Sample
Bacterial strains
Culture samples (colorectal cancer mucosal tissues) were taken from the Beijing Shijitan Hospital. Isolation and culture were performed as described [22]. A strain of Parvimonas micra was isolated from the sample and named Pm-42. DH5α strain were purchase from Sangon Biotech Co., Ltd. (Shanghai, China).
Colorectal cancer mucosa and adjacent normal tissue
The 22 patients with CRC included in this study were from Shijitan Hospital, Beijing. We took 2 cm2 colorectal cancer mucosal tissue and adjacent normal tissue samples, at least 5 cm away from the cancerous tissue.
Cell lines
LoVo and HT-29 cells were purchased from Beina Biological Co., Ltd. (Henan, China) .
Cell counting
LoVo or HT-29 cells were seeded into a 24-well plate. A total of 1 × 104 cells per well were incubated at 37 °C for 4 h in 5% CO2. Then, Pm-42 or DH5α strains were added to the plate at a multiplicity of infection (MOI) of 10. The plates were incubated in an anaerobic incubator for 2 h and then at 37 °C for 24 and 48 h in 5% CO2. The cells seeded in the wells were counted with a cell counter (Invitrogen, Countess 3 FL). PBS was used as a blank control.
MTT cell proliferation assay
LoVo or HT-29 cells were seeded in a 96-well plate. A total of 2 × 104 cells per well were incubated at 37 °C for 4 h in 5% CO2; Pm-42 or DH5α cells were added to the plate at an MOI of 10, that is, 2 × 105 per well. To evaluate proliferation, the MTT cell proliferation and cytotoxicity assay kit (Solarbio, M1020) was used 24 and 48 h after the coincubation, and absorbance was detected at 490 nm. PBS was used as a blank control.
Real-time cell analysis (RTCA)
A total of 2 × 104 LoVo or HT-29 cells were seeded into the wells of a plate. After 8 h, Pm-42 or DH5α was added to the well at an MOI of 10. The plate was placed on the machine for 48 h. PBS was used as a blank control.
Animal experiments
Xenograft model
We purchased 6-week-old female nude mice from Vital River Laboratory Animal Technology (Beijing, China). LoVo or HT-29 cells were incubated with Pm-42 or DH5α strains at an MOI of 10 for 24 h. The cells were collected and washed three times with PBS, mixed with Pm-42 or DH5α at an MOI of 1:1, and injected subcutaneously into nude mice. The number of cells administered to each mouse was 3 × 106 cells/mouse. Three hours after the injection, the mice were given an intraperitoneal injection of piperacillin antibiotic therapy (150 mg/kg body weight). PBS was used as a blank control. Body weight and tumor volume were subsequently measured every two days. On the 24th day after modeling, the mice were killed and the blood was taken, and then the serum was extracted for cytokine detection. Cytokines were detected with the Bio-Plex 200® system (Bio-Rad, United States).
Tumor-bearing nude mice treated with the antagomir (GenePharma, China) were subcutaneously injected with cells using the same protocol. After nine days of cell injection, 5 nmol antagomir was injected locally into the tumor every three days for a total of five injections. Body weight and tumor volume were measured every two days throughout the experiment.
P. micra gavage administration in APCMin/+ mice
APCMin/+ mice were 5-week-old females purchased from GemPharmatech Biotechnology Co., Ltd. (Nanjing, China). Mice were fed streptomycin (2 mg/mL) in water for three days before gavage. Mice were given 109 CFU bacteria once a day by gavage for 12 weeks. Blood and feces were collected at the midpoint or endpoint of gavage. Serum biochemistry was performed using a biochemical detector (Beckman Coulter, AU480), and cytokines were detected with the Bio-Plex 200® system (Bio-Rad, United States).
Pathological and immunohistochemical detection
Fresh tissues were fixed in 4% paraformaldehyde for over 24 h. Tissues were embedded in paraffin. For pathological detection, the paraffin-embedded colonic sections (5 μm thick) were stained with hematoxylin and eosin. For immunohistochemical (IHC) detection, the sections were stained with anti-ERK1/2 (Cell Signaling Technology, 4695 T), anti-c-Fos (Abcam, ab222699), and anti-PTPRR (Abcam, ab180134) antibodies.
Cells transcriptome sequencing
LoVo cells were incubated with Pm-42 or PBS for 48 h at an MOI of 10. After incubation, the cells were collected, and washed three times with PBS. Cellular RNA was extracted with the PureLink RNA Mini Kit (Invitrogen, 12183018a). The RNA samples were sent to Novogene Biological Technology Company (Beijing, China) for transcriptome sequencing. Raw data were uploaded to GenBank (BioProject PRJNA859257).
Cells microRNA sequencing
HT-29 cells were incubated with Pm-42 or PBS for 48 h at an MOI of 10. After incubation, the cells were collected and washed three times with PBS. Cellular RNA was extracted by using Trizol (Ambion, 15,596,026). Finally, the RNA samples were sent to Novogene Biological Technology Company (Beijing, China) for microRNA sequencing. Raw data were uploaded to GenBank (BioProject PRJNA859413 and PRJNA859414).
Cell exosomes sequencing
HT-29 cells were cultured for 24 h. The medium was replaced with serum-free media. The cells were incubated with Pm-42 or PBS at an MOI of 10 for 48 h. Cell supernatants were collected, and exosomes were extracted using the Extracellular vesicle extraction kit (Raojing Gene, China), followed by RNA extraction with the miRNeasy Mini Kit (QIAGEN, 217004). Library construction was performed with the NEBNext Multiplex Small RNA Sample Prep Set for Illumina (NE, E7300). Raw data were uploaded to GenBank (BioProject PRJNA859256).
DNA extraction and 16S rDNA sequencing of mice feces
Fresh feces were collected from mice at the end of gavage. DNA was extracted from feces by using the QIAamp PowerFecal Pro DNA kit (QIAGEN, 51804). The extracted DNA was sent to Novogene Biological Technology Company (Beijing, China) for 16S rDNA sequencing. Raw data were uploaded to GenBank (BioProject PRJNA859250).
Western blot
Total protein of cells or tissues was separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins in SDS-PAGE were transferred onto a nitrocellulose membrane (Millipore). The membrane was incubated first with primary antibodies and then with secondary antibodies. The primary and secondary antibodies used are as follows: anti-Ras (Abcam, ab108602), anti-ERK1/2 (Cell Signaling Technology, 4695 T), anti-p-ERK1/2 (Cell Signaling Technology, 4370 T), anti-c-Fos (Abcam, ab222699), anti-PTPRR (Abcam, ab180134), anti-β-actin (Ray antibody, EM2001), and a fluorescent secondary antibody (LICOR, 926–32,211 and 926–32,210). The band intensity ratio of the target protein to β-actin was calculated. Fold change = (target protein/β-actin)/mean value of the band intensity ratio in the blank group. The experiment was performed in three independent replicates.
Quantitative polymerase chain reaction (qPCR)
Cell samples were directly collected and washed with PBS three times. Mucosal tissue samples were homogenized (HODER, China). RNA was extracted with the Trizol reagent. Mucosal tissues were homogenized and treated with lysozyme (Thermo Scientific, 89,833) for four hours. Total DNA was extracted with the QIAamp DNA mini Kit (QIAGEN, 51306). Total RNA, containing mRNAs and microRNAs, was converted to cDNA using reverse transcription reagents (Vazyme, R323–01 and MR101–01) according to the manufacturer’s protocols. qPCR-based detection of P. micra, mRNA and microRNA was performed using SYBR Green qPCR Master Mix (Vazyme, Q511–02) in the LightCycler 480 II Real-Time PCR System (Roch, Switzerland). Data were presented as fold change using the 2-△△CT method. Primer sequences are listed in Supplemental Table 1.
Transfection of plasmid, mimics, inhibitor, and negative control into the cells
Cells were seeded into wells and transfected with the pcDNA3.1(+)-hPTPRR or pcDNA3.1(+) using a transfection reagent (Vazyme, T101–01), or transfected with the mimics, inhibitor, or negative control (NC) using a transfection reagent (Polyplus, 101,000,028). Bacteria were added for coincubation using the method used in MTT cell proliferation assay, 24 h after transfection.
Prediction of the miR-218-5p target genes and the binding sites to the target genes
Target genes were screened using a target gene prediction database. The starBase database integrates the prediction results of seven databases to predict microRNA target genes (https://starbase.sysu.edu.cn/). Target genes predicted in ≥ 4 databases are screened out. The binding sites for miR-218-5p and protein tyrosine phosphatase receptor R (PTPRR) were predicted using TargetScan (https://www.targetscan.org/).
Luciferase reporter assay
To construct the dual luciferase plasmid, we cloned: 1) the 3′-untranslated region (3′-UTR) of the target site (of the target gene corresponding to the microRNA) and its adjacent sequences into the pmirGlo vector as the wild type and 2) mutated 3′-UTR target site (of the target gene corresponding to the microRNA) into the pmirGlo vector as the mutant type. HT-29 or LoVo cells were cultured in 12-well plates until the cell density reached ~ 60%. The cells were cotransfected with the pmirGlo plasmid and mimics or mimics negative control using a transfection reagent (Vazyme, T101–01), and the luciferase assay was performed using the Dual Luciferase Reporter Assay Kit (Vazyme, DL101–01) according to the manufacturer’s protocols. Relative luciferase activity = (Firefly/Renilla) / mean (Firefly/Renilla) of the mimics NC.
The Cancer genome atlas database analysis
The Cancer Genome Atlas (TCGA) is a project overseen by the National Cancer Institute and the National Human Genome Research Institute to apply high-throughput genome analysis techniques to help understand cancer (https://portal.gdc.cancer.gov/). We used the Xiantao academic (https://www.xiantao.love/) to search the TCGA database for transcriptome sequencing and microRNA sequencing data related to colorectal cancer and to analyze the data and prepare figures. Screened TCGA database ID numbers can be found in Supplemental Table 2.
Statistical analysis
For comparisons between multiple sets of data, the normality and homogeneity of variance of each data set were first confirmed. If the data were distributed normally and showed homogeneity of variance, analysis of variance (ANOVA) or two-tailed unpaired Student’s t-test was used, whereas if they were not normally distributed or showed heterogeneity of variance, a rank-sum test was used (P < 0.05). For the comparison of P. micra abundance and genes (or microRNA) expression in tumor and adjacent normal tissues, a paired T test was used for statistical analysis. P. micra abundance and gene (or microRNA) expression were statistically calculated using Spearmans’s correlation analysis. Data are expressed as mean ± SD from 3 independent experiments. All P values were 2-tailed and P values of < 0.05 were considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001). All statistical analyses were done using the SPSS Statistics 20.0 software.