Dysregulation of lncRNA‐CCRR contributes to brain metastasis of breast cancer by intercellular coupling via regulating connexin 43 expression

Abstract Cardiac conduction regulatory RNA (CCRR) is down‐regulated in the pathogenesis of heart failure (HF), which accordingly suppresses cardiac conduction while promoting arrhythmogenicity. Meanwhile, CX43 was reported to play a role in the pathogenesis of metastatic breast cancer and melanoma brain colonization. In this study, we studied the role of long non‐coding RNA CCRR and its interaction with CX43 in brain metastasis of breast cancer. Breast cancer patients were grouped according to the metastasis status. Real‐time PCR and IHC assay were used to measure the expression of lncRNA‐CCRR and CX43 in patients. Western blot was conducted to observe the effect of lncRNA‐CCRR on the expression of CX43 in MDA‐MB‐231BR and BT‐474BR cells. Compared with the non‐metastasis group, the mRNA expression of tissue lncRNA‐CCRR, cerebrospinal fluid (CSF) lncRNA‐CCRR, tissue CX43 and tissue protein expression of CX43 were both evidently up‐regulated in metastasis patients, especially in patients with brain metastasis. The expression of lncRNA‐CCRR was positively correlated with the up‐regulated expression of CX43. Moreover, CX43 expression was significantly lower in MDA‐MB‐231WT cells compared with that in MDA‐MB‐231BR cells. Also, the overexpression of lncRNA‐CCRR evidently increased dye transfer rate from astrocytes to MDA‐MB‐231BR/BT‐474BR cells but reduced lncRNA‐CCRR expression and suppressed the transmigration of MDA‐MB‐231BR/BT‐474BR cells in a blood‐brain barrier (BBB) model. In this study, we demonstrated that the presence of lncRNA‐CCRR could up‐regulate the expression of CX43, which promoted gap junction formation in brain metastasis of breast cancer. Accordingly, the communication between breast cancer cells and astrocytes was also promoted.


| INTRODUC TI ON
About 10% of patients with cancer tend to develop brain metastasis. [1][2][3] Also, small lesions of brain metastasis still can trigger nerve impairment, so that the average survival of brain metastasis patients is short, 1 and the two primary reasons of brain metastasis are lung adenocarcinomas and breast adenocarcinomas. 2 Lung adenocarcinoma metastasis tends to develop within months of medical diagnosis to exert an effect on a number of organs outside the brain, suggesting that aggressive pre-metastatic functions will foster cancer cell colonization at different organs at the same time. 4 In breast cancer, distant relapse often occurs after an extended period of remission, indicating that breast cancer cells do not have the total capability for metastasis initially in distant organs. 5,6 Instead, breast cancer cells obtain this capability for metastasis under the selective stress pressure of microenvironments in various organs. 1 Lately, long non-coding RNA (lncRNA) has actually emerged as a key player in the regulation of gene expression. 7,8 LncRNAs are also recognized as regulatory molecules featured by lack of proteincoding capability. However, lncRNAs may take part in numerous vital biological processes as well as pathophysiological activities. 9,10 As the first lncRNA determined to show the ability to manage cardiac conduction, lncRNA AK045950 is also called cardiac conduction regulatory RNA (CCRR), which is actually down-regulated in a heart failure (HF) mouse model as well as people with HF by slowing down cardiac conduction as well as enhancing arrhythmogenicity. 11 However, CCRR was needed for keeping appropriate connexin 43 (CX43) distribution in intercalated discs and prevent the backward trafficking as well as subsequent degradation of CX43, a mechanism likely acted to disrupt gap junctions. 11 CX43, as a gap junction channel protein, was additionally discovered to become dysregulated in various forms of cancers such as stomach, cervical, rectal and prostate cancers. In breast cancer cells, the down-regulation of CX43 substantially enhanced cancer cell growth, while CX43 overexpression possibly suppressed cancer cell growth and restored their differentiation potential to suppress tumour. 12 In addition, the ubiquitylation of CX43 resulted in gap junction accumulation at cell membrane to trigger a concomitant boost of intercellular interaction. 13,14 The brain is a big target of metastasis, with astrocytes acting as the predominant mediator.
The function of astrocytes, most abundant type of cells in the brain, has been studied in brain metastasis. Breast as well as lung cancer cells can express PCDH7 to promote the assembly of astrocytes-cancer cell gap junctions comprised of CX43. After engaging the gap junction network in astrocytes, cancer cells metastasizing to brain utilize these gap junctions to transfer cGAMP into astrocytes, activating the STING signalling as well as the synthesis of proinflammatory cytokines IFNα and TNFα. 15 It has been reported that CCRR is down-regulated in a mouse model of HF and in patients with HF, and this down-regulation slows cardiac conduction and enhances arrhythmogenicity. 11 Meanwhile, CX43 was reported to play a role in the pathogenesis of metastatic breast cancer and melanoma brain colonization. 16 In this study, we hypothesized that lncRNA-CCRR could interact with CX43 and regulate its expression, and the dysregulated CX43 influenced gap junction formation in brain metastasis of breast cancer, which accordingly influenced the communication between breast cancer cells and astrocytes.

| Patient recruitment
In this study, 64 breast cancer patients were grouped to the following three groups according to their metastasis status: (1) brain me-   5′-ACCACCCTGTTGCTGTAGCCAA-3′) was used as the calibrator.

| Western blot analysis
Western blot analysis was carried out to measure the protein expression of CX43 in each sample. In brief, total protein was isolated from cell and tissue samples by making use of a radioimmunoprecipitation (RIPA) buffer (Thermo Fisher Scientific) following the standard experimental protocol provided on the operation manual by the buffer manufacturer. Then, the concentration of protein in each sample was determined by utilizing a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific) following the standard experimental protocol provided on the operation manual by the assay kit manufacturer. In the next step, the proteins were resolved by using 10% sodium dodecyl sulphate-PAGE and then transferred onto a polyvinylidene difluoride membrane via electroblotting. In the next step, non-specific binding was blocked with Tris-buffered saline with Tween-20 + 5% non-fat milk before the membrane was probed overnight at 4°C with primary anti-CX43 antibody (dilution 1:1000, OriGene Technologies) following the standard incubation protocol provided on the operation manual by the antibody manufacturer, followed by subsequent incubation with HRP-conjugated secondary antibodies (dilution 1:5000, Thermo Fisher Scientific) for 1 hour at ambient temperature. After image development by making use of an enriched chemiluminescence assay kit (Thermo Fisher Scientific) following the standard experimental protocol provided on the operation manual by the assay kit manufacturer, the protein expression of CX43 in each sample was calculated.

| IHC
Immunohistochemical staining was done to visualize the protein expression of CX43 in each sample. In brief, tissue samples were fixed in formalin, embedded in paraffin, deparaffinized and then hydrated by utilizing gradient alcohol. The samples were then blocked in PBS + 0.2% Triton X-100 + 10% horse serum for 1 hour at room temperature. After that, the samples were incubated against primary anti-CX43 antibodies and biotin conjugated secondary antibodies following the standard incubation protocol provided on the operation manual by the antibody manufacturer (Dako). Dab was used as the dye for counter-staining.

| Dye transfer assay
Due to the fact that the communication between non-tumour astrocytes and breast carcinoma cells influences brain metastasis, the effect of lncRNA-CCRR on dye transfer rate was investigated via the dye transfer analysis. The dye transfer assay was done using a calcein-AM/DiD assay kit (Takara) following the standard experimental protocol provided on the operation manual by the assay kit manufacturer. The results were evaluated on a FACS Canto II Flow Cytometer (BD Biosciences) and analysed using the FlowJo software (Treestar).

| Transwell assay
To observe the effect of lncRNA-CCRR expression on the transmigration in an organotypic BBB model established based on co-cultivation of PBECs with astrocytes, a transwell assay was performed on MDA-MB-231BR cells, respectively, grouped as: (1) negative control group; (2) co-astrocyte + NC siRNA group; and (3) co-astrocyte + lncRNA-CCRR siRNA group. The transwell assay was performed using the BBB model co-

| Statistical analysis
The statistical analysis was carried out using the SPSS software package (version 19.0, SPSS). The data was shown as mean ± SD.
One-way analysis of difference (ANOVA) was done to compare the difference among different groups. A Student's t test was done to compare the difference between two groups. A value of P < .05 suggested statistical significance.

| Expression of lncRNA-CCRR and CX43 was higher in patients with metastasis
The breast cancer patients were grouped according to their me- Accordingly, compared with those in the non-metastasis group, tissue expression of lncRNA-CCRR ( Figure 1A) and CSF expression of lncRNA-CCRR ( Figure 1B) were both evidently up-regulated in metastatic patients, while the patients with brain metastasis showed even higher lncRNA-CCRR expression compared with patients with no brain metastasis. Also, the relative expression of CX43 mRNA in tissues ( Figure 1C) and CX43 protein in tissues ( Figure 2) also exhibited the same trend.

| Overexpression of lncRNA-CCRR promoted the expression of CX43
To

| Expression of CX43 was increased in cells with brain metastasis
To further explore the influence of brain metastasis on the effect

| Overexpression of lncRNA-CCRR inhibited cell communication between non-tumour astrocytes and breast carcinoma cells
Due to the fact that the communication between non-tumour astrocytes and breast carcinoma cells influences brain metastasis, the effect of lncRNA-CCRR on dye transfer rate was investigated via the dye transfer analysis. As shown in Figure 5A,B, the overexpression of lncRNA-CCRR evidently increased the dye transfer rate F I G U R E 2 IHC assay indicated that the expression of tissue CX43 protein was evidently up-regulated in metastasis patients, especially in patients with brain metastasis

| Reduced lncRNA-CCRR expression suppressed transmigration in a BBB model
To observe the effect of lncRNA-CCRR expression on the transmigration in an organotypic BBB model established based on co-cultivation of PBECs with astrocytes, as shown in Figure 6A, results were demonstrated in BT-474BR cells ( Figure 6B).

| Reduced lncRNA-CCRR expression suppressed the expression of cellular CX43
The levels of CX43 among different MDA-MB-231BR ( Figure 7A) and BT-474BR cell groups ( Figure 7B) were also compared. The expression of CX43 was significantly elevated in the co-astrocyte + NC

| D ISCUSS I ON
In this study, we collected tissue samples from breast cancer patients with or without brain metastasis and found that, compared with that in the non-metastasis group, the expression of tissue proximity to the vascular tissues in human brain. 16,26 CX43 is found in metastatic tumour cells at the communication interface with astrocytes. 20 The past results showed that the capacity of cancer stem cells (CSCs) to metastasis to the brain may be triggered by the higher CX43 expression in CSCs. Furthermore, it was recently shown that the transfer of cGAMP to astrocytes from cancer cells through CX43-containing enhances the brain metastasis of cancer, suggesting that the gap junctions between astrocytes and tumour cells can be used as a good target in the treatment of metastatic cancer. 15,27 In this study, we found that the overexpres-

| CON CLUS ION
In this study, we demonstrated that the presence of lncRNA-CCRR could up-regulate the expression of CX43, which promoted gap junction formation in brain metastasis of breast cancer. Accordingly, the communication between breast cancer cells and astrocytes was also promoted.

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.