Graphical abstract graphic file with name fx1.jpg [47]Open in a new tab Highlights * • Knocking out Mogat2 promotes intestinal tumor growth in Apc^Min/+ mice * • Mogat2 knockout in mice alters the composition of gut microbiota * • Gut microbiota from Mogat2^−/− mice promotes intestinal tumor growth * • Mogat2 suppresses colorectal cancer cell proliferation by the NF-κB signaling pathway __________________________________________________________________ Molecular biology; Cancer Introduction Colorectal cancer (CRC) is one of the major malignancies and accounts for approximately 9% of all cancer cases.[48]^1^,[49]^2 Approximately 80% of patients with colorectal cancer have inactivating mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, which acts as a negative regulator of canonical Wnt signaling.[50]^3 CRC involves a complex mechanism of carcinogenesis, encompassing various risk factors for its initiation, promotion, progression, relapse, and metastasis. These factors include environmental elements such as obesity, being of an older age, male gender, lifestyle, physical inactivity, smoking, and genetic predisposition.[51]^4^,[52]^5^,[53]^6 Despite great efforts in researching CRC carcinogenesis, the detailed molecular mechanisms have not been fully elucidated. Perturbed fat metabolism has been found to be critical in many biological steps regulating tumor initiation and progression.[54]^7^,[55]^8 Increasing evidence has investigated the role of the fat metabolism in cancer.[56]^9^,[57]^10^,[58]^11^,[59]^12 Triacylglycerol (TAG) is a major source of calories, which can be hydrolyzed to monoacylglycerols (MAG) and fatty acids (FA) in the small intestinal lumen. The hydrolysis products are then resynthesized into TAG in enterocytes via two pathways: the glycerol-3-phosphate pathway and the MAG pathway, which accounts for approximately 75% of TAG resynthesis.[60]^13^,[61]^14^,[62]^15 Mogat2 is a member of the DGAT2 family, which is highly expressed in the endoplasmic reticulum of enterocytes in the small intestine. Its function is to catalyze the conversion of MAG to DAG, which is the first step in TAG production.[63]^16^,[64]^17^,[65]^18 It has been reported that Mogat2-deficient mice prevent high-fat diet-induced obesity, glucose intolerance, hypercholesterolemia, and fatty liver.[66]^13^,[67]^16^,[68]^19^,[69]^20 However, the function of Mogat2 remains poorly characterized in colorectal cancer progression, and its pathological significance needs to be determined. In this study, we observed that knockout of Mogat2 in the Apc^min/+ spontaneous intestinal adenoma mice model enhanced intestinal adenoma progression and reduced the survival time of the mice. We also discovered significant differences in intestinal microbial communities between C57BL/6J (WT) and Mogat2^−/− mice; Apc^min/+ mice and Apc^Min/+; Mogat2^−/− mice. FMT experiments also suggested that the fecal samples of Mogat2^−/− mice can accelerate intestinal tumorigenesis in Apc^min/+ mice. Furthermore, we found that overexpression of Mogat2 in CRC cells can inhibit cellular proliferation and tumor growth by NF-κB pathway in vivo. These findings identify a protective role of Mogat2 in colorectal cancer, extending our current understanding beyond its role in fat metabolism regulation. Results Knock out of monoacylglycerol acyltransferase-2 promotes intestinal tumor growth in Apc^Min/+ mice To investigate the clinical significance of Mogat2 in colorectal cancer, we initially analyzed the expression levels of Mogat2 using the Gene Expression Profiling Interactive Analysis (GEPIA) tool ([70]http://gepia.cancer-pku.cn/). Analysis of the GEPIA database indicated a significant decrease in Mogat2 expression levels in tumor specimens from patients with Colon Adenocarcinoma and Rectum Adenocarcinoma (CRC) compared to normal colon tissues ([71]Figure 1A, left). We further found that MOGAT2 expression was positively correlated with overall survival in patients with CRC ([72]Figure 1A, right). To explore the role of Mogat2 in tumorigenesis, we generated Apc^Min/+; Mogat2^−/− mice by crossing Apc^Min/+ mice with Mogat2^−/− mice ([73]Figure 1B). Our experiments revealed that Apc^Min/+; Mogat2^−/− mice exhibited significant weight loss compared to Apc^Min/+ mice ([74]Figure 1C). After excising the small intestines from 16-week-old Apc^Min/+ and Apc^Min/+; Mogat2^−/− mice, we counted the intestinal tumors with a stereoscopic microscope to detect differences in tumor growth. The Apc^Min/+; Mogat2^−/− mice had a higher mean volume, a greater number of small intestine tumors, ([75]Figures 1D and 1E) and a shorter survival time compared to the Apc^Min/+ mice ([76]Figure 1F), indicating that Mogat2 deletion promotes intestinal tumorigenesis. Furthermore, the pictures of hematoxylin-eosin staining show many tumor cells invading the basement membrane of the small intestine in Apc^Min/+; Mogat2^−/− mice, but there is no tumor cells invading the basement membrane of the small intestine in Apc^Min/+ mice ([77]Figure 1I), it showed severe malignant tumor progression in Apc^Min/+; Mogat2^−/− mice. Figure 1. [78]Figure 1 [79]Open in a new tab Deletion of Mogat2 promotes tumorigenesis in Apc^Min/+ mice (A) The mRNA level of Mogat2 in cancer tissues and non-cancerous tissues from the GEPIA database (left) and Kaplan-Meier analysis of the overall survival of patients with CRC with high or low Mogat2 expression from the GEPIA database (right). (B) Representative dissecting gross image of Apc^Min/+ and Apc^Min/+; Mogat2^−/− mice. (C) Body weight of the Apc^Min/+ (n = 8) and Apc^Min/+; Mogat2^−/− mice (n = 10) (D) Representative dissecting gross image of small intestines and colons from the Apc^Min/+ and Apc^Min/+; Mogat2^-/- mice. (E) Total number and volume of tumors of the Apc^Min/+ (n = 13) and Apc^Min/+; Moagt2^−/− mice (n = 10) in the small intestine mice calculated according to the diameter. (F) Kaplan-Meier plot analysis of the survival of APC^Min/+ (n = 31) and Apc^Min/+; Mogat2^−/− (n = 21) mice. (G) Representative blood image of Apc^Min/+ and Apc^Min/+; Mogat2^−/− mice. The number of red blood cells and the concentration of hemoglobin in Apc^Min/+ (n = 16) and Apc^Min/+; Mogat2^−/− mice (n = 22). (H) The level of LPS in the serum of Apc^Min/+ (n = 5) and Apc^Min/+; Mogat2^−/− mice (n = 5). (I) H&E staining of the intestines from the Apc^Min/+ mice and Apc^Min/+; Moagt2^−/− mice, five views were randomly selected from each section to calculate the percentage of positive cells per crypt. (J) Immunohistochemical staining for Ki67 in the intestinal tumor tissues from Apc^Min/+ and Apc^Min/+; Moagt2^−/− mice, five views were randomly selected from each section to calculate the percentage of positive cells per crypt. (K) Quantitative PCR analysis of Mogat2 mRNA level in the MC38 overexpressing cells. Mogat2 mRNA level was normalized to β-actin, Column, mean; bar, s.d. (from triplicates). (L) Immunoblotting analysis of Mogat2 protein levels in the MC38 overexpressing cells and MC38 vector cells. Alpha-tubulin was used as the loading control. (M) Image of the colony formation assay of the MC38 cells with or without Mogat2 overexpression. (N) Representative images showing primary tumors in cecum. Significance was established by unpaired t test (E, G, H, J, K) and two-way ANOVA (D). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Additionally, we examined the red blood cells and hemoglobin levels of Apc^Min/+ mice and found that Apc^Min/+; Mogat2^−/− mice had decreased levels of both red blood cells and hemoglobin ([80]Figure 1G). Intestinal barrier dysfunction can aggravate the development of colitis and CRC, we detect the Lipopolysaccharide (LPS) in the serum of mice, we also found a higher serum level of LPS in Apc^Min/+; Mogat2^−/− mice than Apc^Min/+ mice ([81]Figure 1H). This finding suggests that the Apc^Min/+; Mogat2^−/− mice display more severe anemic conditions. Nonetheless, metastasis to the liver was absent in both groups ([82]Figure S1A). By utilizing Ki-67 expression to quantify the proliferating cells within the polyps, we noted a greater proportion of proliferating cells in the polyps of Apc^Min/+; Mogat2^−/− mice (see [83]Figure 1J). It is important to highlight that no disparities were observed in the counts of white blood cells including monocytes, lymphocytes, and neutrophils, nor platelets within the blood ([84]Figure S1B). MC38 is a tumorigenic yet poorly metastatic mouse colon cancer cell line. To explore the effects of Mogat2, we generated MC38 cells with stable overexpression of Mogat2. The overexpression of Mogat2 was confirmed using real-time quantitative PCR and western blotting ([85]Figures 1K and 1L). Further, colony formation assays demonstrated that Mogat2 overexpression inhibited tumor growth ([86]Figure 1M). To investigate the progression of CRC in response to Mogat2 overexpression, we employed a orthotopic mouse model. This involved injecting either MC38 cells with a vector control or MC38 cells overexpressing Mogat2 into the cecum of syngeneic C57BL/6J mice. MC38-vector cells led to the formation of three tumors in five mice. In contrast, fewer tumor was observed in mice injected with Mogat2-overexpressing MC38 cells ([87]Figure 1N). Therefore, Mogat2 overexpression in MC38 cells significantly inhibited tumor incidence and growth. Loss of monoacylglycerol acyltransferase-2 significantly alters the composition of gut microbiota in wild type mice To better understand the effect of Mogat2 on gut microbiota, we performed 16S rRNA gene sequencing of fecal samples from WT mice and Mogat2^−/− mice. Beta-diversity analysis, utilizing principal coordinate analysis, showed a notable separation between the two groups ([88]Figure 2A). A Venn diagram identified 95 overlapping operational taxonomic units (OTUs), with 3 OTUs specific to the WT group and 5 OTUs exclusive to the Mogat2^−/− group ([89]Figure 2B). When assessing community richness through alpha-diversity using the Chao and Ace indices, no statistically significant difference was observed between the groups (p = 0.64, p = 0.557) ([90]Figure 2C). An analysis of the gut microbiota composition at the phylum level revealed that Mogat2 deficiency decreased the relative abundance of Bacteroidetes (51.8% in the WT vs. 32.6% in the Mogat2^−/−, p < 0.05), while increasing the relative abundance of Saccharibacteria (0.27% in the WT vs. 0.89% in the Mogat2^−/−, p < 0.05) and Verrucomicrobia (0.98% in the WT vs. 11.3% in the Mogat2^−/−, p < 0.05) ([91]Figure 2D). Comparative analysis at the microbial genus level revealed that Mogat2 deficiency significantly elevated the abundance of several genera, including Akkermansia, Ruminiclostridium_6, Prevotellaceae_NK3B31_group, and Clostridium_sensu_stricto_1 ([92]Figure 2E). Collectively, these findings underscore the pivotal role of Mogat2 in regulating the composition of gut microbiota in mice. Figure 2. [93]Figure 2 [94]Open in a new tab Alterations in Microbiota Dysbiosis Observed in Mogat2^−/− mice (A) Principle coordination analysis (PCoA) was performed based on OTU abundance. (B) Venn diagram analysis compared the fecal samples of WT mice and Mogat2^−/− mice. (C) Alpha-diversity analysis using the Chao and Ace indexes to compare the community richness between the two groups. (D) The taxonomic composition distribution at the phylum level of fecal microbiota was compared between the two groups. (E) The taxonomic composition distribution at the genus level of fecal microbiota was compared between the two groups (6 mice per group). ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. Loss of monoacylglycerol acyltransferase-2 alters the composition of gut microbiota in Apc^Min/+ mice To further investigate whether Mogat2 knockout affects the gut microbiota during CRC, we analyzed the microbial composition of the Apc^Min/+ mice and Apc^Min/+; Mogat2^−/− mice. The PCoA analysis demonstrated a significant separation between the two groups ([95]Figure 3A). Venn diagram analysis revealed that 96 OTUs were shared between the groups, while 6 OTUs were present only in the Apc^Min/+ mice group, and 14 OTUs were present specific in the Apc^Min/+; Mogat2^−/− group ([96]Figure 3B). The quantification of microbial diversity via the Chao and Ace diversity indexes showed significant differences between the two groups (p=0.0065 and p=0.0016), respectively ([97]Figure 3C). Figure 3. [98]Figure 3 [99]Open in a new tab Microbiota dysbiosis alterations in Apc^Min/+ mice with Mogat2 deletion (A) Principle coordination analysis (PCoA) based on OTU abundance. (B) Venn diagram analysis of fecal samples from Apc^Min/+ and Apc^Min/+; Mogat2^−/− mice. (C) Alpha-diversity comparison between two groups using Chao and Ace indexes. (D) The taxonomic composition distribution among the two groups on the phylum-level of the fecal microbiota. (E) The taxonomic composition distribution among the two groups at the genus-level of the fecal microbiota. (6 mice per group). ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. Compared to Apc^Min/+ mice, the Apc^Min/+; Mogat2^−/− mice exhibited a significant alteration in their microbial composition. Specifically, the Apc^Min/+; Mogat2^−/− mice showed a significant increase in the abundance of Verrucomicrobia (0.03% vs. 3.24%, p < 0.01) and Actinobacterio (0.04% vs. 0.15%, p < 0.05) phyla compared to Apc^Min/+ mice, while the proportion of the Deferribacteres phylum was markedly decreased (0.68% vs. 0.04%, p < 0.05) in the Apc^Min/+; Mogat2^−/− mice ([100]Figure 3D). At the genus level, Apc^Min/+; Mogat2^−/− mice exhibited a significant increase in Akkermansia (3.25%) compared to Apc^Min/+ mice (0.03%) (p < 0.05). Additionally, other genera, such as Bacteroides, Prevotellaeae_NK3B31_group, and Clostridium_sensu_stricto_1, also increased in abundance in the Apc^Min/+; Mogat2^−/− mice ([101]Figure 3E). Gut microbiota from monoacylglycerol acyltransferase-2 knockout -mice aggravates the progression of intestinal adenoma To evaluate the impact of gut microbiota derived from Mogat2^−/− mice on intestinal adenoma in 16-week-old Apc^Min/+ mice, pseudo-germ-free (PGF) mice and FMT was conducted ([102]Figures 4A and 4B). A higher total adenoma count was observed in Apc^Min/+ mice that received fecal samples from Mogat2^−/− mice, compared to those that received fecal samples from WT mice, indicating that the gut microbiota from Mogat2^−/− mice promoted the progression of intestinal adenoma in Apc^Min/+ mice ([103]Figure 4C). Furthermore, the rate of Ki-67-positive cells in the FMT-Mogat2 group was significantly higher than in the FMT-WT group ([104]Figure 4D). Moreover, the goblet cells play an important role in maintaining intestinal health during steady-state, we found the average number of goblet cells in each crypt was significantly lower in the FMT-Mogat2 group than in the FMT-WT group ([105]Figure 4E). These results suggest that gut microbiota from Mogat2^−/− mice could promote the proliferation of intestinal tumor cells. Figure 4. [106]Figure 4 [107]Open in a new tab Gut microbiota from Mogat2^−/− mice enhances the progression of intestinal adenoma in Apc^Min/+ mice (A) An experimental design indicating the establishment process. (B) Pseudo-germ-free mice developed caecal enlargement. (C) The total number and volume of tumors in each group were calculated (n = 8). (D) Immunohistochemical staining for Ki67 in the intestinal tumor tissues from FMT-WT mice and FMT-mogat2 mice was performed. (E) The number of colon goblet cells was evaluated by AB-PAS staining. Significance was established by unpaired t test. (C, D, E). ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗p < 0.05. Overexpression of monoacylglycerol acyltransferase-2 inhibits colorectal cancer cell growth in vitro and in vivo The Mogat2 protein expression levels were examined in seven CRC cell lines. Western blot assays showed that Mogat2 had low expression in SW620 and HT29 cells ([108]Figure 5A). To further investigate the role of Mogat2 in CRC development, Mogat2 was stably overexpressed in the human CRC cell lines HT29 and SW620, and the overexpression of Mogat2 was confirmed by real-time quantitative PCR and western blotting ([109]Figures 5B and 5C). Overexpression of Mogat2 reduced the proliferation markers FoxM1 and MYC, as confirmed by Q-PCR ([110]Figure 5D), and inhibited cell proliferation and colony formation, as evidenced by CCK8 and colony formation assays ([111]Figures 5E and 5F). To validate the function of Mogat2 in vivo, 4×10^6 SW620 cells and Mogat2-overexpressing SW620 cells were subcutaneously injected into the dorsal flank of nude mice, and the tumor sizes were measured 17 days later ([112]Figure 5G). The results showed that Mogat2-overexpressing cells significantly decreased tumor volume and weight, suggesting that Mogat2 acts as a tumor suppressor in CRC. To further reveal the molecular mechanism by which Mogat2 inhibits the progression of CRC, RNA-seq analysis was performed on SW620 cells transfected with control and overexpression for Mogat2. The results showed that 70 genes were up-regulated and 131 genes were down-regulated in Mogat2-overexpressing SW620 cells compared with SW620 vector cells ([113]Figure 5H). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that Mogat2 regulates the NF-κB pathway ([114]Figure 5I). We then detected the protein level of P65, p-P65, P50, IKBα, and p-IKBα in vector and overexpression cell lines. The P65, p-P65, P50, and p-IKBα protein levels were decreased and IKBα was increased in Mogat2 overexpressing cells ([115]Figure 5J), these results revealed that Mogat2 could inhibit the NF-κB signaling activation. Figure 5. [116]Figure 5 [117]Open in a new tab Over-expression of Mogat2 inhibits the proliferation of CRC cells (A) The protein level of Mogat2 in CRC cells was measured. (B and C) Quantitative PCR performed to detect Mogat2 mRNA expression in HT29 and SW620 cells. Mogat2 mRNA level was normalized to β-actin, Column, mean; bar, s.d. (from triplicates). (D) The mRNA level of the proliferation genes FoxM1 and Myc were measured in SW620 cells upon Mogat2 overexpression, FoxM1 and Myc mRNA level was normalized to β-actin, Column, mean; bar, s.d. (from triplicates). (E) The result of the CCK8 proliferation assay, Student’s t test was used to perform the statistical analyses. (F) The colony formation assay conducted in HT29 and SW620 cells upon Mogat2 overexpression, Student’s t test was used to perform the statistical analyses. (G) The xenograft tumor growth of SW620 cells was measured upon Mogat2 overexpression. ∗, p < 0.05, ∗∗, p < 0.01, and ∗∗∗∗, p < 0.0001. (H) Differentially expressed genes in Mogat2-overexpressing SW620 cells compared to Vector SW620 cells from the RNA-Sequencing (RNA-Seq) analysis. (I) KEGG pathway enrichment analysis in CRC cells with Mogat2 overexpression. (J) NF-κB signaling makers in Mogat2 overexpressing SW620 cells and SW620 Vector cells were evaluated by immunoblotting. Discussion The Apc^Min/+ mouse model carries a mutation at codon 850 of the human Apc gene, resulting in the constitutive activation of Wnt signaling in the intestine.[118]^21 Due to the mutation in multiple intestinal neoplasia, Apc^Min/+ mice spontaneously develop multiple adenomatous polyps in the small intestine and fewer polyps in the colon.[119]^22^,[120]^23 Therefore, Apc^Min/+ mice remain a classic mouse model for studying human intestinal tumorigenesis.[121]^24^,[122]^25^,[123]^26^,[124]^27 The Apc^Min/+; Mogat2^−/− mice model was established to investigate the effect of Mogat2 on spontaneous intestinal adenocarcinoma in mice with an APC mutation. Our study revealed that the number and volume of intestinal tumors significantly increased after knocking out Mogat2, along with an accelerated pathological progression of the tumors in Apc^Min/+; Mogat2^−/− mice. To sum up, our studies highlight the inhibiting roles of Mogat2 in CRC. The gut microbiome is recognized as a mediating factor that contributes to the connection between environmental factors and the development of colorectal cancer.[125]^28^,[126]^29^,[127]^30^,[128]^31 In recent decades, increasing evidence has shown that the gut microbiota plays a pivotal role in the carcinogenic process.[129]^32 Microbiota has been implicated in around 20% of cancers, particularly CRC.[130]^33^,[131]^34^,[132]^35 The human gut microbiota is predominantly categorized into three major phyla: Firmicutes (30%–50%), Bacteroidetes (20%–40%), and Actinobacteria (1%–10%). Notably, alterations at the phylum level, including an increase in Bacteroidetes and a decrease in Firmicutes and Proteobacteria, have been reported in relation to colorectal cancer incidence.[133]^33 In our study, we observed that the loss of Mogat2 did not result in alterations in the abundance of Bacteroidetes, Firmicutes, and Proteobacteria in Apc^Min/+ mice. However, the loss of Mogat2 led to an increase in Verrucomicrobia at the phylum level and a heightened presence of Akkermansia muciniphila at the genus level. Akkermansia muciniphila, a gram-negative anaerobic bacterium, is one of the most prevalent species within the human gut microbiota, constituting1 to 5% of the microbial population in the human intestine and is the only known member of the Verrucomicrobia phylum found in the human intestinal tract.[134]^36^,[135]^37 Akkermansia muciniphila has recently been identified as a promising probiotic candidate.[136]^37^,[137]^38 Importantly, it is worth noting that the abundance of Akkermansia muciniphila is significantly lower in individuals with metabolic disorders including diabetes and obesity.[138]^38 However, there are studies that have demonstrated a significant decrease in the mRNA and protein expression levels of tight junction proteins in the over-colonized Akkermansia muciniphila mouse model.[139]^39^,[140]^40 Qin’s group revealed that the excessive colonization of Akkermansia muciniphila disrupts the delicate equilibrium between mucin secretion and degradation. This disruption leads to a thinning of the intestinal mucous layer and compromises the integrity of the intestinal barrier. As a consequence, this situation can aggravate the development of colitis and CRC.[141]^40 Previous investigations into Akkermansia muciniphila have established its status as a beneficial bacterium that plays a crucial role in maintaining intestinal homeostasis.[142]^41 Treatment with Akkermansia muciniphila has been shown to reverse metabolic disorders induced by a high-fat diet, effectively countering issues such as increased fat mass, metabolic endotoxemia, adipose tissue inflammation, and insulin resistance.[143]^41 However, excessive colonization of Akkermansia muciniphila disrupts the balance between mucin secretion and degradation, leading to damage to the intestinal barrier. Ultimately, this disruption exacerbates the development of colitis and colorectal cancer.[144]^40 In the present study, the deletion of Mogat2 was also found to increase the abundance of Clostridium_sensu_stricto_1. Clostridium_sensu_stricto_1 has been reported to exhibit a significant association with colon inflammation and the integrity of the intestinal barrier. This association is indicated by a positive correlation between the mRNA expression of colonic TLR4, TLR5, and NF-κB.[145]^39 To further investigate the role of the gut microbiota in adenoma progression, fecal microbiota transplantation was performed.[146]^42 This transplantation indicated that the gut microbiota from Mogat2^−/− mice could promote the development of intestinal adenomas. NF-κB has been widely implicated in the development and progression of cancer.[147]^43 NF-κB acts as a collective term for a family NF-κB of five related Rel proteins: RelA/p65, c-Rel, RelB, NF-κB1 (also known as p50, p105 or p50/p105) and NF-κB 2 (also known as p52, p100 or p52/100). In colorectal cancer, NF-κB plays a critical role in cell proliferation, apoptosis, angiogenesis, and metastasis.[148]^44^,[149]^45 In order to investigate the role of Mogat2 in CRC progression in vivo, we constructed cell lines with stable overexpression of Mogat2 in HT29 and SW620 cells. Our experiments revealed that Mogat2 overexpression inhibited cellular proliferation and colony formation. The interaction of Mogat2 and NF-κB pathway was not reported in the previous study. In the present study, we found that P65, p-P65, P50, and p-IKBα, key factors in the NF-κB pathway, were decreased when overexpression of Mogat2. The findings of this study highlight that the overexpression of Mogat2 could inhibit the NF-κB pathway in CRC cells. Previous studies have considered Mogat2 as an intervention target to address obesity and related metabolic diseases.[150]^20^,[151]^46^,[152]^47 However, our findings reveal that Mogat2 assumes a tumor-suppressive role in the formation of intestinal tumors: directly inhibit CRC cells growth via NF-κB pathway, as well keep the gut microbiota homeostasis. Loss of Mogat2 not only accelerates intestinal tumor growth but also expedites the pathological processes involved. The disrupted gut microbiota observed in Mogat2^−/− mice further enhances the advancement of intestinal adenomas in Apc^min/+ mice. These results offer new perspectives to understand cancer development and novel strategies for potential target therapy for CRC. Ethics approval and consent to participate The animal experiments were approved by the institutional ethics committee of Sun Yat-sen University, Guangzhou, China (Approval No: L102012016008E). Availability of data and materials Some of the clinical survival data can be found in the GEPIA database ([153]http://gepia.cancer-pku.cn/). All the other data can be found in the article or the supplementary data. Consent for publication Not applicable. Limitations of the study Our data demonstrate that Mogat2 plays a tumor-suppressive role in the formation of intestinal tumors and that gut microbiota from Mogat2^−/− mice aggravates the progression of intestinal adenoma. However, the study has several limitations. First, it is necessary to identify the specific strains and metabolites that facilitate adenoma progression. Second, while overexpression of Mogat2 in CRC cells appears to inhibit the NF-κB pathway, further research is required to understand how Mogat2 interacts with NF-κB signaling. Third, the detailed molecular mechanisms explaining how monoacylglycerol acyltransferase-2 suppresses colorectal cancer carcinogenesis in APC^min+/− mice through the modulation of gut microbiota are yet to be elucidated. STAR★Methods Key resources table REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies __________________________________________________________________ Rabbit monoclonal anti-Ki67 Cell Signaling Technology Cat# 12202; RRID: [154]AB_2620142 Rabbit polyclonal anti-MGAT2 Abcam Cat# ab228950; RRID: [155]AB_3105860 Rabbit polyclonal anti- Alpha Tubulin Proteintech Cat# 11224-1-AP; RRID: [156]AB_2210206 Rabbit monoclonal anti-P65 Cell Signaling Technology Cat# 8242; RRID: [157]AB_10859369 Rabbit monoclonal anti-Phospho-P65 Cell Signaling Technology Cat#3033; RRID: [158]AB_331284 Rabbit polyclonal anti- P105/P50 Cell Signaling Technology Cat#3035; RRID: [159]AB_330564 Rabbit monoclonal anti- IKBα Cell Signaling Technology Cat#4812; RRID: [160]AB_10694416 Rabbit monoclonal anti- Phospho- IKBα Cell Signaling Technology Cat#2859; RRID: [161]AB_561111 __________________________________________________________________ Chemicals, peptides, and recombinant proteins __________________________________________________________________ RPMI-1640 medium Gibco Cat#C11875500BT Protease Inhibitor Cocktail Sigma Cat#ZH-P8340 Puromycin Invitrogen Cat#A1113802 Ampicillin Macklin Cat#A830931 Neomycin Macklin Cat#N799581 Metronidazole Macklin Cat#M813526 Vancomycin Macklin Cat#V871983 __________________________________________________________________ Critical commercial assays __________________________________________________________________ ELISA kit Cloud-clone Cat#MEB526Ge BCA Protein Assay Kit Thermo Scientific Cat#23227 Lenti-Pac HIV Kit GeneCopoeia Cat#LT002 Cell Counting Kit-8 Abmole Cat#M4839 __________________________________________________________________ Experimental models: Organisms/strains __________________________________________________________________ Mogat2 homozygous knockout mice the Jackson Laboratory Cat#011085; RRID: IMSR_JAX:011085 The Apc^min/+ mice GemPharmatech Cat#T001457; RRID: IMSR_GPT:T001457 __________________________________________________________________ Software and algorithms __________________________________________________________________ Adobe Illustrator Adobe [162]https://www.adobe.com/ GraphPad Prism 8.4.2 GraphPad [163]https://www.graphpad.com/ ImageJ 1.53 ImageJ [164]https://fr.vessoft.com/software/windows/download/imagej [165]Open in a new tab Resource availability Lead contact Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Dr. Jiliang Qiu (qiujl@sysucc.org.cn). Materials availability This study did not generate new unique reagents and all materials in this study are commercially available. Data and code availability * • All data reported in this paper will be shared by the [166]lead contact upon request. * • This paper does not report original code. * • Any additional information required to reanalyze the data reported in this paper is available from the [167]lead contact upon request. Experimental model and study participant details Animals Mogat2 homozygous knockout mice were obtained from the Jackson Laboratory in Bar Harbor, USA, while the Apc^min/+ mice were obtained from GemPharmatech in Nanjing, China. All of the mice were in a C57BL/6J background. To generate Apc^min/+;Mogat2^+/- mice, male Apc^min/+ mice were bred with female Mogat2^-/- mice. Subsequently, the male Apc^min/+; Mogat2^+/- mice were backcrossed with female Mogat2^-/- mice to obtain Apc^Min/+; Mogat2^-/- mice.[168]^25^,[169]^48 The mice were housed under specific pathogen-free conditions at the Animal Center of Guangdong Pharmaceutical University and all animal experiments were conducted with the approval of the Ethics Committee for Laboratory Animals of Sun Yat-sen University Cancer Center. Method details Analysis of intestinal tumors Upon euthanizing and dissecting the mice, the colon and small intestines were carefully removed and opened longitudinally. Subsequently, the luminal contents were meticulously flushed with phosphate-buffered saline (PBS) solution and the small intestines divided into three equal segments: the proximal, middle and distal. The numbers and diameters of the tumors in the small intestines were measured using an inverted microscope. The small intestine tumor volumes were calculated using the formula: volume=4/3πab^2 (where ‘a’ represents half of the long diameter and ‘b’ represents half of the short diameter).[170]^25^,[171]^49 The intestines were rolled with the mucosa facing outwards, fixed in 10% buffered formalin, processed and embedded in paraffin. Orthotopic-transplantation model of mouse colon-cancer cell line MC38 The mouse colon-cancer cell line MC38 was grown in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum. Twelve 6-week-old male C57BL/6J mice were anesthetized with 5% isoflurane and then placed on a sterile heating pad. The abdomen, from which all hair was removed the previous day using depilatory cream, was prepared for sterile surgery by cleansing with ethanol and then betadine three times. A small midline abdominal incision was made using a scalpel to cut through the skin and scissors to carefully cut through the underlying musculature without damaging any organs. The cecum was exteriorized and placed on a piece of sterile gauze, then hydrated with sterile saline. Under microscopic guidance, a 100 μl suspension of 1 × 10^6 MC38 cells and Mogat2 overexpressed cells in PBS were carefully injected into the cecal wall. Afterwards, the cecum was then repositioned inside the peritoneum, and the muscle and skin incisions were closed with sutures.[172]^50 Hematoxylin and Eosin (H&E) and Immunohistochemistry (IHC) Paraffin-embedded sections of intestines (4 μm in thickness) were stained with H&E and Alcian Blue Periodic Acid Schiff (AB-PAS) staining, following standard protocol. For immunohistochemistry, the intestinal tissue slices were incubated with their corresponding primary antibodies. Specifically, anti-Ki67 (Cell Signaling Technology) was used at 1:200 dilution overnight at 4°C. The following day, the slices were treated with the secondary antibody for 30 minutes at 37°C, followed by DAB reaction and hematoxylin staining. Enzyme-Linked Immunosorbent Assay (ELISA) The serum level of Lipopolysaccharide (LPS) in the mice was measured using commercial ELISA kits (Cloud-clone) according to the manufacturer’s instructions. Immunoblotting The cells were washed with cold PBS and lysed using a lysis buffer (Millipore) that was supplemented with a Protease Inhibitor Cocktail (Sigma) at 4°C for 20 minutes. The resulting lysates were centrifuged at 12000 rpm/min for 15 minutes at 4°C and the supernatants collected. Protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific). Afterwards, the protein was loaded onto an SDS-PAGE gel after boiling with a loading buffer at 99°C for 10 minutes, and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with TBST containing 5% non-fat milk for 1 hour at room temperature, followed by overnight incubation with an MOGAT2 antibody (Abcam) at 4°C. Finally, the membrane was incubated with a secondary antibody for 1 hour at room temperature. Stable overexpression of Mogat2 in HT29 and SW620 cells Cell lines stably expressing either Mogat2 or the vector were constructed using pLVX-3X-Flag-Puro plasmids from the GeneCopoeia company. Lentiviruses for overexpression were produced in 293FT cells according to the procedure outlined in the Lenti-Pac HIV Kit (GeneCopoeia). Infectious lentiviruses were harvested 48 hours after transfection and filtered through a 0.45 mm filter (Millipore). Cells transduced with lentivirus were then cultured in 2 μg/mL of puromycin (Invitrogen) for two weeks to facilitate selection. The transfection efficiency was validated using Western Blot and qPCR, confirming the successful overexpression of Mogat2. Cell culture and reagents Colorectal carcinoma cell lines LOVO, RKO, DLD1, HT29, HCT-116, SW620, and SW480 were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum at 37°C in a humidified chamber containing 5% CO2. Cell proliferation assay and colony formation assay Cell proliferation was assessed using the Cell Counting Kit-8 (Abmole) according to the manufacturer’s protocol. Specifically, 1 × 10^3 cells were seeded in 96-well plates, with each well containing 0.1 mL of medium. At designated time points (24, 48, 72, 96 hours), each well was incubated with 10 μL CCK8 solution for 2 hours at 37°C. Subsequently, an absorbance reading at 450 nm was taken using the Gen5 microplate reader (BioTek). For the colony formation assay, a total of 2 × 10^3 cells were grown in each well of a 6-well plate and cultured for approximately 2 weeks. Afterwards, the cells were fixed with methanol for 30 minutes and subsequently stained with 2% crystal violet for 30 minutes. The number of colonies was then determined. These experiments were independently repeated three times. DNA extraction and polymerase chain reaction (PCR) amplification Fresh fecal samples were promptly collected and kept on ice before being stored at -80°C for subsequent use. The extracted DNA underwent evaluation on 1% agarose gel, and its concentration and purity were assessed using a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific). The hypervariable region V3-V4 of the bacterial 16S rRNA gene was amplified with primer pairs 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The amplification process for the 16S rRNA gene involved initial denaturation at 95°C for 3 minutes, followed by 27 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 45 seconds, with a final single extension step at 72°C for 10 minutes. The PCR procedure was carried out in triplicate. The PCR product was subsequently extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences) according to manufacturer’s instructions. The Quantus™ Fluorometer (Promega) was then used to quantify the resulting purified DNA. Establishment of pseudo-germ-free (PGF) mouse model and FMT Apc^min/+ mice at 6 weeks old were randomly divided into two groups. To induce a pseudo-germ-free state, their drinking water was supplemented with an antibiotic cocktail (1 g/L ampicillin, 1 g/L neomycin, and 1 g/L metronidazole, and 0.5 g/L vancomycin). All the antibiotics used were procured from Macklin in Shanghai, China. Fresh fecal samples collected from WT mice (n=8) and Mogat2^-/- mice (n=8) were then mixed at equal weights, respectively. The mixed feces were diluted in sterile PBS at a ratio of 1:10 (w/v), vigorously shaken for 3 minutes, and allowed to settle by gravity for 2 minutes. The mixture was then subjected to initial filtration, centrifugation, and homogenization. The resulting microbial suspensions were combined with 30% glycerol and stored at -80°C until used for gavage into Apc^Min/+ mice. For each gavage, 200 μL of the microbial suspension was administered to each mouse, and repeated twice a week over a 4-week period. Quantification and statistical analysis The results were analyzed using GraphPad Prism 8 and shown as mean ± SD from at least three independent experiments. The Kaplan-Meier method was used to calculate the survival curves, and the log-rank test was used to determine the statistical significance. Differences between groups were analyzed using two-tailed Student’s t-tests and two-way analysis of variance (ANOVA). Data were considered statistically significant when P < 0.05. Acknowledgments