细胞核和线粒体微卫星不稳在大肠癌发生中的作用及两者的关系

目的:探讨细胞核和线粒体DNA微卫星不稳在大肠癌发生中的作用及两者的关系.方法:直接测序法检测大肠癌线粒体控制区DNA微卫星不稳定位点(mi tochondr i a lmicrosatellite instability,mtMSI);微卫星扫描方法检测细胞核BAT25、BAT26微卫星位点不稳定性(nucle-ar microsatellite instability,nMSI).分析大肠癌mtMSI发生率在性别、年龄、部位、分级各组间以及与nMSI的相关性.结果:40份大肠癌组织检出mtMS I 11例(27.5%),其中仅1个微卫星位点mtMSI阳性者11份(17.5%),有2个微卫星位点mtMSI阳性者2例(5%).有9份于BAT25或BAT26位点检出nMSI,阳性率为22.5%.大肠癌mtMSI发生率在性别、年龄、部位、分级各组间无显著性差异( P>0.05),但与nMSI有显著相关性( P<0.05).结论:mtMSI在部分大肠癌的发生中起重要作用,大肠癌mtMSI与nMSI有相关性.     

Dietary polyphenols and colorectal cancer risk:The Fukuoka colorectal cancer study

AIM:To investigate the associations between dietary intake of polyphenols and colorectal cancer. METHODS:The study subjects were derived from the Fukuoka colorectal cancer study, a community-based case-control study. The study subjects were 816 cases of colorectal cancer and 815 community-based controls. The consumption of 148 food items was assessed by a computer-assisted interview. We used the consumption of 97 food items to estimate dietary intakes of total, tea and coffee polyphenols. The Phenol-Explorer database was used for 92 food items. Of the 5 foods which were not listed in the Phenol-Explorer Database, polyphenol contents of 3 foods (sweet potatoes, satoimo and daikon) were based on a Japanese study and 2 foods (soybeans and fried potatoes) were estimated by ORAC-based polyphenol contents in the United States Department of Agriculture Database. Odds ratios (OR) and 95%CI of colorectal cancer risk according to quintile categories of intake were obtained by using logistic regression models with adjustment for age, sex, residential area, parental history of colorectal cancer, smoking, alcohol consumption, body mass index 10 years before, type of job, leisure-time physical activity and dietary intakes of calcium and n-3 polyunsaturated fatty acids.RESULTS:There was no measurable difference in total or tea polyphenol intake between cases and controls, but intake of coffee polyphenols was lower in cases than in controls. The multivariate-adjusted OR of colorectal cancer according to quintile categories of coffee polyphenols (from the first to top quintile) were 1.00 (referent), 0.81 (95%CI:0.60-1.10), 0.65 (95%CI:0.47-0.89), 0.65 (95%CI:0.46-0.89) and 0.82 (95%CI:0.60-1.10), respectively (P trend = 0.07). Similar, but less pronounced, decreases in the OR were also noted for the third and fourth quintiles of total polyphenol intake. Tea polyphenols and non-coffee polyphenols showed no association with colorectal cancer risk. The sitespecific analysis, based on 463 colon cancer cases and 340 rectal cancer cas     

Prognostic significance of microsatellite instability in sporadic colorectal cancer

Background and aims Colorectal cancers exhibiting microsatellite instability (MSI) appear to have unique biological behavior. The influence of MSI on the prognosis of sporadic colorectal cancers is controversial and requires further investigation. The aim of this study was to analyze the association between MSI status and clinicopathological features and prognosis in sporadic colorectal cancer patients.Patients and methods Of the 322 consecutive colorectal cancer patients operated upon at the Seoul National University Hospital between January and December 1998, we examined the clinicopathological features and prognosis of 248 patients with sporadic primary colorectal cancer. The MSI status of these 248 patients has been reported in a previous study. Of the 248 patients, 23 (9.3%) had MSI+ tumors. The patients clinicopathological parameters were obtained from their medical records, and follow-up and survival data were obtained from medical records and phone calls.Results MSI+ sporadic colorectal cancers were found predominantly in the proximal colon (p<0.001) and were associated with poor differentiation (p=0.030), a lower preoperative serum carcinoembryonic antigen (CEA) level (p=0.012), and less frequent systemic metastasis (p=0.034) than MSI– tumors. Low tumor grade (p=0.022), low tumor T-stage (p=0.002), no lymph node metastasis (p<0.001), no systemic metastasis (p<0.001), adjuvant chemotherapy (p<0.001) and MSI+ status (p=0.038) were independent favorable prognostic factors for survival in sporadic colorectal cancer patients.Conclusion MSI status was an independent favorable prognostic factor for survival in sporadic primary colorectal cancer patients.     

遗传性非息肉病性结直肠癌的微卫星不稳定研究

目的　探讨国人北方人群HNPCC的微卫星不稳定 (microsatelliteinstability ,MSI)发生情况及其意义。方法　 44例患者来源于 3 0个HNPCC (hereditarynonpolyposiscolorectalcancer)家系 ,这些家系主要分布于北方 5省市。所有患者均符合BGl 3 (Bethes dal 3 )HNPCC诊断标准。以荧光标记法检测 44例患者的石蜡包埋组织微卫星稳定性。结果　 44例患者中高度微卫星不稳定 (highfrequencymicrosatelliteinstability ,MSI H)为 81.81( 3 6/ 44 ) ,低度微卫星不稳定 (lowfrequencymicrosatelliteinstability ,MSI L)为 6.82 ( 3 / 44 ) ,微卫星稳定 (microsatellitestable ,MSS)为 11.3 ( 5 / 44 ) ;所选择的 5个微卫星位点中Bat2 5和Bat2 62个位点MSI H的表达率较高 ,分别为 10 0 %和 97.2 2 %。符合AmsterdamⅡ和符合BGl 3标准的HNPCC患者的MSI H表达率分别为 85 .2 9%和 81,81% ,仅符合BGl 3标准 ,而不符合AmsterdamII的 10个患者中 ,7个发现MSI H。结论　HNPCC肿瘤的MSI H发生率高 ,MSI检测方法简便、易行 ,可作为错配修复基因种系突变初筛方法 ,Bethesdal 3标准可更多地收集到可疑的HNPCC患者     

结直肠锯齿状病变的微卫星状态研究

目的 比较结直肠锯齿状病变与传统腺瘤、腺癌的微卫星状态的差异,以期间接验证传统型锯齿状成瘤通路的存在.方法 收集北京军区总医院病理科保存的75例大肠息肉及肿瘤组织蜡块标本,其中锯齿状腺癌(Sca)15例,非锯齿状腺癌(N-Sca)20例,传统型锯齿状腺瘤(TSA)20例,普通腺瘤20例.抽提基因组DNA,采用荧光标记引物扩增BAT25、BAT26两个位点,随后使用DNA自动测序仪检测其微卫星状态,并对实验结果进行统计学分析.结果部分标本扩增失败,对于成功扩增的68例标本:18例TSA中6例为高度微卫星不稳定型(MSI-H),12例为低度微卫星不稳定型(MSI-L)/微卫星稳定型(MSS);18例普通腺瘤均为MSS;13例Sca中3例为MSI-H,10例为MSI-L/MSS;19例N-Sca中仅1例为MSI-H,18例为MSI-L/MSS.统计学分析表明,普通腺瘤组、N-Sca组MSI-H发生率明显低于TSA组和Sca组(P<0.05),而后两组间差异无统计学意义(P>0.05).结论 与普通腺瘤、N-Sca相比,MSI-H更多见于TSA、Sca,由此推断存在一条有别于传统"腺瘤-癌"发生模式的传统型锯齿状成瘤通路,但尚需大规模前瞻性研究确认.     

Relationship between microsatellite instability and telomere shortening in colorectal cancer

PURPOSE: Two pathways have been proposed for the development of colorectal cancers: loss of heterozygosity and replication error. Colorectal cancers arising through the replication error pathway, like most hereditary nonpolyposis colorectal cancers, show microsatellite instability. It has been also reported that telomere shortening frequently occurs in colorectal cancers and that telomerase is often activated strongly in them. The aim of this study was to examine whether any relationships can be found among microsatellite instability, telomere length, and telomerase activity in colorectal cancers. METHODS: Genomic DNA was extracted from 55 invasive cancers and corresponding normal mucosas. Five microsatellite loci were analyzed by polymerase chain reaction. Telomere length was examined by Southern blot analysis. Telomerase activity was assayed by telomeric repeat amplification protocol with minor modifications. RESULTS: Microsatellite instability was found in 8 (14.5 percent) of 55 tumors, and all of them showed short telomeres. Furthermore, four high-frequency microsatellite instability tumors that showed microsatellite instability at more than two loci exhibited remarkably short telomeres. The microsatellite instability correlated significantly with frequency of telomere shortening (P=0.0183; Fisher's exact probability test), but not with strength of telomerase activity. CONCLUSION: The relationship identified by this study between microsatellite instability and telomere shortening might suggest some association between the DNA mismatch repair system and the telomere maintenance mechanism in colorectal cancers.     

结直肠癌线粒体DNA拷贝数与其微卫星不稳定关系的研究

目的 探讨结直肠癌(CRC)的线粒体DNA(mtDNA)拷贝数异常与临床指标及微卫星不稳定(MSI)的关系.方法 选取50例CRC及相应癌旁组织标本,分别提取基因组DNA.对非编码区微卫星位点进行测序;对线粒体ND1基因进行荧光定量PCR,进而计算出mtDNA拷贝数;然后与临床指标和非编码区MSI进行比较分析.结果 CRC组织的mtDNA平均拷贝数/细胞数为312±185,而相应的癌旁组织为525±125,前者显著低于后者(P<0.001).mtDNA拷贝数高低与非编码区MSI有明显相关性(P<0.001),与性别、年龄、病理分型、TNM分期无相关性(P>0.05).结论 CRC的mtDNA拷贝数明显降低,这种降低与线粒体的MSI相关.     

Pathology features in Bethesda guidelines predict colorectal cancer microsatellite instability: a population-based study

BACKGROUND & AIMS: The revised Bethesda guidelines for Lynch syndrome recommend microsatellite instability (MSI) testing all colorectal cancers in patients diagnosed before age 50 years and colorectal cancers diagnosed in patients between ages 50 and 59 years with particular pathology features. Our aim was to identify pathology and other features that independently predict high MSI (MSI-H). METHODS: Archival tissue from 1098 population-based colorectal cancers diagnosed before age 60 years was tested for MSI. Pathology features, site, and age at diagnosis were obtained. Multiple logistic regression was performed to determine the predictive value of each feature, as measured by an odds ratio (OR), from which a scoring system (MsPath) was developed to estimate the probability a colorectal cancer is MSI-H. RESULTS: Fifteen percent of tumors (162) were MSI-H. Independent predictors were tumor-infiltrating lymphocytes (OR, 9.1; 95% confidence interval [CI], 5.9-14.1), proximal subsite (OR, 4.7; 95% CI, 3.1-7.3), mucinous histology (OR, 2.8; 95% CI, 1.7-4.8), poor differentiation (OR, 1.9; 95% CI, 1.2-3.1), Crohn's-like reaction (OR, 1.9; 95% CI, 1.2-2.9), and diagnosis before age 50 years (OR, 1.9; 95% CI, 1.3-2.9). MsPath score >or=1.0 had a sensitivity of 93% and a specificity of 55% for MSI-H. CONCLUSIONS: The probability an individual colorectal cancer is MSI-H is predicted well by the MsPath score. There is little value in testing for DNA mismatch repair loss in tumors, or for germline mismatch repair mutations, for colorectal cancers diagnosed in patients before age 60 years with an MSPath score <1 (approximately 50%). Pathology can identify almost all MSI-H colorectal cancers diagnosed before age 60 years.     

Mitochondrial microsatellite instability in gastric cancer and its precancerous lesions

AIM: To evaluate the role of mitochondrial microsatellite instability (mtMSI) in gastric carcinogenesis. METHODS: MtMSI was measured with PCR-single strand conformation polymorphism (PCR-SSCP) in 68 cases of advanced gastric cancer, 40 cases of chronic gastritis, 30 cases of intestinal metaplasia and 20 cases of dysplasia. RESULTS: MtMSI was observed in 12.5% (5 of 40) of chronic gastritis, 20.0% (6 of 30) of intestinal metaplasia, 25.0% (5 of 20) of dysplasia and 38.2% (26 of 68) of gastric cancer. These findings showed a sequential accumulation of mtMSI in the histological progression from chronic gastritis to gastric cancer. An association of mtMSI with intestinal histological type and distal location was found (P=0.001 and P=0.002), whereas no significant correlation was found between mtMSI and age at diagnosis, sex, tumor size, depth of invasion, lymph node spread and clinical stages (P>0.05). CONCLUSION: MtMSI may play an early and important role in the gastric carcinogenesis pathway, especially in the intestinal type and distal gastric cancer.     

微卫星不稳定状态与结直肠癌患者临床病理特征的相关性研究

目的探究微卫星不稳定(MSI)状态与结直肠癌患者临床病理特征的相关性。方法采用回顾性队列研究方法,收集2015年1月至2019年12月南京医科大学第一附属医院结直肠外科1280例结直肠癌手术患者的临床病理资料,其中男性800例,女性480例;中位年龄为63岁;右半结肠癌337例,左半结肠癌398例,直肠癌545例。PCR方法检测肿瘤标本的微卫星状态。依据MSI状态,将患者分为高度微卫星不稳定性(MSI-H)组和微卫星稳定性(MSS)/低度微卫星不稳定性(MSI-L)组。观察人口学特征,手术标本病理学检查,微卫星状态等指标。计数资料以绝对数或百分比表示,组间比较采用卡方检验或Fisher精确检验。等级资料采用秩和检验。非正态分布资料,采用中位数M(P25,P75)表示,组间比较采用曼-惠特尼U检验。结果在1280例患者中,112例(8.7%)为MSI-H,79例(6.2%)为MSI-L,1089例(85.1%)为MSS。MSI-H组与MSS/MSI-L组患者在术前血清CEA(χ²=6.943,P<0.05)、肿瘤部位(Z=-9.451,P<0.001)、肿瘤TNM分期(Z=-2.108,P<0.05)、肿瘤T分期(Z=-2.397,P<0.05)、肿瘤N分期(Z=-3.892,P<0.001)、肿瘤分化(χ²=6.663,P<0.05)、肿瘤黏液成分(χ²=78.833,P<0.001)、肿瘤最大直径(χ²=39.656,P<0.001)、癌结节(χ²=8.759,P<0.05)、神经侵犯(χ²=10.238,P<0.05)、淋巴结转移率(LNR)(χ²=5.880,P<0.05)、淋巴结检出数(Z=-5.019,P<0.001)、阳性淋巴结检出数(Z=-3.667,P<0.001)等方面差异有统计学意义,主要表现为,MSI-H组患者CEA<4.7 ng/mL、右半结肠癌、低TNM分期、高T分期、低N分期、低分化、黏液成分、肿瘤直径≥4 cm、无癌结节、无神经侵犯、低LNR所占比例显著高于MSS/MSI-L组患者。MSI-H组淋巴结检出数显著多于MSS/MSI-L组,而阳性淋巴结检出数显著较少。结论MSI-H结直肠癌患者具有较为特殊的临床病理特征,其中既有预后良好的,亦有预后不良的特征。     

Role of detection of microsatellite instability in Chinese with hereditary nonpolyposis colorectal cancer or ordinary hereditary colorectal cancer

AIM: To detect microsatellite instability (MSI) in patients with hereditary nonpolyposis colorectal cancer or ordinary hereditary colorectal cancer and to provide criteria for screening the kindreds with hereditary nonpolyposis colorectal cancer at molecular level. METHODS: MSI was detected in the specimens from 20 cases with HNPCC, 20 cases with ordinary hereditary colorectal cancer and 20 cases with sporadic colorectal cancer by means of polymerase chain reaction-single strand conformation polymorphism. RESULTS: The positive rate of MSI was 85% (17/20) in HNPCC group, 40% (8/20) in ordinary hereditary colorectal cancer group and 10% (2/20) in the sporadic colorectal cancer group respectively. The differences were significant. The mean ages of the three groups were 43.6, 52.2, and 61.8 years respectively, which increased gradually. The incidence of right hemicolon cancer was 64.7%, 37.5%, and 0% respectively, which decreased gradually and had significant difference. The expression ratio of BAT26 and BAT25 was 94.1% respectively, which was highest in the 5 gene sites studied. The incidence of poorly differentiated adenocarcinoma was 70.6% in HNPCC group among high frequency microsatellite instability (MSI-H), which was higher than the other two groups, which had 50% and 50% respectively. CONCLUSION: The incidence of MSI-H is higher in HNPCC group. The detection of MSI is simple and economical and has high correlation with the clinicopathologic feature of HNPCC and can be used as a screening method to detect the germ line mutation of the mismatch repair gene.     

Mismatch repair status in sporadic colorectal cancer: immunohistochemistry and microsatellite instability analyses

Background and Aim: The aim of the present study was to evaluate associations between mismatch repair (MMR) status and clinicopathological characteristics and prognosis using immunohistochemistry (IHC) and microsatellite instability (MSI) analyses in a prospective cohort of a large number of accumulated samples. Methods: Tumor tissue samples obtained during curative surgery (n = 2028) were analyzed using both MLH1/MSH2 IHC and MSI assays. Clinicopathological parameters and survival outcomes were compared according to IHC and MSI results. The median follow‐up period was 43 months (range: 1–85 months). Results: IHC identified 207 tumor samples (10.2%) with a loss of either MLH1 or MSH2 expression. The MSI analysis identified 203 tumor samples (10%) with high‐frequency MSI (MSI‐H). Patients with MMR defects were younger, and had tumors characterized by right‐colon predilection; large‐size, infrequent lymph node metastasis; poorly‐differentiated or mucinous histology, and synchronous adenomas (P < 0.001–0.008). Patients with MSI‐H status had higher 4‐year disease‐free survival rates than patients with microsatellite stable status (90.8% vs 80.6%, P = 0.001). A multivariate analysis showed that MSI‐H status was a good prognostic factor for recurrence (hazard ratio: 0.48, 95% confidence interval: 0.30–0.83, P = 0.007). Conclusions: Patients with MMR defects had distinct clinicopathological characteristics, including a lower risk of recurrence. IHC and MSI analyses provided complementary information regarding specific clinicopathological parameters and prognosis.     

CpG island methylation in sporadic colorectal cancers and its relationship to microsatellite instability

BACKGROUND & AIMS: Methylation of CpG islands is increasingly recognized as an important event in colorectal carcinogenesis. We evaluated the extent of CpG island methylation in 426 sporadic colorectal cancers to define its relationship to microsatellite instability and to describe its clinicopathologic and genetic features. METHODS: Fresh cancer tissue was obtained from 417 consecutive individuals undergoing curative surgery for sporadic colorectal cancer. Methylation of p16 and hMLH1 promoters was determined by methylation-specific polymerase chain reaction (PCR), whereas methylation at MINT 1, 2, 12, and 31 loci was assessed by bisulfite PCR. Microsatellite instability and K-ras and p53 status were determined using microsatellite PCR, restriction enzyme-mediated PCR, and immunohistochemistry, respectively. RESULTS: Individual loci were commonly methylated, but locus-specific phenotypic changes were not seen. CpG island methylation was associated with right-sided location, female sex, and older age, as well as high tumor grade, mucinous type, wild-type P53, microsatellite instability, and K-ras mutations. More than half of tumors showing CpG island methylation were microsatellite stable. Compared with microsatellite unstable cancers, they were more commonly left-sided, had fewer intraepithelial lymphocytes, presented later, and had a worse outcome. CONCLUSIONS: Colorectal cancers with CpG island methylation have distinct clinicopathologic features and in some cases lead to sporadic microsatellite unstable cancers.     

Dinucleotide microsatellite repeats are essential for the diagnosis of microsatellite instability in colorectal cancer in Asian patients

AIM: The molecular diagnosis of microsatellite instability (MSI) in colorectal cancer (CRC) is based on the analysis of five microsatellite markers. Among them, the two mononu-cleotide microsatellite repeats are considered more informative for this analysis than the three dinucleotide ones. The aim of this study is to establish the most relevant markers for MSI analysis in colorectal cancers from Asian patients. METHODS: The MSI analysis of 143 CRC cases in a routine molecular diagnostic laboratory was reviewed. Analysis by fluorescence-based PCR of the five recommended microsatellites was performed, followed by data interpretation according to internationally accepted guidelines. The results were analyzed to address (1) the rate of success in the analysis of histopathological samples not specifically prepared for molecular analysis; (2) the relative importance of individual markers in the diagnosis of high-MSI (H-MSI). RESULTS: MSI analysis was unsuccessful in 34 cases (24%), but for tissues archived in recent years the unsuccessful rate was 5%. We found the D2S123 marker the most vulnerable to inadequate tissue preservation, failing to amplify in 58 instances. Approximately 30% (32/109) of the cases were H-MSI, while 7/109 (6%) were low-MSI. A detailed analysis of the H-MSI cases revealed that the dinucleotide repeats (and D5S346 in particular) were more relevant than the mononucleotide repeats in assigning the correct MSI status. CONCLUSION: The analysis of dinucleotide repeats is essential for the establishment of MSI status in Asian CRC patients.     

The inhibition effect and its molecular mechanism of human cervical cancer oncogene siRNA on hepatocarcinoma cells

OBJECTIVE: To evaluate the effect and the possible mechanism of HCCR siRNA on cell proliferation and apoptosis of hepatocarcinoma cells. METHODS: pRIV2-siHCCR plasmids, which express small interfering RNA of HCCR were constructed and transfected into HepG2 cells. The mRNA and protein expressions of HCCR were detected by real time PCR and Western blot. The proteins p15, p16, p27, p53, and PTEN were detected by Western blot. The cell proliferation and apoptosis were observed by MTT and FACS. RESULTS: The plasmid pRIV2-siHCCR was constructed successfully. Real time PCR and Western blot analysis showed that the HCCR siRNA effectively inhibited HCCR expression in HepG2 cells after pRIV2-siHCCR transfection. MTT method confirmed that HepG2 cell proliferation was suspended, while the cell apoptosis was increased much more than that in the control group. After the transfection with the plasmid of pRIV2-siHCCR into HepG2 cells, the expression of p53 protein was decreased, and P15 increased; and levels of PTEN, p16, and p27 were evidently not changed. CONCLUSION: After being transfected with HCCR siRNA expression plasmid, the cell proliferation of HepG2 was arrested, while the apoptosis of HepG2 cells increased. Our results demonstrate the potential role of p53 and p15 in HCCR signaling.