CpG Island Methylator Phenotype(CIMP) is An Independent Prognostic Marker with Tumor-Promoting Functions in Colorectal Cancer

The CpG island methylator phenotype(CIMP)with extensive promoter methylation isa distinct epigenotype incolorectal cancer (CRC). Changes in microbiota and epigenetic dysregulation mightbe the key underlying mechanism. collected afterproctocolectomy. The 16S rRNA gene sequencing was carried to determine the differences in microbiota.Subsequently,BRAF mutation,the statusof microsatellite instability (MSI, also known as mismatch repair deciency) and CIMP were also tested. The Chi-square test was carriedoutto analyze the relationship between molecular changes (MSI and CIMP) and the development of CRC.

Based on the above information, we aimto determine whether the CIMP status provides further insight into clinical behavior and survival in patients with CRC. Since the CIMP status associates with MSI and mutation of BRAF, we have quanti ed the expression levels in tumor tissues. Besides, we have also checked the structure of the microbiota community and the status of MSI and CIMP, and the mutation of the BRAF.It is expected that this study can reveal the pathological process of how theabove molecular phenotypes could affect the prognosis of CRC patients.

Method
Study population and design A total of 180 individuals scheduled for colorectal resection at Wenzhou Medical University, Wenzhou, China, were recruited to the study. All patients with stages I-III cancer underwent a standard curative surgery, while stage IV CRC tissues were collected from patients who received palliative surgery to relieve serious cancer-related contradiction. The patients were not treated with antibiotics in the month before surgery but were administered antibiotics intravenously in a few hours after resection. Patients who had received or regularly used non-steroidal anti-in ammatory drugs, statins, or probiotics were excluded. Besides, those who had chronic bowel disease, infections, food allergies, and dietary restrictions were also excluded. Surgeries were performed at the A liated Hospital of Wenzhou Medical University between 2014 and 2017, and the treatments were given according to the National Comprehensive Cancer Network Guidelines.

Sample preparation
Tumor samples were obtained from the 180 patients who had undergone proctocolectomy. These CRC tissue samples and adjacent normal tissue samples (at least 5cm from the tumor site) of these 180 patients were obtained from the gastrointestinal cancer specimen bank of the A liated Hospital of Wenzhou University. To be speci c, surgically resected specimens were collected immediately after tumor removal and stored at -80°C. The TNM stages were determined according to the American Joint Committee on Cancer system and all specimens were graded histologically according to the World Health Organization classi cation criteria. Written informed consents of joining the specimen bank were obtained from all the patients before surgery, and the protocols used in the study were approved by the Ethics Committee of A liated Hospital of Wenzhou Medical University. Clinical and pathologic data were reviewed from the gastrointestinal cancer database of A liated Hospital of Wenzhou Medical University. DNA extraction CTAB method was used to extract DNA from all tumor samples with minimal modi cation. The concentration of DNA was measured by uorometer or microplate reader and sample integrity was tested by agarose gel electrophoresis (1% concentration of agarose Gel; 150 V; 40 min electrophoresis time). All DNA samples were stored at -20°C.

PCR and sequencing analysis
The V4 region of the bacterial 16S rRNA gene was ampli ed by polymerase chain reaction (PCR) using universal primers 319F and 806R. The reaction mix consisted of Phusion High-Fidelity PCR Master Mix (NEB, Ipswich, MA, USA) and appropriate primer/probe pairs. The PCR program was as follows: Denaturation at 98°C followed by 30 cycles of 45 s at 95°C (denaturation) in 3 min, thenannealing at 55°C and 45 s at 72°C (extension) in 45 s, with a nal extension at 72°C for 7 min. The PCR products were puri ed with AMPure XP beads (Agencourt Bioscience) to remove the unspeci c products before library construction. The library was quantitated in two ways. The average molecule length was measured using the Agilent 2100 bioanalyzer instrument (Agilent DNA 1000 Reagents) and then quanti ed by real-time quantitative PCR (qPCR; EvaGreen TM). The sequencing of quali ed libraries was performed at the BGI-Huada Genomics institute in Shenzhen using MiSeq System, with the sequencing strategy PE250 (PE251+8+8+251) or PE300 (PE301+8+8+301) (MiSeq Reagent Kit).
Representative OTUs were aligned to the optimized sequences and the abundance of OTUs per sample was obtained for further analysis. Ribosomal Database Project (RDP) Classi er v.2.2 was used to taxonomically classify OTU representative sequences in the following databases: Greengene V201305 [21] and RDP (Release9 201203) [22].

Promoter Methylation Analysis
The De nition of CpG is the land promoter hypermethylation of at least 2 out of 5 methylation markers (CACNA1G, CDKN2A, NEUROG1, CRABP1and MLH1), as proposed by Weisenberger et al [23]., to detect CIMP in tumor tissue of CRC. Methylation of these markers was determined by bisul te modi cation of 500 ng genomic DNA using a commercially available kit (Zymo Research), to create a methylation index with the CIMP markers and subsequent methylation-speci c PCR (MSP) [24,25]. Since MSP is effective, speci c, and simple, it was used to detect CIMP. Also, the research shows that there are no differences between MSP and other technologies such as MethyLight [26].

MSI Analysis
As a second-generation genetic marker, MSI has been widely applied in tumor gene diagnosis and genetic analysis because of its high polymorphism, stability, and Mendelian co-dominant inheritance. It is determined through the multiplex uorescent PCR combined with capillary electrophoresis(CE)using the MSImarkersNR-21 BAT-26 NR-27 BAT-25 NR-24, and MONO-27, as reported by Suraweera et al [27]. This method has high e ciency, stability, high sensitivity, thus it provides reliable analysis results. After ampli ed by Multiplex uorescent PCR, the CE method using dual internal standards (molecular weight markers shorter and longer than the PCR fragment of interest) was implemented to measure microsatellite length. To validate the CE method in detecting MSI, a human tumor sample and a control DNA sample collected from the patients were also tested.

Statistical analysis
Statistical analyses were conducted using the SPSS 20 statistical software. The two-sided v2 test was used to determine the associations between CIMP status and different clinicopathological features. The signi cance was determined using the logrank test. Cox proportional hazard models were used to carry out multivariate survival analyses. Metastases (http://metastats.cbcb.umd.edu/) and R (v3.0.3) were used to determine which taxonomic groups were signi cantly different.

Associations between Molecular phenotype and clinicopathologic characteristics
The clinical characteristics of the patients with different molecular phenotypes were compared, as shown in Table 1.Wecould see that the patients belonging to different groups might be correlated with the patients' outcomes in CRC. Based on the stages of the tumor samples, we found that tumor location(P = 0.022), differentiation(P = 0.047), and tumor size(P = 0.031) were signi cantly associated with CIMP status, Besides, correlations between age(P = 0.026), gender(P = 0.045), remote Metastasis(P = 0.005) and MSI status were also signi cant. In addition, gender (P = 0.046)and differentiation (P = 0.043)were found associated with BRAF mutation (Table 1).   (Table 3). Furthermore, positive CIMP, MSI status,and differentiation were associated with poor 3-year DFS both in the univariate Cox regression analyses and the multivariate analysis (Table 4).

Diversity and structural changes of the tumor microbiota in CRC patients with different prognosis outcomes
The overall microbiota at the phylum level is shown in Fig. 2A (Fig. 2B).
In specie level, we found a higher level of B. fragilis ( [29][30][31]. Acid provides a methyl group, and a methyl group is added to the fth carbon atom of cytosine, making a chemical modi cation reaction of 5-methylcytosine. This process mainly occurs on CpG dinucleotides,thus epigene silencing is caused by DNA methylation. CIMP, as an epigenetic phenotype of CRC, has different clinical, pathological, and biological characteristics, such as being related to proximal colon, female, poor differentiation, MSI, BRAF high-frequency mutations, tp53 low-frequency mutations [32][33][34], and CIMP-H, MSI-H and TGFBR2 single nucleotide mutation. During the development of CRC, CIMP that progresses to dentate adenomas shows frequent promoter methylation, while at adenomas not [35].
To our best knowledge, this is the rst study to compare the CIMP status, MSI,and BRAF among groups of cancer tissues divided by different post-operation prognoses. CIMP has been extensively studied in CRC because it re ects epigenetic aberration in tumor cells [36,37]. CIMP, as an epigenetic phenotype of CRC, has different clinical, pathological, and biological characteristics, such as related to proximal colon, female, poor differentiation, this was also demonstrated in our study.
Divergent ndings ofthe prognostic values of CIMP have been reported. Some studies suggest an adverse effect of CIMP on the survival of CRC patients [38][39][40][41][42], whereas other studies reported no relationship between CIMP status and prognosis in CRC [43][44][45][46]. These contradictory results can be explained at least in part by the absence of a consensus CIMP panel since some studies used the classical panel de ned by Toyota et al [37]., whereas others used the new CIMP panel [13]. Overall, MSI often manifests as a Pathological type with poor prognoses such as poor differentiation or mucinous adenocarcinoma [47], and remote metastasis was also found in our study. Besides, we also examined the BRAF mutation on patients, CIMP-high status was independently associated with a bad differential, whereas BRAF mutation was associated with a signi cantly increasing numbers. It might explain why these two molecular phenotypes were associated with bad outcomes in many studies [48,49].
The speci c mechanisms by which gut microbiota affects the development of CRC are still not well understood. One of the most promising theories is the microbe-driven intestinal mechanism. Interestingly, F. nucleatum, B. fragilis, and F. prausnitzii are all key players in modifying intestinal in ammation levels. To our knowledge, CIMP is believed to promote carcinogenesis through methylation mediated transcriptional silencing in tumor suppressor genes [32]. However, the role of microbiota in the progress of CIMP and the mechanism underlying its sustained in uence remains unknown. In fact, the relationships between CIMP, MSI, BRAF mutations, and the gut microbiota in colorectal cancer arecomplex. To decipher the complex association of CIMP, MSI, and BRAF mutation on patients' microbiota, we collect the patients' CRC tissues in four groups. The results showed the relationship in CIMP status between different groups, which was consistent with previous studies. Besides, the higher incidence was observed in there currence group, with a greater abundance of F. nucleatum. Some studies have shown that F. nucleatum is associated with epigenetic changes such as the status of MSI and CIMP [50] ,which also corresponds with the results in this study. In addition, another study has also shown that patients with a high level of F. nucleatum have a signi cantly shorter survival time, which was similarly obtained in this study [51]. But their sample size was relatively small. A more recent study has reported a similar result using a larger database of CRC cases in the USA, revealing a correlation between a high amount of tissue F. nucleatum DNA and higher CRC-speci c mortality. Evidence has also shown that overexpression of BRAF were markers of the poor prognosis [52,53], Moreover, MSI, the primary causes of which is hypermethylation of the MLH1 promoter, was also associated with the clinical outcomes of CRC [4,6].A relevant study hasshown that microbiota has a relation with MMR, which is the most important mechanism for the appearance of MSI [54]. Furthermore, overexpression of BRAF was also considered the marker of poor prognosis which was consistent with our ndings of prognostic values of F. nucleatum,B.
fragilis,and F.prausnitzii [55] . This might explain why BRAF mutation could accompany MSI as reported in the previous studies. Finally, it is important to mention that our study had some limitations. The major one is the small size after dividingthe cohort into 4 subgroups.Although we have studied the related risk factors and survival rate, we have not explored the correlation between these phenomena and cancer medication.Besides, some mechanisms between them and the intestinal micro-ecology are just a taste.

Conclusions
Our results underline the interest that CIMP can be useful to predict prognosis in CRC patients, and at present, it is believed that MSI is closely related to the occurrence and development of CRC. So far, MSI has beena tumor-related factor recognized worldwide. Microbiota, such as B. fragilis and F. prausnitzii, participate in in uencing the course/progression of CRC in patients subjected to energy restriction in their early childhood, which further validates the association of F. nucleatum with epigenetic changes and gene mutations.The future goal is to use precision medicine to analyze the patient's genes, select the most suitable individualized treatment plan for the patient, and make the most accurate judgment on the patient's prognosis.

Declarations
The authors do not have any con icts of interest with the content of the manuscript.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
The ethical committee approved the procedure.

CONSENT FOR PUBLICATION
Written informed consent for publication was obtained from all participants.

AVAILABILITY OF DATE AND MATERIALS
The raw/processed data required to reproduce these ndings cannot be shared at this time as the data also forms part of an ongoing study.

COMPETING INTERESTS
None.  Kaplan-Meier survival curves for overall survival (OS) and disease free survival (DFS) in 180 CRC patients in relation to CIMP, BRAF and MSI. P values were obtained by log-rank test.