Paper intensive reading (六)：Altered Interactions between the GM and Colonic Mucosa Precede Polyposis

论文题目：Altered Interactions between the Gut Microbiome and Colonic Mucosa Precede Polyposis in  Mice

link：https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127985

Google citation: 26

Published: June 29, 2015

Published in PLOS ONE

Author: Joshua S. Son,Shanawaj Khair,...,Basil Rigas,Ellen Li 

Organization: Department of Medicine, Stony Brook University, Stony Brook, NY, United States of America

Abstract:

Mutation of the adenomatous polyposis coil (APC腺瘤样结肠息肉易感基因蛋白 gene), an early event in the adenoma-caricinoma(腺瘤癌) sequence, is present in 70%~80% of sporadic(零散的) human colorectal adenomas(腺瘤) and carcinomas(癌). To test the hypothesis that mutation of the APC gene alters microbial interactions with host intestinal mucosa(粘膜) prior to the development of polyposis(息肉病), culture-independent methods (targeted qPCR assays and Illumina sequencing of the 16S rRNA gene V1V2 hypervariable region) were used to compare the intestinal microbial composition of 30 six-week old C57BL/6   and 30 congenic(同类系的) wild type (WT) mice. The results demostrate that similar to 12-14 week old  mice with intestinal neoplasia(肠道肿瘤), 6 week old  mice with no detectable neoplasia, exhibit an increased relative abundance of Bacteroidetes spp in the colon(结肠). Parallel mouse RNA sequence analysis, conducted on a subset of proximal colonic RNA samples (6 , 6 WT) revealed 130 differentially expressed genes (DEGs, fold change >=2, FDR < 0.05). Hierarchical(分层的) clustering of the DEGs was carried out by using 1-r dissimilarity measurement, where r strands for the Pearson correlation, and Ward minimum variance linkage, in order to reduce the number of input variables. When the cluster centroids (medians) were included along with APC genotype as input variables in a negative binomial (NB, 负二项式) regression model, four of seven mouse gene clusters, in addition to APC genotype, were significantly associated with the increased relative abundance of Bacteroidetes spp. Three of the four clusters include several downregulated genes encoding immunoglobulin(免疫球蛋白) variable regions and non-protein coding RNAs. These results support the concept that mutation of the APC gene alters colonic-microbial interaction prior to polyposis. It remains to be determined whether interventions directed at ameliorating(改善) dysbiosis in  mice, such as through probiotics, prebiotics or antibiotics, could reduce tumor formation.

Outline:

1. Introduction

2. Materials and Methods

2.1 Animal Type and Housing

2.2 Tissue and Luminal Content Sample Collection

2.3 DNA and RNA Extraction of Intestinal Tissue and Luminal Content Samples

2.4 Quantitative PCR (qPCR) for targeted bacterial clades(分化支，进化支)

2.5 16S rRNA Amplicon(扩增子) Library Construction and Illumina V1V2 Sequencing Analysis

2.6 Histological(组织学的) Analysis of the Intestinal Sections

2.7 Comparison of Mouse Proximal Colonic mRNA Expression in and Wild type mice

3. Results

3.1 16S rRNA sequence analysis of proximal colonic mucosal samples from   and WT mice

3.2 Differentiallly expressed genes in 6 week-old   mice

4. Discussion

Content:

1. Introduction

肠道菌群失调在人类结肠癌中早有报道。但是肠道失调是肿瘤形成的结果，还是促进了肿瘤形成，即因果关系，尚不明确。

One of the most prominent genetic mutations associated with the pathogenesis of sporadic and hereditary colorectal cancers (CRC) lies in the tumor suppressing adenomatous polyposis coli (APC) gene. 与散发性和遗传性结肠癌最相关的基因突变是发生在APC（腺瘤性息肉病）基因。APC本来是一个抑癌基因，如果APC发生突变，就会引发息肉病变，最后演变发展成癌症。

 familial adenomatous polyposis (FAP) 家族性结肠息肉综合征

APC突变会引发FAP，但是FAP患者只要及早检查跟进和接受手术治疗，大部分结肠癌是可以预防的。

APC mutations also represent an early event in the adenoma-carcinoma sequence and are present in about 70–80% of sporadic human colorectal adenomas and carcinomas.

 multiple intestinal neoplasia (Min) 多发性肠肿瘤

The multiple intestinal neoplasia (Min) mouse model of FAP carries a truncation mutation at codon 850 of the Apc gene. 多发性肠肿瘤的FAP小鼠模型携带APC基因的850号密码子的突变。

需要明确的是APC(Min/+)突变小鼠特指APC基因第850号密码子出现无义突变（TTG---> TAG）的C57BL/6J品系小鼠，其它APC基因突变或敲除的小鼠即使出现肠道多发性腺瘤也不宜称之为APC(Min/+)突变小鼠。

Studies comparing the number of intestinal polyps in germ-free and conventionally raised C57Bl/6 APCMin/+ mice suggest that the gut microbiome may promote development of intestinal neoplasia. 肠道菌群可能促进肠道肿瘤的发展。

One study reported decreased incidence of polyps in only the mid small intestinal segment, however a subsequent study reported a significant reduction of intestinal adenomas in both the small and large intestine of germ-free mice compared with conventionally raised mice. 有一项研究表明无菌小鼠只有在小肠中段的息肉发病率降低，而另一项研究表明无菌小鼠的小肠和大肠的息肉发生率均明显降低。

Antibiotic treatment of C57BL/6 APCMin/+MSH2-/-mice, which carry both the APC mutation and an HNPCC DNA mismatch repair mutation, reduced the number of polyps in both the small and large intestine. 对于携带这两种突变的小鼠，抗生素治疗可以减少小肠和大肠的息肉数量。

We hypothesize that mutation of the APC gene results in alterations in host-microbiota interactions prior to tumor formation. 我们假设：APC基因的突变导致了宿主与微生物群相互作用的改变，进而形成肿瘤。

为了验证假设：gut microbial composition was compared between 6 week-old C57Bl/6 APCMin/+, prior to the development of detectable neoplasia, and congenic WT mice. 比较了6周大的C57Bl/6 APCMin/+小鼠（在可检测到的肿瘤发展之前）和同类系的野生型小鼠。

2. Materials and Methods

2.1 Animal Type and Housing

为了减轻小鼠的旅行压力，让小鼠适应环境了两周。在小鼠安乐死的时候使用了二氧化碳。

Three shipments of 10 four-week-old female C57BL/6J APCMin/+ and 10 four-week-old female C57BL/6J WT mice were received from The Jackson Laboratory (Bar Harbor, ME) between June 2012 and May 2013. 3批，10只雌性，10只雄性。

 APCMin/+ mice and WT mice were housed separately in groups of three to four in specific pathogen free (SPF) cages for two weeks prior to euthanization. 实施安乐死之前，置于无特定病原体的笼子，三到四只一个笼子。

所有操作符合动物实验规范。

WT mice是从哪里来？还是只有携带APC基因突变的小鼠才特别从那个实验室发过来的？

2.2 Tissue and Luminal Content Sample Collection

The segments analyzed included the ileum, cecum, proximal colon, and distal colon. 所分析的节段包括回肠、盲肠、近端结肠和远端结肠。

Each small intestinal segment was washed in sterile(无菌) phosphate(磷酸盐) buffered saline to remove the luminal(腔内) content. 冲洗

A 1.0–1.5-cm section was obtained from the proximal ends of duodenum(十二指肠), jejunum(空肠), distal ends of ileum(回肠远端), proximal colon(近侧结肠), and distal colon(末端结肠), and placed into RNAlater solution (Life Technologies, Grand Island, NY, USA) for RNA/DNA studies. 

Stools were collected from nine 12–14 week APCMin\+ female mice with intestinal neoplasia [18] and six WT female mice, and placed into RNAlater. 不是说小鼠6周的时候都进行了安乐死？怎么又收集了9只12~14周大的携带APC基因突变小鼠的粪便了？然后6只野生雌性小鼠的粪便，也没说年龄。

2.3 DNA and RNA Extraction of Intestinal Tissue and Luminal Content Samples

Total RNA and DNA, (host and associated bacterial mixed community) were extracted from the duodenum, jejunum, ileum, cecal pouch, proximal colon and distal colon tissues using TRI Reagent (Sigma, St. Louis, MO) according to the manufacturer’s recommendations. 

 For distal colonic luminal content samples, DNA was extracted using the UltraClean Fecal Kit (Mo BIO Laboratories, Inc., Carlsbad, CA).

2.4 Quantitative PCR (qPCR) for targeted bacterial clades(分化支，进化支)

PCR: Polymerase Chain Reaction 聚合酶链式反应 是一种用于放大扩增特定的DNA片段的分子生物学技术，它可看作是生物体外的特殊DNA复制，PCR的最大特点是能将微量的DNA大幅增加。

QPCR的英文全名是Real-time Quantitative PCR Detecting System。即实时荧光定量核酸扩增检测系统，也叫实时定量基因扩增荧光检测系统，简称QPCR。

QPCR assays were performed using established primers for Bacteroides(拟杆菌属)–Prevotella(普氏菌属)–Porphyromonas(卟啉单胞菌属), Lachnospiraceae(毛螺菌科), and total bacteria on all tissue and distal colonic luminal content samples as previously described. 

 The relative abundance of taxa within the Bacteroidetes phylum measured by ΔCt = Ct (threshold cycle)total bacteria - CtBacteroides–Prevotella–Porphyromonas.

The relative abundance of taxa within the Lachnospiraceae clade (i.e., Clostridia Group Xi’an clade) was measured by ΔCt = Ct (threshold cycle)total bacteria - CtLachnospiraceae as previously described.

All assays were carried out in triplicate. 所有的检测均是一式三份。

Plasmid(质粒) quantification standards were prepared from representative clones of the target organisms. 

The Mann-Whitney test using GraphPad Prism 5 (La Jolla, California) was performed to compare APCMin/+ and WT ΔCt values. 

The Bonferroni correction was made to correct for multiple comparison, thus significance required p< 0.025.

Bonferroni校正：如果在同一数据集上同时检验n个独立的假设，那么用于每一假设的统计显著水平，应为仅检验一个假设时的显著水平的1/n。

2.5 16S rRNA Amplicon(扩增子) Library Construction and Illumina V1V2 Sequencing Analysis

Bacterial profiles were determined by broad-range amplification and sequence analysis of 16S rRNA genes following our previously described methods. 按照我们之前描述的方法，通过对16S rRNA基因进行大范围扩增和序列分析确定细菌谱。

测序之前的各种准备工作的详细描述。

Paired-end sequences were sorted by sample via barcodes in the paired reads with a python script .

Sorted paired end sequence data were deposited in the NCBI Short Read Archive under BioProject Accession Number: PRJNA270112 (www.ncbi.nlm.nih.gov/bioproject/PRJNA270112)PRJNA270112. 原始数据，那么我也可以根据这个数据进行一步一步的分析。

• The sorted paired reads were assembled using phrap and paired reads that did not assemble were discarded. assemble? 用的phrap算法？
• Assembled sequence ends were trimmed over a moving window of 5 nucleotides until average quality met or exceeded 20.
• Trimmed contigs with more than 1 ambiguity or shorter than 200 nt were discarded.
• Potential chimeras(嵌合体) identified with Uchime (usearch6.0.203_i86linux32) using the Schloss Silva reference sequences were removed from subsequent analyses.
• Assembled sequences were aligned and classified with SINA (1.2.11) using the 418,497 bacterial sequences in Silva 115NR99 as reference configured to yield the Silva taxonomy. 再处理Assembled sequences
• Operational taxonomic units (OTUs) were produced by clustering sequences with identical taxonomic assignments. 
• OTU counts were normalized between samples by dividing sequence counts by the total number of sequences generated per sample.  OTU的归一化
• Phylum-level and family-level OTU tables were generated by collapsing lower level OTUs into higher-level categories. 
• OTUs with a relative abundance <0.0001 and with a prevalence <0.01 were culled and relative abundances then transformed using the square root function.
• The software package Explicet (v2.9.4, www.explicet.org) was used to display OTU data and estimate alpha diversity indices (i.e., SChao1, Shannon complexity [H], and Shannon Evenness [H/Hmax]) through 1000 replicate samplings of rarefied datasets.
• the effects of APCMin/+ genotype on individual OTU abundances were examined using the negative binomial (NB) regression model  采用负二项回归模型研究OTU相对丰度
• The NB and zero-inflated NB models are chosen based on AIC criterion
• The p values for the genotype effects on each OTU were then adjusted by the Benjamini-Hochberg procedure to calculate the FDR. 
• Significance was set as FDR<0.05. In addition, a 10-fold cross-validation was performed to validate the significant OTUs that were identified.
• The square root transformation was then applied to the relative abundances to correct for the skewness and to reduce the coefficient of variation. 
• Comparisons of overall microbial composition between APCMin/+mice versus the wild type mice were subsequently conducted using the permutation Hotelling T2 test with 10,000 permutations using the R package ‘Hotelling’.
• Principle coordinate analysis (PCoA) was conducted at the lowest taxonomic level (genus) using the wcmdscale function implemented by the vegan R package and using Morisita-Horn dissimilarity scores.

2.6 Histological(组织学的) Analysis of the Intestinal Sections

病理学相关方面的分析

2.7 Comparison of Mouse Proximal Colonic mRNA Expression in and Wild type mice

• IL-1β mRNA expression relative to actin mRNA, was measured in cecal, proximal colonic, and distal colonic intestinal tissue RNA samples in all 30 APCMin/+ and 30 WT mice as previously described

• IL-1β和IL-1βmRNA检测的意义分别是什么? 前者是检测细胞因子的蛋白质.而后者是检测该基因是否有表达.蛋白质可以被分泌到细胞外,所以可以在体液中检出,而mRNA只有在产生该细胞因子的细胞内才有.

• RNA extracted from the proximal colon of 9 week-old WT mice treated with 3% DSS in water for 7 days was used as a positive control for the assay. 

• The IL-1β ΔCt values (ΔCt = Ctactin-CtIL1β) were compared between APCMin/+ and WT groups using the Mann-Whitney test. Significance was set at a threshold of p <0.05.

• 1、T检验是用于正态分布资料的小样本（样本容量小于30）的两个平均值差异程度的检验方法。 2、Mann-Whitney U 检验是与独立样本t检验相对应的方法，当正态分布、方差齐性等不能达到t检验的要求时，可以使用该检验。

• Aliquots (1 μg) of proximal colon RNA samples from 6 APC were subjected to paired-ends 100 bp Illumina sequencing. 

• Between 81 and 314 million reads were generated for each of the RNA samples. 产生的reads

• The RNA-Seq data were deposited in NCBI's Gene Expression Omnibus database with accession number GSE67634.

• The short reads were aligned to the GRCm38 genome (http://useast.ensembl.org/Mus_musculus/Info/Annotation) using STAR (Spliced Transcripts Alignment to a Reference), and then converted to raw gene counts using featureCounts.

• The edgeR package was used to identify differentially expressed (FDR<0.05) genes (DEGs) between the APCMin/+ and wild type mice, using additional cutoff of 2-fold differential expression between groups.

• Hierarchical clustering based on the reads per kilobase of exon per million mapped reads (RPKM) value of the 130 DEGs was carried out by using 1-r dissimilarity measurement and Ward linkage, and the cluster number (n = 7) was chosen based on inspection of the coefficient of determination (R2) plot as previously described.

• Second, a negative binomial (NB) regression model was fit with gene clusters as following

• All models were fitted with R package: glmmADMB

3. Results

• The relative abundance of Bacteroidetes spp is increased in APCMin/+mice colonic mucosa and luminal content prior to the development of intestinal neoplasias. 在肿瘤形成前，拟杆菌门丰度增加。
• In preliminary targeted qPCR studies, we observed that the relative abundance of Bacteroidetes spp. in fecal DNA was higher in 12–14 week-old APCMin/+ female mice compared to age-matched WT female mice (ΔCt = -2.4 vs. ΔCt = -5.1, p = 0.0004).
• To test the hypothesis that the increase in the relative abundance of Bacteroidetes spp. preceded polyposis, we compared the relative abundance of this clade in the ileal, cecal, proximal colonic, distal colonic mucosa and the distal colonic luminal content in 6 week-old mice. 6周龄(肿瘤形成前)小鼠在各处的拟杆菌门的丰度
• Because gender effects have been previously reported on the number and location of polyps, we restricted our analysis to female mice. 分析仅限于雌性老鼠，因为之前已经有过性别上的比对研究了。
• 先确认没有肿瘤形成，然后We observed a significant increase in the relative abundance of Bacteroidetes spp. in proximal colonic, distal colonic and distal luminal contents between the APCMin/+ and WT-control mice, but no significant difference in the ileal or cecal mucosal samples. 回肠或盲肠粘膜样本没有检测到显著变化，其他部位均检测到了。
• In contrast we observed no significant difference is Lachnospiriceae spp., a prominent group of Firmicutes(厚壁菌门), except in the luminal content of the distal colon, which exhibited significantly higher loads in WT mice (p = 0.003).

3.1 16S rRNA sequence analysis of proximal colonic mucosal samples from   and WT mice

• Illumina sequencing of the 16S rRNA gene V1V2 hypervariable region was carried out for the proximal colonic DNA samples. 近端结肠DNA样本

• A total of 13,248,412 high-quality sequences were generated

• The median Good’s coverage score was ≥ 99.9987% at the rarefaction(稀疏) point of 9,381 sequences, indicating deep sequence coverage of the intestinal microbiome.

• The 16S rRNA sequencing results confirmed the targeted qPCR results in demonstrating a significant increase in the relative abundance of taxa within the Bacteroidetes phylum (FDR = 0.0009) in the APCMin/+ mice (Fig 1A and Table 2).  证实了之前提到的丰度观察结果。

• The relative abundance of the Cyanobacteria phylum and the Chloroplast family, which is the most prevalent family in this phylum), was decreased in APCMin/+ mice (FDR = 0.047). However this observation was confirmed in only 4 out of 10 cross-validations. batch effect？

• Principle Coordinate Analysis(PCoA) 和principal components analysis (PCA) 这两个原来不是一个东西啊。。。

• PCA (principle component analysis)主成分分析：常用的降维算法，该方法是在样本*特征矩阵上直接进行特征转换，前提假设是数据点变化大的维度信息量更大，设法保留数据里的变异让点的位置尽量不要变动。

• PCoA(principle coordination analysis)主坐标分析：是探索数据相似度或者相异度可视化方法。该方法是在样本距离矩阵上进行变换，尽量在低维空间保持样本在高维空间的距离关系。

3.2 Differentiallly expressed genes in 6 week-old   mice

•  To examine how the APC mutation could alter the host colon gene expression, we conducted parallel RNA-sequence analysis on 6 APCMin/+ mice and 6 WT mice, (sampling all three cohorts).
• A total of 130 host genes (fold change > 2 fold, FDR <0.05) were selected using edgeR
• The DEGs were grouped into seven clusters as described in Methods (see Table 4), with 106 upregulated genes distributed among three clusters and the 24 downregulated genes distributed among four clusters.
• While APC genotype had a dominant effect on the relative abundance of Bacteroidetes, detection of additional associations with mouse colonic gene expression, suggest that alterations in host colonic gene expression play a role in influencing mucosal associated microbial composition.
•  These values were both very low compared to that measured in DSS treated mice (-4.9), indicating that IL-1β was not highly expressed in the colons of either mouse group in our study.

4. Discussion

• This study demonstrates that alterations in the gut microbiome, characterized by an increased relative abundance of Bacteroidetes spp. observed in association with intestinal neoplasias, actually precedes the development of microscopically detectable intestinal neoplasias in 6 week old APCMin/+ mice.
• The relative abundance of the phylum Tenericutes observed in this study is higher than reported by some studies of C57Bl/6 mice, but similar to another study using C57Bl/6 mice purchased from the same vendor 从同一供应商拿的小鼠的研究结果相近
• In this study, the APCMin/+ and WT mice were housed in separate cages, which could influence the reported microbial compositions, possibly related to coprophagic behavior. 共同预防行为可能影响小鼠肠道菌群
• In summary, our results support the concept that APC haplo-insufficiency of the host colonic epithelial cell alters colonic microbial interactions prior to polyposis.
• It is thus conceivable that such microbiome changes contribute to the pathogenesis of colon cancer. 
• An important corollary to such a notion would be that the colonic microbiome represents an important (and druggable) target for the prevention of colon cancer. 重要推论，靶向肠道菌群或可治愈结肠癌
• Indeed, interventions directed at the microbiome (germ free and antibiotic treatment) have been reported to modulate tumor formation in mouse models of colon cancer 这个推论相关的研究已经被报道过了
• However, it remains to be determined whether interventions directed at ameliorating dysbiosis in APCMin/+ mice, such as through probiotic, prebiotic or antibiotic interventions, could reduce tumor formation. 至于干预效果还是需要继续研究