Oestrogen promotes tumorigenesis of bladder cancer by inducing the enhancer RNA—eGREB1

Abstract In recent years, studies have shown that enhancer RNAs (eRNAs) can be transcribed from enhancers. Increasing evidence has revealed that eRNAs play critical roles in the development of various cancers. Oestrogen‐associated eRNAs are closely related to breast cancer. In view of the gender differences in bladder cancer (BCa), we suppose that oestrogen‐associated eRNAs are also involved in tumorigenesis of BCa. In our study, we first demonstrated that eGREB1 derived from the enhancer of an oestrogen‐responsive gene—GREB1 was up‐regulated in BCa tissues, and the expression level of eGREB1 is positively associated with the histological grade and TNM stage of BCa. Knockdown of eGREB1 by CRISPR‐Cas13a could inhibit cell proliferation, migration and invasion and induce apoptosis in BCa cells T24 and 5637. Besides, we exhibited the promoting effect of oestrogen on BCa cells. What's more, down‐regulation of eGREB1 could improve the malignant biological characteristics of BCa cells induced by oestrogen. In conclusion, our data indicated that eGREB1 plays oncogenic role and oestrogen may promote the occurrence and progression of BCa by inducing eGREB1 production. Our findings provide new insights into the prevention of BCa and develop a novel therapeutic target for the treatment of BCa.

Global statistics has shown that sex hormones also play an important role in the initiation and invasion of BCa, as the incidence of BCa in males has increased 3.3 times higher than that of females, but the latter tend to be more aggressive and cause twice mortality than males with BCa. 7,8 However, the molecular mechanisms of oestrogen in BCa remain elusive.
Enhancers are cis-regulatory elements with the ability to increase target gene transcription independent of the distance and direction relative to the promoters. 9 It is generally accepted that enhancers and promoters are physically close together to form enhancerpromoter looping (E:P looping) to enhance the expression of target genes. 10,11 In recent years, an amazing discovery revealed more complicated roles of enhancers in gene transcriptional activation.
Enhancers could transcribe into enhancer RNAs (eRNAs) uni-or bi-directionally similar to the promoters when respond to various stimulations. 12 GREB1, gene regulated in breast cancer 1, is a chromatin-binding coactivator that stabilizes the combination of oestrogen receptor (ER) and other cofactors. 18 Gosh et al 19 were the first to show that GREB1 was an oestrogen regulated gene and played an important role in hormone-responsive cancer. GREB1 promoted oestrogenstimulated proliferation of breast cancer, 19 prostate cancer 20 and endometriosis. 21 Oestrogen could induce the transcription of the corresponding enhancers of GREB1, giving rise to GREB1 eRNA (eGREB1) production in MCF-7. 12,16 In view of the continuous existence of gender differences in BCa, oestrogen seems to be especially important in BCa progression. It is necessary to elucidate whether eGREB1 participated in the action of oestrogen on BCa.
In this study, we examined eGREB1 expression in BCa tissues and investigated the effect of eGREB1 knockdown on the proliferation, migration, invasion and apoptosis of BCa cells T24 and 5637. Moreover, we explored whether eGREB1 knockdown could influence oestrogen-induced biological behaviours of BCa cells. Our work aims to clarify the potential molecular mechanisms of oestrogen-induced BCa and provides new insights for the prevention of BCa.

| Patient samples
We collected 38 tumour tissues and matched normal bladder tissues from patients diagnosed with bladder urothelial carcinoma. All samples were snap-frozen in liquid nitrogen immediately after partial or radical cystectomy. We got the written informed consents from all the BCa patients included in this study. The Institutional Review Board of Shenzhen Second People's Hospital approved the study.

| Cell culture and treatments
Human bladder cancer cell lines (T24, 5637) were purchased from the America Type Culture Collection (ATCC, Manassas, VA, USA).

| Real-time quantitative PCR (RT-qPCR)
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. cDNA was converted from total RNA using a Revertra Ace qPCR RT Kit

| Transwell assay
The cells were treated with Cas13a-eGREB1 or Cas13a-NC for After that, each chamber with the invaded cells was soaked into 1 mL 33% acetic acid for 10 min to wash out the crystal violet. One hundred microlitres of 33% acetic acid was added into each well of 96-well plates, and then, the absorbance was measured at 570 nm by a microplate reader (Bio-Rad, Hercules, CA, USA). Experiments were performed in triplicate.

| Caspase 3 ELISA assay
Bladder cancer 5637 and T24 cells were transfected with Cas13a vectors in petri plates. Twenty-hour hours after transfection, apoptosis was detected by measuring the activity of caspase 3 using the caspase 3 enzyme-linked immunosorbent assay (ELISA) assay kit (Cusabio, Wuhan, China) according to the manufacturer's instructions. OD values were measured at 450 nm using a microplate reader (Bio-Rad).

| Statistical analysis
All data were presented as mean ± standard deviation (SD). The eGREB1 expression differences between BCa tissues and matched normal tissues were analysed using paired samples t test. The CCK-8 assay was analysed using ANOVA. Other data were analysed by the independent samples t test. All these statistical analyses were performed using SPSS 17. A P value of less than 0.05 was considered to be statistically significant.

| eGREB1 was up-regulated in BCa tissues and it was related to clinical-pathological features
The relative expression level of eGREB1 in a total of 38 patients with BCa was measured by RT-qPCR. Compared with matched normal tissues, eGREB1 is significantly up-regulated in 73.7% (28 of 38) of BCa tissues (P = 0.018, Figure 1A  Thus, only CRISPR-Cas13a is suitable for the knockdown study on eGREB1.

| Oestrogen induced the production of eGREB1 in BCa cells
To explore whether oestrogen could induce eGREB1 transcription in BCa cells, we analysed eGREB1 expression after E2 stimulation for 1 h in T24 and 5637. Our results showed the relative expression level of eGREB1 increased by 2.87-fold in T24 (P < 0.001) and 2.74fold in 5637 (P < 0.01), respectively ( Figure 1E). We demonstrated that eGREB1 expression was significantly raised in BCa cells when stimulated by oestrogen.

| eGREB1 knockdown attenuated the cancer-promoting effect of oestrogen
We revalidated the cancer-promoting effect of oestrogen on BCa.
Subsequently, BCa cells were transfected with Cas13a-eGREB1 or Cas13a-NC vectors and treated with oestrogen meanwhile. We compared their biological behaviours using the methods described above. For the group of eGREB1 knockdown with oestrogen treatment, the cell proliferation was obviously decreased ( Figure 3A, B, P < 0.01 in T24 and 5637); the cell migration ratio were reduced by 11.68% in T24 and 18.28% in 5637 ( Figure 3C-E, P < 0.001 in T24 and 5637); the invasive ability of the cells is also prominently weakened ( Figure    Experiments were performed in triplicate. Data were shown as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001) knockdown by siRNA is barely satisfactory. Instead, targeted knockdown of eGREB1 by CRISPR-Cas13a is extremely meaningful. The Cas13a construct could localize to the nucleus and cleave transcripts precisely and efficiently based on the nuclear localization sequence. 38 In summary, we demonstrated that CRISPR-Cas13a is more applicable to knockdown of eRNAs.
To further verify the function of eGREB1 in BCa, we used CRISPR-Cas13a vectors to reduce eGREB1. Cell proliferation inhibition, decreased motility and increased apoptosis were observed in T24 and 5637 cells. These findings indicated that eGREB1 may play positive roles in the occurrence and progression of BCa. Then, we explored the effects of oestrogen on the biological characteristics of BCa and found that oestrogen could promote cell proliferation, migration and invasion and inhibit cell apoptosis, which was consistent with previous studies. 29,39 Last but most important is that knockdown of eGREB1 is able to reverse oestrogen-induced biological effects on BCa cells. In this way, we suppose that the cancerpromoting effect of oestrogen may be achieved at least in part through the induction of eGREB1.
In conclusion, our study demonstrated the crucial role of eGREB1 in BCa and oestrogen may promote the development of BCa by inducing eGREB1 production for the first time. There are many molecular hypotheses of the association between oestrogen and BCa. Current