Boris knockout eliminates AOM/DSS‐induced in situ colorectal cancer by suppressing DNA damage repair and inflammation

Abstract The Brother of Regulator of Imprinted Sites (BORIS, gene symbol CTCFL) has previously been shown to promote colorectal cancer cell proliferation, inhibit cancer cell apoptosis, and resist chemotherapy. However, it is unknown whether Boris plays a role in the progression of in situ colorectal cancer. Here Boris knockout (KO) mice were constructed. The function loss of the cloned Boris mutation that was retained in KO mice was verified by testing its activities in colorectal cell lines compared with the Boris wild‐type gene. Boris knockout reduced the incidence and severity of azoxymethane/dextran sulfate‐sodium (AOM/DSS)‐induced colon cancer. The importance of Boris is emphasized in the progression of in situ colorectal cancer. Boris knockout significantly promoted the phosphorylation of γH2AX and the DNA damage in colorectal cancer tissues and suppressed Wnt and MAPK pathways that are responsible for the callback of DNA damage repair. This indicates the strong inhibition of colorectal cancer in Boris KO mice. By considering that the DSS‐promoted inflammation contributes to tumorigenesis, Boris KO mice were also studied in DSS‐induced colitis. Our data showed that Boris knockout alleviated DSS‐induced colitis and that Boris knockdown inhibited the NF‐κB signaling pathway in RAW264.7 cells. Therefore Boris knockout eliminates colorectal cancer generation by inhibiting DNA damage repair in cancer cells and relieving inflammation in macrophages. Our findings demonstrate the importance of Boris in the development of in situ colorectal cancer and provide evidence for the feasibility of colorectal cancer therapy on Boris.

because of the nonspecific treatment. In total, 80% of CRC patients have no prior family history, although mutations in the MLHs, APC, and PMS2 genes are known in the development of CRC. Targeted therapy on cancer-specific targets would be an ideal strategy. 7,8 BORIS (gene symbol CTCFL) expresses specifically in cancer cells but not in normal cells. BORIS expression specificity in cancer cells is advantageous for therapy. 9 It has been reported that BORIS is a potential immunotherapeutic target for the treatment of cervical carcinoma and lung cancer. 10 BORIS promotes colorectal cancer cell proliferation, while inhibiting apoptosis. It will be interesting to investigate if BORIS gene knockout completely eliminates colorectal cancer.
BORIS protein includes three parts: the N terminal section, the middle zinc finger domains, and the C terminal section. 11 The 11 middle zinc finger domains are conserved with its paralog CTCF (CCCTC-binding factor), which is essential for normal cells. The N terminal section is predicted to be the functional part for oncogenesis. 12 Truncation of BORIS, which lacks zinc finger domains, directed BORIS to the cytoplasm and still inhibited colorectal cancer apoptosis.
Further deletion of the BORIS N section might destroy this function.
Boris knockout (KO) mice were constructed and used in this study to investigate AOM/DSS-induced colorectal cancer. The loss of amino acids (aa) after 137 aa of the Boris protein damaged the Boris function in promoting cancer cell proliferation and chemotherapy resistance.
Boris knockout seriously eliminated in situ colorectal cancer generation under the induction of AOM and DSS. In total, 90% of the animals with Boris knockout had no polyp, and the other Boris knockout individuals had obviously smaller polyps compared with wild-type animals. The Boris knockout also suppressed inflammation caused by DSS treatment. [13][14][15] High-throughput sequencing of KO mice colon tissues compared with wild mice revealed that the Boris knockout inhibited the MAPK pathway. In addition, Boris knockout promoted apoptosis of colon tissue and relieved DSS-induced colitis. Our study demonstrated the importance of Boris in colorectal cancer development and provided evidence for the feasibility of targeted therapy on Boris.

| Cellproliferationandapoptosisassays
The cell viability, colony formation ability, and apoptosis assays were applied to detect the difference among the transfected Caco-2 cells. In total, 1000 cells plated into each well of 96-well plates were subjected to plasmid transfection followed by the treatment of 5-FU or cisplatin for 24 h. The cell viability was measured by MTT (Solarbio, 298-93-1) according to the instructions. In total, 1000 cells plated into each well of 12-well plates were transfected and cultured for 7 days. Then cells were stained with crystal violet to examine colony formation. Caspase-Glo® 3/7 lysis substrate (Promega, G8090) was used to measure the apoptosis extent.

| RNAisolationandquantitativereal-timePCR (qPCR)
Total RNAs were isolated by TRIzol reagent (Life Technologies) according to the manual. cDNAs were reverse transcribed using the Hifair® III First Strand cDNA Synthesis Kit (gDNA digester plus) (Yeasen, 11121ES60) and applied for PCR/qPCR analysis. qRT-PCR was performed using the 2× T5 Fast qPCR Mix (SYBR Green, Yeasen, 11201ES08) and a CFX connect real-time PCR detection system (Bio-Rad). GAPDH was used as an internal reference for normalization. The primers used in this study are listed in Table SI.

| Antibodyandgeneralreagents
Catalogs and working dilutions of the antibodies were as follows:

| Immunoblotassays
Lysis of intestine tissues and cells was prepared using lysis buffer containing PMSF and RIPA buffer (EMD Millipore Corp, 20-188) and then immediately subjected to western blot assay. The procedure is as follows: proteins were fractionated using SDS-PAGE, transferred to PVDF membranes, blocked in TBS containing 5% (wt/vol.) nonfat milk for 1 h rocking at room temperature, and then incubated overnight at 4°C with the indicated primary antibody. After washing with survival mouse number, n = 16) and are presented as means ± SEM. The significant difference between the groups was determined using an unpaired two-tailed Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001.
TBST three times and 10 min each time, a secondary antibody was added and the sample incubated for 2 h at room temperature. Signals were detected after washing with TBST three times for 10 min each time; signals were presented using the Ultrasensitive ECL Kit and BIO-RAD ChemiDoc XRS+ system.

| Statisticalanalysis
The software GraphPad Prism 8 (GraphPad Software) was used for all statistical analyses. Two-tailed Student's t-test or ANOVA-Tukey test was used whenever appropriate. The variance was assessed by  calculating the SEM in each group; p-values less than 0.05 were considered statistically significant.

| ConstructionofBoris knockout mice and verification of functional loss of Boris
Boris KO mice were constructed using the CRISPR/Cas gene deletion systems to delete the genomic DNA between exons 2 and 3 of the Boris gene. Boris genotypes were identified using PCR and sequencing, as shown in Figure S1A. The remolding of Boris KO resulted in amino acid loss after 137 aa ( Figure 1A). Because the deletion did not remove all of the Boris amino acids from the genome, it is unknown whether the remaining 137 aa of Boris still retained functions. The Boris mutant was cloned from homozygous Boris KO mice to test its function in colorectal cancer cells (Figures 1 and   S1B). 16 Our previous studies demonstrated that BORIS knockdown significantly inhibited proliferation and promoted the apoptosis of colorectal cancer cells. Therefore colony formation, cell proliferation, and apoptosis assays were performed to verify the function of the Boris mutant in Caco-2 cells compared with the mouse Boris wildtype gene and human BORIS. The results showed that the Boris mutant did not enhance cell proliferation in the MTT assay ( Figure 1C).
Consistently, the Boris mutant from the KO mice did not promote colony formation ( Figure 1D). Furthermore, the Boris mutant's effect on 5-FU or cisplatin resistance was confirmed. The Boris mutant lost its function to inhibit apoptosis in caspase 3/7 assays and colorectal cancer proliferation in cell viability assays. (Figure 1E-H).

| Boris knockout eliminates AOM/DSS-induced in situ colorectal cancer
To investigate the function of Boris on in situ colorectal cancer generation, 17,18 the AOM/DSS-induced mouse colorectal cancer model was established (Figure 2A). The positive CRC model group had significant weight loss during the first, second, and third cycles of DSS treatment compared with the negative control group. In contrast, the Boris KO group showed a significant remission for weight loss ( Figure 2B). Then, to determine the incidence of colorectal cancer, we calculated the number and size distribution of tumors. The colon length of the Boris KO and Boris WT groups was significantly shorter than that of the control group, as shown in Figure  The AOM/DSS-WT group had more tumors (larger than 2 mm) than the AOM/DSS-Boris KO group ( Figure 2F). Figure 2G

| Wntsignalpathwayisdownregulatedby Boris knockout
The Cancer Genome Atlas (TCGA) database showed that BORIS expression increased significantly in human colon adenocarcinoma (COAD) tissues in the clinic ( Figure 3A). 8 This was consistent with our findings that Boris was significantly increased in colorectal cancer tissues in mice treated with AOM/DSS ( Figure 3B,C). 19 It has been reported that most CRC occurrence is related to the activation of the Wnt signaling pathway. Therefore, we measured the level of β-catenin in the cytoplasm and nucleus of colon tissues ( Figure 3D).
Compared with the WT control, 732 differential genes were F I G U R E 5 Boris regulates DNA damage. (A) Western blot was used to determine DNA damage in CRC tissues induced by AOM/DSS. The transcript expression levels of MYC (B), BRCA1 (C), and POLH (D) of the colon tissues were detected using qRT-PCR. Heatmap (E) revealed a transcript expression profile of 12 genes associated with DNA damage or apoptosis. (F) TUNEL assay shows the CRC tissue apoptosis extent among the negative control without treatment, WT with AOM/DSS treatment, and Boris KO with AOM/DSS treatment. (G) P-γH2AX and Boris expression levels were detected in NIH3T3 cells, which were treated with 5-FU or cisplatin after transfection or Boris knockdown. Data are shown as means ± SEM. The significant difference was determined using an unpaired two-tailed Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001. detected, including 448 upregulated genes and 284 downregulated genes (fold change>2; Figure 4B). The MAPK signaling pathway and the apoptosis-related pathway were enriched in the KEGG pathway analysis of the differential genes ( Figure 4C). In Gene Ontology (GO) analysis, immune-related complexes, such as the MHC class II protein complex and interleukin-6 receptor complex, were enriched in cellular component analysis ( Figure 4D). As expected, the MAPK pathway genes, including Fgfr1, Mapk8ip3, Cacnb3, and so forth, F I G U R E 6 Boris knockout relieves colitis. The procedure of the DSS-induced mouse colitis model. There was a difference in body weight (A), DAI (B), colon length (C), histology score (D, E), and spleen weight (F) between Boris KO and WT mice. (G) Boris is highly expressed in the colon of the mouse with colitis; IL6 (H) and TNFα (I) were suppressed by Boris knockout. Data are expressed as means ± SEM. The significant difference was determined using an unpaired two-tailed Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001.
were regulated by Boris ( Figure 4E). Furthermore, western blot verification revealed that JNK and P38 phosphorylation in Boris KO colon tissues was significantly lower than in WT colon tissues ( Figure 4F). In conclusion, Boris was found to regulate CRC through the MAPK signaling pathway. 20,21 Because the MAPK pathway reflects the occurrence of both DNA damage and inflammation, 22 we propose that Boris affects CRC by regulating both DNA damage and inflammation.

| BorisregulatesDNAdamage
We proposed that Boris knockout leads to DNA damage repair, thereby relieving colorectal cancer development. Phospho-γH2AX levels, which reflect DNA damage, were significantly higher in the Boris KO tissue than in the other two groups. However, there was less phospho-γH2AX in the WT tissue than in the negative control ( Figure 5A). Our results demonstrated that Boris enhances DNA F I G U R E 7 Boris promotes inflammation in macrophages. After overexpression using Boris plasmids (A) and Boris knockdown (B), the transcripts of Il6, Tnfα, and Boris were detected by qPCR in RAW264.7 cells. (C) The phosphorylation of P65 and IκBα and the total amounts of P65 and IκBα were detected by western blotting of RAW264.7 cells when the expression of Boris was modulated by transfection of overexpression plasmid or siRNA. The NF-κB pathway was induced by Boris overexpression or by LPS treatment. Data are shown as means ± SEM. The significant difference was determined using an unpaired two-tailed Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001.
repair under the treatment of AOM and DSS. 23,24 Therefore, 12 genes responsible for DNA damage repair were examined. Three genes, MYC, BRCA1, and POLH, were significantly downregulated after Boris knockout compared with the WT control ( Figure 5B-E).
This suggests that DNA damage repair was affected by the Boris knockdown. The TUNEL assay for detecting DNA damage levels revealed that the extent of DNA damage in Boris KO colon tissues was significantly greater than in the other two groups ( Figure 5F). 16,23,25 Based on our findings from previous studies that BORIS promotes DNA damage repair in epithelial cells H1299, HCT116 and Caco-2, 26 here we used fibroblasts that usually exist in the tumor microenvironment and are responsible for tissue repair to confirm the effect of Boris on DNA damage. BORIS was transfected into NIH3T3 cells that were treated with 5-FU or cisplatin to induce DNA damage. Consistently Boris overexpression suppressed phospho-γH2AX compared with the negative control ( Figure 5G). In summary, we demonstrated that Boris enhanced DNA damage repair and the loss of Boris led to cancer cell apoptosis.

| Boris knockout relieves colitis
Because Boris has been shown to regulate inflammation in AOM/ DSS-induced CRC in pathway analysis (Figure 4) Boris KO significantly impacted colon length ( Figure 6C). When the colitis tissue sections were examined by H&E staining, we found that crypt structure destruction or disappearance, goblet cell loss, and inflammatory infiltration of varying degrees occurred in WT groups, but a milder pathological degree was present in KO groups.
In addition, the spleen weights differed significantly between the WT and Boris KO groups ( Figure 6F). The expression of Boris in colitis was increased dramatically by the induction of DSS in WT mice, and the inflammatory factors of Il6 and Tnfα decreased in colitis in the Boris KO group ( Figure 6H,I). These findings showed that Boris expression was induced in colitis and that Boris knockout alleviated colitis and inflammation in animals.

| Borispromotesinflammationinmacrophages
Boris locates in the nucleus and cytoplasm of cancer cells to promote tumorigenesis. As DSS-induced colitis was reported to be induced by the activation of the NF-κB signaling pathway, we used LPS to activate the cells and the NF-κB pathway in macrophages and to verify the relationship between Boris and NF-κB activation. 27 Boris promoted the expression of inflammation factors in RAW264. 7 cell ( Figure 7A,B).
The western blot results revealed that the P-P65/P65 and P-IκBa/IκBa ratios in the LPS-stimulated siNC group were significantly higher than in the control and Boris KO groups. Conversely, levels in the empty vector group without LPS treatment was lower than in the group overexpressing Boris without LPS ( Figure 7C). These findings suggested that Boris provokes inflammation in macrophages.

| DISCUSS ION
In this study, we constructed in situ colorectal cancer induced by AOM/DSS and colitis induced by DSS in Boris KO mice. Our findings revealed that Boris is important for the development of CRC and colitis (Figures 2 and 6). Boris regulates the Wnt signaling pathway ( Figure 3). By comparing human BORIS, mouse Boris, and the Boris mutant cloned from Boris KO mice, we discovered that murine Boris had the same functions as human BORIS in promoting cancer cell proliferation and resistance to 5-FU and cisplatin treatment.
Furthermore, the Boris mutant lost all these activities ( Figure 1).
The tumor formation process induced by AOM/DSS was similar to that of humans. 28 We observed that high expression of Boris was induced in both clinical CRC samples and mouse in situ colorectal tissues ( Figure 3A-C). This suggests that Boris relates to the progression of CRC. Our data confirmed that the loss of BORIS inhibited CRC in mice. Considering the mechanism that the Wnt signaling pathway was usually activated in AOM plus DSS-induced colorectal cancer, the subcellular location of β-catenin was examined by mod- According to transcriptome analysis of mice colorectal tissues, the MAPK pathway is regulated by Boris. By further examination of colorectal cancer tissues from mice, we determined that Boris knockout led to the suppression of the MAPK pathway, which is presented as phosphorylation withdrawal of ERK, JNK, and p38 ( Figure 4F). 20 The MAPK pathway is known to be activated by DNA damage. However, our data showed that Boris knockout led to DNA damage ( Figure 5). It is contradictory that the loss of Boris promoted DNA damage but also inhibited the response of the MAPK pathway.
Considering that MAPK responded to DNA damage and promoted subsequent cell proliferation and tissue repair, Boris deficiency might inhibit the MAPK pathway and cause a wide range of prohibition of colon tissue repair and the maximum extent of inhibition on colorectal cancer. 29 It has been reported that the Wnt pathway promotes DNA damage repair in colorectal cancer cells. Our results that Boris knockdown inhibits the Wnt pathway in Figure 3 also support our prediction that Boris deficiency caused a wide range of prohibition of colon tissue repair.
In conclusion, BORIS was highly expressed in CRC and promoted the development of tumors. Boris knockout alleviated in situ CRC generation by relieving colitis and suppressing DNA damage repair.
Our findings provide new insights into the development of CRC and provide new strategies for CRC therapy.