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

論文題目：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腺瘤樣結腸息肉易感基因蛋白 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(同類系的) wild type (WT) mice. The results demostrate that similar to 12-14 week old 

 mice with intestinal neoplasia(腸道惡性良性腫瘤), 6 week old 

 mice with no detectable neoplasia, exhibit an increased relative abundance of Bacteroidetes spp in the colon(結腸). 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(分層的) 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, 負二項式) 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，進化支)

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

2.6 Histological(組織學的) 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

腸道菌群失調在人類結腸癌中早有報道。但是腸道失調是惡性良性腫瘤形成的結果，還是促進了惡性良性腫瘤形成，即因果關系，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. 與散發性和遺傳性結腸癌最相關的基因突變是發生在APC（腺瘤性息肉病）基因。APC本來是一個抑癌基因，如果APC發生突變，就會引發息肉病變，最後演變發展成癌症。

 familial adenomatous polyposis (FAP) 家族性結腸息肉綜合征

APC突變會引發FAP，但是FAP患者隻要及早檢查跟進和接受手術治療，大部分結腸癌是可以預防的。

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) 多發性腸惡性良性腫瘤

The multiple intestinal neoplasia (Min) mouse model of FAP carries a truncation mutation at codon 850 of the Apc gene. 多發性腸惡性良性腫瘤的FAP小鼠模型攜帶APC基因的850号密碼子的突變。

需要明确的是APC(Min/+)突變小鼠特指APC基因第850号密碼子出現無義突變（TTG---> TAG）的C57BL/6J品系小鼠，其它APC基因突變或敲除的小鼠即使出現腸道多發性腺瘤也不宜稱之為APC(Min/+)突變小鼠。

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. 腸道菌群可能促進腸道惡性良性腫瘤的發展。

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. 有一項研究表明無菌小鼠隻有在小腸中段的息肉發病率降低，而另一項研究表明無菌小鼠的小腸和大腸的息肉發生率均明顯降低。

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. 對于攜帶這兩種突變的小鼠，抗生素治療可以減少小腸和大腸的息肉數量。

We hypothesize that mutation of the APC gene results in alterations in host-microbiota interactions prior to tumor formation. 我們假設：APC基因的突變導緻了宿主與微生物群互相作用的改變，進而形成惡性良性腫瘤。

為了驗證假設：gut microbial composition was compared between 6 week-old C57Bl/6 APCMin/+, prior to the development of detectable neoplasia, and congenic WT mice. 比較了6周大的C57Bl/6 APCMin/+小鼠（在可檢測到的惡性良性腫瘤發展之前）和同類系的野生型小鼠。

2. Materials and Methods

2.1 Animal Type and Housing

為了減輕小鼠的旅行壓力，讓小鼠适應環境了兩周。在小鼠安樂死的時候使用了二氧化碳。

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隻雌性，10隻雄性。

 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. 實施安樂死之前，置于無特定病原體的籠子，三到四隻一個籠子。

所有操作符合動物實驗規範。

WT mice是從哪裡來？還是隻有攜帶APC基因突變的小鼠才特别從那個實驗室發過來的？

2.2 Tissue and Luminal Content Sample Collection

The segments analyzed included the ileum, cecum, proximal colon, and distal colon. 所分析的節段包括回腸、盲腸、近端結腸和遠端結腸。

Each small intestinal segment was washed in sterile(無菌) phosphate(磷酸鹽) buffered saline to remove the luminal(腔内) content. 沖洗

A 1.0–1.5-cm section was obtained from the proximal ends of duodenum(十二指腸), jejunum(空腸), distal ends of ileum(回腸遠端), proximal colon(近側結腸), and distal colon(末端結腸), 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. 不是說小鼠6周的時候都進行了安樂死？怎麼又收集了9隻12~14周大的攜帶APC基因突變小鼠的糞便了？然後6隻野生雌性小鼠的糞便，也沒說年齡。

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，進化支)

PCR: Polymerase Chain Reaction 聚合酶鍊式反應 是一種用于放大擴增特定的DNA片段的分子生物學技術，它可看作是生物體外的特殊DNA複制，PCR的最大特點是能将微量的DNA大幅增加。

QPCR的英文全名是Real-time Quantitative PCR Detecting System。即實時熒光定量核酸擴增檢測系統，也叫實時定量基因擴增熒光檢測系統，簡稱QPCR。

QPCR assays were performed using established primers for Bacteroides(拟杆菌屬)–Prevotella(普氏菌屬)–Porphyromonas(卟啉單胞菌屬), 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. 所有的檢測均是一式三份。

Plasmid(質粒) 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：如果在同一資料集上同時檢驗n個獨立的假設，那麼用于每一假設的統計顯著水準，應為僅檢驗一個假設時的顯著水準的1/n。

2.5 16S rRNA Amplicon(擴增子) 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. 按照我們之前描述的方法，通過對16S rRNA基因進行大範圍擴增和序列分析确定細菌譜。

測序之前的各種準備工作的較長的描述。

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. 原始資料，那麼我也可以根據這個資料進行一步一步的分析。

• 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(嵌合體) 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. 再處理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的歸一化
• 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  采用負二項回歸模型研究OTU相對豐度
• 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(組織學的) Analysis of the Intestinal Sections

病理學相關方面的分析

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檢測的意義分别是什麼? 前者是檢測細胞因子的蛋白質.而後者是檢測該基因是否有表達.蛋白質可以被分泌到細胞外,是以可以在體液中檢出,而mRNA隻有在産生該細胞因子的細胞内才有.
• 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檢驗是用于正态分布資料的小樣本（樣本容量小于30）的兩個平均值差異程度的檢驗方法。 2、Mann-Whitney U 檢驗是與獨立樣本t檢驗相對應的方法，當正态分布、方差齊性等不能達到t檢驗的要求時，可以使用該檢驗。
• 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. 産生的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. 在惡性良性腫瘤形成前，拟杆菌門豐度增加。
• 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周齡(惡性良性腫瘤形成前)小鼠在各處的拟杆菌門的豐度
• Because gender effects have been previously reported on the number and location of polyps, we restricted our analysis to female mice. 分析僅限于雌性老鼠，因為之前已經有過性别上的比對研究了。
• 先确認沒有惡性良性腫瘤形成，然後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. 回腸或盲腸粘膜樣本沒有檢測到顯著變化，其他部位均檢測到了。
• In contrast we observed no significant difference is Lachnospiriceae spp., a prominent group of Firmicutes(厚壁菌門), 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. 近端結腸DNA樣本
• 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).  證明了之前提到的豐度觀察結果。
• 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) 這兩個原來不是一個東西啊。。。
• PCA (principle component analysis)主成分分析：常用的降維算法，該方法是在樣本*特征矩陣上直接進行特征轉換，前提假設是資料點變化大的次元資訊量更大，設法保留資料裡的變異讓點的位置盡量不要變動。
• PCoA(principle coordination analysis)主坐标分析：是探索資料相似度或者相異度可視化方法。該方法是在樣本距離矩陣上進行變換，盡量在低維空間保持樣本在高維空間的距離關系。

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 從同一供應商拿的小鼠的研究結果相近
• 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. 共同預防行為可能影響小鼠腸道菌群
• 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. 重要推論，靶向腸道菌群或可治愈結腸癌
• Indeed, interventions directed at the microbiome (germ free and antibiotic treatment) have been reported to modulate tumor formation in mouse models of colon cancer 這個推論相關的研究已經被報道過了
• 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. 至于幹預效果還是需要繼續研究