JARID1B was markly upregulated in CRC tissues and was closely related to CRC progression.
To investigate the role of JARID1B in the development of colorectal cancer, we investigate JARID1B expression levels in colorectal cancer, 54 paired fresh CRC tissue samples and matched adjacent non-tumor tissues were used. The qRT-PCR revealed that the mRNA expression level of JARID1B was notably elevated in CRC tissue samples compared with the corresponding adjacent tissues (Fig. 1a, b). Furthermore, western blotting showed that JARID1B protein expression in 54 paired CRC tissues was consistent with the qRT-PCR results (Fig. 1c, d). Consistently, immunohistochemistry (IHC) results showed that JARID1B protein were highly expressed in 75.38% (98 of 130) tissues, while weakly positive staining was observed in the adjacent non-tumor tissues(Fig. 1e). Then, we explored whether JARID1B expression would affect clinic pathologic parameters. As shown in Table 1, the JARID1B high expression group demonstrated a larger tumour size, higher CEA levels and greater T classification compared with the JARID1B low expression group. Furthermore, the Kaplan-Meier survival curves demonstrated that patients with high JARID1B expression levels in 130 immunohistochemical results had a significant poor overall survival (Fig. 1f), while univariate and multivariate analysis of overall survival in CRC patients also indicated that JARID1B high expression was a risk factor for 5-year survival (Table 2). Altogether, the findings demonstrated that JARID1B expression was up-regulated in CRC and implicated in the progression of CRC.
Table 1
Relationship between JARID1B protein expression and clinicopathological features in CRC patients.
Parameters | Total 54a | Relative JARID1B protein expressionb | p value |
Gender | | | | 0.53 |
Male | 36 | 2.11 ± 0.85 | |
Female | 18 | 2.34 ± 0.89 | |
Age (years) | | | | 0.71 |
≤ 60 | 20 | 2.14 ± 0.93 | |
> 60 | 34 | 2.22 ± 0.83 | |
Tumor size(cm) | | | | 0.00** |
< 5 | 23 | 1.61 ± 0.80 | |
≥ 5 | 31 | 2.62 ± 0.62 | |
Preoperative CEA level | | | | 0.00** |
< 5 ng/mL | 40 | 1.86 ± 0.72 | |
≥ 5 ng/mL | 15 | 3.13 ± 0.41 | |
Histologic grade | | | | 0.98 |
well differentiated | 42 | 2.17 ± 0.89 | |
poorly differentiated | 12 | 2.26 ± 0.80 | |
T classification | | | | 0.00** |
T1 + T2 | 17 | 1.19 ± 0.54 | |
T3 + T4 | 37 | 2.65 ± 0.53 | |
TNM stage | | | | 0.38 |
I | 4 | 1.57 ± 0.55 | |
II | 14 | 2.28 ± 1.02 | |
III | 28 | 2.18 ± 0.88 | |
IV | 8 | 2.37 ± 0.51 | |
Lymphatic invasion | | | | 0.52 |
Negative | 21 | 2.12 ± 0.91 | |
Positive | 33 | 2.24 ± 0.84 | |
Distant metastasis | | | | 0.88 |
Negative | 45 | 2.16 ± 0.92 | |
Positive | 9 | 2.33 ± 0.50 | |
a: 54 fresh tissue samples from June 2018 to December 2018, b:protein level in Tumor tissues/protein level in Adjacent tissues(mean ± SD),**p ≤ 0.01 |
Table 2
Univariate and multivariate analysis of overall survival in CRC patients. (Cox proportional hazards regression model)
Parameters(130 CRC patients)a | Univariate analysis | | Multivariate analysis |
HR | 95%CI | p value | HR | 95%CI | p value |
Age ( ≦ 60/>60) | 0.70 | 0.34–1.41 | 0.32 | | | | |
Gender(Male/Female) | 1.13 | 0.57–2.22 | 0.73 | | | | |
Histologic grade (well/poor) | 0.66 | 0.29–1.51 | 0.33 | | | | |
Tumor size(cm)(< 5/≧5) | 1.78 | 0.90–3.51 | 0.10 | | | | |
CEA(ng/ml) (< 5/≧5) | 1.09 | 0.75–1.60 | 0.64 | | | | |
Lymphatic invasion (Positive/Negative) | 1.95 | 1.04–3.67 | 0.04* | | 1.52 | 0.52–4.43 | 0.03* |
Distant metastasis (Positive/Negative) | 2.03 | 1.09–3.78 | 0.03* | | 1.65 | 0.62–4.38 | 0.01** |
T classification (T3 + T4/T1 + T2) | 2.65 | 1.20–5.85 | 0.02* | | 1.80 | 0.67–4.84 | 0.02* |
TNM(III,IV/I,II) | 2.49 | 1.24-5.00 | 0.01** | | 2.12 | 1.13–3.98 | 0.02* |
JARID1B protein expression (High/Low) | 2.29 | 1.41–3.73 | 0.00** | | 2.53 | 1.50–4.27 | 0.00** |
a: Immunohistochemical results of 130 paraffin-embedded tissues from December 2012 to December 2017, *p ≤ 0.05,**p ≤ 0.01 |
JARID1B promoted proliferation and tumorigenesis of CRC in vivo and in vitro.
To investigate the potential biological function of JARID1B in CRC development, we initially examined JARID1B expression levels in in CRC cells (SW620, HCT116, LOVO, SW480, DLD-1) by qRT-PCR, and western blotting, compared to the non-malignant cell line (HCoEpic). The results showed that JARID1B expression notably elevated in CRC cells (Fig. 2a, b). As shown in Table 1, the high JARID1B expression group demonstrated a larger tumour size, so that we speculate that JARID1B may be functional in CRC proliferation.. Next, we investigated the relationship between JARID1B expression and CRC cells proliferation. Colony formation, 5-ethynyl-20-deoxyuridine (EdU) and Real Time Cellular Analysis(RTCA) assays revealed that the proliferation capacity of DLD-1 cells with short hairpin RNA (shRNA)-mediated JARID1B knockdown was markly lower than the control group (Fig. 2c, d, e). In contrast, JARID1B over-expression significantly enhanced the proliferation ability of LOVO cells (Supplementary Fig. 1a, b). Furthermore, an in-vivo experiment showed that the JARID1B knockdown group had smaller volumes and lower weights than the control group (Fig. 2f, g, h). That is, knockdown of JARID1B significantly inhibited tumor size and weight in vivo. These data demonstrated that JARID1B could promote CRC cells proliferation in vivo and in vitro.
JARID1B influence CRC proliferation through regulating the Wnt/β-catenin signaling pathway
In subsequence, we explored the mechanism how JARID1B regulated CRC cells proliferation. Studies have indicated that Wnt signaling pathway played an important role in controlling the proliferation of CRC cells. Therefore, we speculated that JARID1B might regulate CRC proliferation through Wnt/β-catenin signaling pathway. First, the Gene Set Enrichment Analysis(GSEA) based on TCGA COAD RNA expression dataset revealed that JARID1B level was positively correlated with Wnt signaling pathway activity (Fig. 3a). Then, we investigated the effect of JARID1B on the activation of Wnt/β-catenin pathway in CRC cells. In JARID1B-silenced DLD-1 cells, we observed that β-catenin and its downstream target genes, c-MYC and CycinD1, were down-regulated(Fig. 3b). The Western blotting results also showed that downregulation of JARID1B expression significantly decreased the expression levels of the total and nuclear β-catenin (Fig. 3c). Consistently, TOP-Flash luciferase assay revealed that JARID1B downregulation inhibited Wnt/β-catenin pathway activity, proving that JARID1B played a key role in activation of Wnt/β-catenin signaling pathway(Fig. 3d). In contrast, upregulation of JARID1B expression significantly increased the expression protein levels of c-MYC, Cycin D1, the total and nuclear β-catenin in LOVO cells (supplementary Fig. 2a, b, c). These results indicated that Wnt/β-catenin signaling pathway may be regulated by JARID1B.
Next, we want to validate that CRC proliferation was mediated by JARID1B via Wnt/β-catenin signaling pathway. Thus, we increased β-catenin expression in JARID1B-knockdown DLD-1 cells. The western blotting results showed that β-catenin overexpression significantly inhibited the decrease in β-catenin, c-MYC and CycinD1 expression in JARID1B–knockdown DLD-1cells and rescued the decreased Wnt/β-catenin pathway activity induced by downregulating JARID1B (Fig. 3e, f). As the results shown, upregulation of β-catenin rescued the proliferation ability in DLD-1cells with JARID1B downregulation in vivo and in vitro(Fig. 3g, h, i). Conversely, we suppressed β-catenin expression in JARID1B-overexpression LOVO cells. The results showed that β-catenin knockdown inhibited the increase of β-catenin, c-MYC and CycinD1 expression and inhibited Wnt/β-catenin pathway activity induced by JARID1B overexpression (supplementary Fig. 2d, e). And the results showed that the enhanced proliferation ability in LOVO cells caused by JARID1B overexpression was remarkably inhibited by downregulation of β-catenin (supplementary Fig. 2f,g). The above mentioned results demonstrated that JARID1B regulated CRC proliferation via the Wnt/β-catenin pathway.
JARID1B significantly inhibited CDX2 expression in CRC.
We further explored how JARID1B regulated Wnt/β-catenin signaling in CRC cells. It has been reported that CDX2 knockdown promoted the proliferation of colorectal cancer cells via Wnt/β-catenin signaling. Thus, we speculated that JARID1B might regulate the expression of CDX2 to enhance the activity of Wnt/β-catenin signaling in CRC cells. Given the role of JARID1B in the epigenetic regulation of transcription, we first performed RNA-seq to identify potential JARID1B target genes involved in cells proliferation. The results showed that CDX2 was one of the most notably upregulated transcripts when JARID1B knockdown (Fig. 4a). Next, qRT-PCR revealed that downregulation of JARID1B significantly increased CDX2 mRNA (Fig. 4b). Western blotting results showed that downregulation of JARID1B expression significantly increased the expression levels of CDX2, while the expression level of β-catenin, c-MYC and cyclinD1 were decreased (Fig. 4c). Conversely, JARID1B overexpression decreased the levels of CDX2 mRNA expression(Fig. 4d). Western blotting results showed that upregulation of JARID1B expression significantly decreased the expression levels of CDX2, meanwhile the expression level of β-catenin, c-MYC and cyclinD1 were increased (Fig. 4e). Furthermore, we examined CDX2 in 54 CRC tissue samples and the corresponding by Western blotting and qRT-PCR. The results show that the expression level of CDX2 was significantly higher in adjacent tissues compared to tumors tissues (Fig. 4f, h). Finally, the statistical analysis results revealed that CDX2 expression was negatively correlated with JARID1B expression in CRC tissues(Fig. 4g, i). Consistently, A Kaplan-Meier analysis indicated that patients with both high JARID1B expression and low CDX2 expression in 130 immunohistochemical results were predicted the worst prognosis (Fig. 4i). Collectively, these data suggested that JARID1B negatively regulated CDX2 expression to increased Wnt/β-catenin signaling activity in CRC.
CDX2 was the key protein for JARID1B-mediated Wnt/β-catenin signaling in CRC cells.
To further validate whether JARID1B activated Wnt/β-catenin signaling pathway through regulating CDX2 expression. After we first decreased the expression of CDX2 in JARID1B–knockdown DLD-1 cells, CDX2, GSK-3β, Axin2 and p-β-catenin, β-catenin, c-MYC and cyclinD1 expression levels were observed. The qRT-PCR results showed that knockdown of CDX2 inhibited the increase of CDX2 mRNA expression induced by JARID1B knockdown (Fig. 5a). The western blotting data showed that CDX2 knockdown deceased GSK-3β, Axin2 and p-β-catenin expression and markedly rescued the change of β-catenin, c-MYC and cycinD1 expression levels caused by JARID1B(Fig. 5b). Co-IP showed that JARID1B knockdown could increase ubiquitinated β-catenin level, which was rescued when CDX2 expression was knockdown (Fig. 5c). Simultaneously, TOP-Flash luciferase assay revealed that reduced Wnt/β-catenin signaling activity induced by JARID1B knockdown was partly abolished by the knockdown of CDX2(Fig. 5d). In contrast, upregulation of CDX2 inhibited the JARID1B overexpression-induced increase in LOVO cells (Fig. 5e, f, g, h).These results demonstrated that CDX2 was required for JARID1B-mediated Wnt/β-catenin signaling pathway in CRC cells.
JARID1B regulated CDX2 expression through demethylation of H3K4me3
We then explored how JARID1B regulated CDX2 expression at the transcriptional level. Studies have shown that JARID1B affected the histone H3 lysine4 (H3K4) demethylase and exhibited a strong transcriptional repression function. We explored whether JARID1B demethylated H3K4me3 at the promoter of CDX2 in CRC cells. We first decreased the expression of JARID1B, and then observed the H3K4me3 protein expression levels and CDX2 promoter activity. Western blotting data showed that JARID1B knockdown markedly increased H3K4me3, CDX2, GSK-3β, Axin2 and p-β-catenin expression, and deceased β-catenin, c-MYC and cycinD1 expression (Fig. 6a). Simultaneously, a luciferase reporter gene assay showed that JARID1B downregulation increased CDX2 promoter activity (Fig. 6c). In contrast, overexpression of JARID1B decreased H3K4me3 protein expression and CDX2 promoter activity (Fig. 6b, c). In addition, to test whether JARID1B expression was correlated with the H3K4me3 modification at the CDX2 gene promoter in CRC cells. Meanwhile, a ChIP assay revealed that JARID1B knockdown increased H3K4me3 levels at CDX2 in DLD-1 cells (Fig. 6d), while overexpression of JARID1B decreased H3K4me3 levels at CDX2 in LOVO cells (Fig. 6d). After combining all the experimental results, we revealed a new important mechanism that JARID1B regulated CDX2 expression through H3K4me3 to indirectly activate Wnt/β-catenin pathway, leading to increased CRC proliferation(Fig. 6e).