与Bit1及E-钙黏蛋白表达降低及P16INK4a表达增高有关,宫颈癌细胞可能通过抑制Bit1获得失巢凋亡抗性并影响EMT的发生从而获得更高的侵袭能力,但P16INK4a并未参与此过程。

EZH2、p16INK4a有助于胆管细胞癌与胆管反应及胆管腺瘤的鉴别诊断

肝内胆管癌与胆管腺瘤以及胆管反应的鉴别诊断有时较为困难。作者曾报道胆管细胞癌存在多梳家族蛋白EZH2的高表达,相反,胆管反应则可高表达衰老相关蛋白p16INK4a。本组采用免疫组化定量方法,对33例肝内胆管癌和具有胆管细胞癌成分的混合性肝细胞-胆管细胞癌及16例胆管腺瘤进行分析,着重探讨EZH2和p16INK4a的免疫组化染色有助于区分胆管细胞癌、胆管腺瘤和胆管反应鉴别诊断中的作用。

宫颈液基细胞中p16INK4A及p57KIP2蛋白表达与CIN1病人预后关系的初步探讨

目的探讨P16INK4A及P57蛋白表达对CIN1病人病程预测的可行性。方法将组织学确定为CIN1病人的剩余TCT标本进行P16INK4A及P57KIP2蛋白免疫细胞化学染色，同时，对病人进行1～1．5年的随访，观察其表达与病人病程转化的关系。结果67例病人P16INK4A阴性表达者21例，2O例治愈（95．2％），1例未愈（4．8％），未见1例恶化；p16INK4A阳性表达共46例，22例治愈（47．8％），16例未愈（34．8％），8例恶化（17．4％），其中表达（＋＋）及（＋＋＋）者恶化比例最高，达30．4％；两者比较有显著性差异（X2=25．777，P〈0．001），提示P16INK4A阳性表达的病例预后较差，对p16INK4A阳性表达者可作LEEP术或活检后进一步评估诊断的正确性以进一步治疗。而P16INK4A（-）者无1例病变进展，提示相对无危险的妇女就可能不须实行过度治疗或频繁随访。P57KIP2阳性表达者59例，40例CIN病变治愈（67．8％）；14例未愈（23．7％），5例恶化（8．5％）；而p57KIP2表达阴性者2例治愈（25％）；3例未愈（37．5％）；3例病变进展（37．5％），两者比较有显著性差异（X2=20．980，P=-0．002）。提示P57H阻阳性表达的病例预后较好，可能不须实行过度治疗或频繁随访。结论将P16INK4A和P57KIP2进行联合检测对判断宫颈CIN1的生物学行为可能具有更重要的意义。

p16INK4A和bcl-2蛋白在CIN和宫颈鳞癌中的表达

目的探讨p16INK4A和bcl-2在宫颈上皮内瘤(CIN)和宫颈鳞癌的表达状态及其在病理诊断中的意义。方法采用免疫组化SP法对39例CIN、24例浸润性宫颈鳞癌和20例正常宫颈组织进行p16INK4A和bcl-2检测。结果正常宫颈组织不表达p16INK4A,CINⅠ阳性表达率很低并为局灶表达,但几乎所有的CINⅡ/Ⅲ和浸润性宫颈鳞癌均弥漫大量表达p16INK4A。正常宫颈组织只有基底细胞层表达bcl-2蛋白,CINⅢ和Ⅰ～Ⅱ期浸润癌bcl-2阳性细胞弥漫分布,阳性表达率分别为53.8%和57.1%。Ⅲ～Ⅳ期浸润癌bcl-2阳性率下降,阳性细胞局限。结论p16INK4A过表达与宫颈鳞癌恶性特性密切相关,检测p16INK4A有助于及时发现高级别CIN和宫颈鳞癌。

食管癌组织中CtBP2与P16^(INK4A)的表达及其相关性分析

目的:探讨Ct BP2与P16INK4A在食管癌组织中的表达,并且分析两者之间的相关性。方法:使用免疫组化法(PV二步法)对我院90例食管癌患者组织中Ct BP2与P16INK4A的表达进行检测,并使用统计软件对两者的相关性进行分析。结果:在不同分期中,Ⅰ期和Ⅱ期患者食管癌组织中的P16INK4A阳性率为55.77%,Ⅲ期以上为18.42%,差异较为显著(=7.176,P<0.05);在淋巴结转移中,CtBP2阴性和阳性的差异比较大(=10.986,P<0.01);P16INK4A与Ct BP2呈现典型的负相关关系,r=-0.417,P<0.01。结论:Ct BP2可以通过抑制细胞周期抑制因子P16INK4A来实现细胞增殖,两者对于细胞的调控是从相反的两个方向进行的。

Detection of Serum Aberrant CDKN2/P16 DNA in Colorectal Cancer

Objective： To search for a biomarker for colorectal cancer. Methods： The MSP, SSCP and deletion tests with serum have been taken simultaneously in 100 cases of colorectal cancer and 2 groups of controls, as well as the specimens of 26 cancer tissues and 22 paracancerous tissues and 29 cases of benign disease tissues for a contrast. Results： The aberrant methylation rate of P16 in the serum was 69.00%, deletion rate 4.00% and suspicious point mutation rate 15.00% in colorectal cancer patients. The data of cancer tissues were the same as those of the serum, but in paracancerous tissue those were significantly lower. In 10 cases, sequencing analysis revealed that there were 3 cases of missense, one case of frameshift and one case of nonsense. Among them, four cases had P16 protein deletion. As a tumor marker, the sensitivity of combined use of three methods was 88.00%, specificity 96.87% and accuracy 90.15%. The combined use of MSP and SSCP could obtain the same results. Conclusion： The content of DNA in serum is minimal, but it reflects the tumor burden of patients. The 10^-3 fragments of DNA could be detected in the serum by MSP. It can be used in the clinical diagnosis or popular investigation, and long-term postoperative follow-up.

MUTATION AND ABNORMAL EXPRESSION OF P16^(INK4a) INHEPATOCELLULAR CARCINOMA

Objective:To investigate the relationship betweenp16INK4a and primary hepatocellular carcinoma (HCC),especially hepatitis B-related HCC. Methods: p16INK4a andits protein in HCC were analyzed with PCR-SSCP and theimmunohistochemistry methods respectively. Results: Thepositive incidence of p16INK4 protein expressing in HCC waslower than that of normal liver tissue (P<0.05), and theabsence of p16INK4 protein was associated with HCCmetastasis (P<0.05). The low frequency of mutation ofp16INK4 exonl and exon2 upstream fragment was found inHCC. Conclusion: Absence of p16INK4 protein in HCC wasnot associated with HBV-infection.

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.

L1壳蛋白和p16INK4a蛋白在不同类型宫颈上皮内瘤变1级中的表达意义

p16属于抑癌基因CDKN2A家族,通过阻断细胞由G0期进入S期抑制细胞的增殖而发挥抑癌作用.在CIN组织中P16INK4a过度表达,可能是人感染高危型人乳头瘤病毒（HR-HPV）后,E7癌蛋白在宫颈细胞内表达,竞争性抑制CDK4与Rb蛋白的结合,使Rb蛋白失活,激活了E2F家族转录因子,基因转录水平上升,从而导致p16INK4a阳性表达[1].

cyclin E、p16ink4、ki67的高度表达对宫颈癌高危人群筛查的意义

目的:探讨宫颈脱落细胞中cyclinE、p1 6ink4、ki6 7的高度表达对宫颈癌高危人群筛查的意义。方法:采用免疫组织化学方法对78例宫颈脱落细胞标本(不明意义的鳞状上皮非典型增生ASCUS 1 2例、宫颈上皮内瘤样病变CIN11 7例、CINⅡ～Ⅲ3 8例、浸润癌1 1例)进行cyclinE、p1 6ink4、ki6 7检测,实验数据进行χ2 检验及四格表Fisher确切概率计算。结果:cyclinE、p1 6ink 4、ki6 7在各级宫颈癌中的阳性率均较ASCUS组差异有非常显著意义(P <0. 0 0 5 ) ;cyclinE在CIN1 的表达率较其它各组也有非常显著意义( 91.4%均P <0.0 1 ) ;p1 6ink4、ki6 7均随着宫颈上皮细胞损伤程度加重而表达上升(P <0.0 0 5 ) ,在浸润癌阳性率达到最高(分别为1 0 0 %和90.9% )。CIN1 组3指标特异度均较高(分别为91.7%、1 0 0 %和91 .7% ) ,灵敏度cyclinE最高( 94. 1 % ) ,p1 6ink4与ki6 7均较低(分别为5 8.8%和4 7.1 % )。结论:采用宫颈脱落细胞检测cyclinE、p1 6ink 4、ki6 7中的1项或多项作为诊断指标应用于宫颈癌高危人群筛查,对降低筛查的假阳性和假阴性率,提高筛查的灵敏度和特异度具有重要意义。

Hela细胞P16^(ink4)cDNA的克隆及序列分析

P16^(ink4)是CDK抑制蛋白,参与调控细胞G1期至S期的转换。目前发现P16`(ink4)基因损伤与多种肿瘤的发生、发展有关,可能是功能上非常重要的抑癌基因。为了研究该基因的功能,以及突变对该基因功能的影响,本文应用RT-PCR方法,从Hela细胞中克隆了P16^(ink4)cDNA。扩增得到556bp片段(包括引物两端酶切位点的16bp)克隆于M13载体,测定了其DNA序列。该序列包括了P16^(ink4)cDNA编码区全部471bp,以及3’端69bp序列。表明P16^(ink4)cDNA已成功地得到克隆。