The Expression Of Bc069792 In Breast Cancer Tissues
The pathological types of these 98 cases of breast cancer were primary invasive carcinoma and non-specified type. RT-qPCR was used to detect the expression of BC069792 in these 98 pairs of breast cancer tissues and normal breast tissues. The results showed that the median expression level of BC069792 in breast cancer tissue was 0.013002, and that in normal breast tissue was 0.063384. Compared with the two groups, the difference in the expression level of lncRNA BC069792 was about 4.87 times different (Fig. 1a, P < 0.0001). The results of ROC curve analysis showed that the expression of BC069792 was able to distinguish breast cancer tissue from normal breast tissue, and the area under the curve was 0.9191 (Fig. 1b). This result suggested that BC069792 could be used as a molecular marker for breast cancer.
In breast cancer tissues, the expression of BC069792 was significantly decreased in high pathological grades, lymph node metastasis and high Ki-67 index groups. Spearman correlation analysis showed that BC069792 was negatively correlated with pathological grade, lymph node metastasis and high Ki-67 index, but had no significant correlation with age, tumor size, and hemorrhage/calcification/necrosis/cystic degeneration (Table 1).
Table 1
The relationship between the expression of BC069792 in breast cancer and the clinicopathological parameters of patients
clinical parameter | quantity | BC069792 | Chi-square test |
LOW expression | High expression | χ2 | P, price | |
age | | | | 1.951 | 0.162 |
<65 | 78 | 59 | 19 | | |
≥ 65 | 20 | 12 | 8 | | |
pathological grading | | | | 8.120 | 0.004 |
Ⅰ | 18 | 7 | 11 | | |
Ⅱ+Ⅲ | 80 | 59 | 21 | | |
tumor size | | | | 1.395 | 0.238 |
<2cm | 26 | 3 | 23 | | |
≥ 2cm | 72 | 16 | 56 | | |
Hemorrhage, calcification, necrosis, and cyst change | | | | 3.623 | 0.057 |
NO | 43 | 11 | 32 | | |
YES | 55 | 6 | 49 | | |
lymphatic metastasis1 | | | | 10.324 | 0.001 |
NO | 54 | 18 | 36 | | |
YES | 37 | 25 | 12 | | |
Ki-67 index | | | | | 0.045 |
<30% | 36 | 0 | 36 | | |
≥ 30% | 62 | 7 | 55 | | |
1Seven cases were not tested for lymph node metastasis. Inspection level a = 0.05. |
The Localization And Stability Of Bc069792
RT-qPCR with GAPDH as the internal reference for the cytoplasm and U6 as the internal reference for the nucleus showed that about 27.6% of BC069792 was in the cytoplasm in the MDA-MB-231 cell line. In the MDA-MB-468 cell line, about 26.4% of BC069792 was expressed in the cytoplasm, and 73.6% was expressed in the nucleus, indicating that BC069792 plays roles in both the nucleus and the cytoplasm. (Fig. 1c)
Actinomycin D is an inhibitor of RNA polymerase II. In order to detect the half-life of BC069792, we performed dosing experiments with actinomycin D in MDA-MB-231 and MDA-MB-468 cells. The content of BC069792 in cells was detected by RT-qPCR at 0h, 0.5h, 1h, 4h, 8h and 12h after dosing. The results showed that compared with C-MYC, BC069792 exists stably for a longer time in MDA-MB-231 and MDA-MB-468 cells (Fig. 1d).
BC069792 inhibits the proliferation, migration, and invasion abilities of breast cancer cells in vitro
The relationship between BC069792 and clinicopathological parameters suggests that BC069792 plays the role of tumor suppressor gene in breast cancer. In order to clarify the specific function of BC069792 in breast cancer, we performed a series of experiments in vitro.
CCK-8 and EdU experiments showed that overexpression of BC069792 significantly inhibited the proliferation of MDA-MB-231 and MDA-MB-468 cells compared with the control group (Fig. 2a, b). After knocking down the expression of BC096792, the proliferation of MDA-MB-231 and MDA-MB-468 cells was slightly increased (Supplementary Fig. 1a, b). The results of the Transwell experiment showed that compared with the control group, the number of BC069792 overexpressing cells crossing the basement membrane was significantly reduced, and the overexpression of BC069792 significantly inhibited the migration and invasion abilities of MDA-MB-231 and MDA-MB-468 (Fig. 2c). Knockdown of BC069792 effectively promoted the migration and invasion of MDA-MB-231 and MDA-MB-468 cells, and the number of cells passing through the basement membrane in the chamber was significantly increased (Supplementary Fig. 1c).
Bc069792 Inhibits The Proliferation, Invasion, And Metastasis Abilities Of Breast Cancer Cells In Vivo
The subcutaneous tumorigenesis model of nude mice was successfully established by subcutaneous injection of lentivirus LV-BC069792 and LV-NC infected MDA-MB-231 cells. After 5 weeks, 5 out of 6 nude mice in the LV-BC069792 group developed tumors, and all 6 nude mice in the LV-NC group had tumors (Fig. 3a). The tumor growth curve showed that the tumor growth rate in the LV-NC group was significantly higher than that in the LV-BC069792 group (Fig. 3b), and the tumor size of the mice in the LV-BC069792 group was significantly smaller than that in the LV-NC group (Fig. 3c). By extracting RNA from tumor tissue of nude mice and performing RT-qPCR experiments, it was confirmed that the expression level of BC069792 in the LV-BC069792 group was significantly higher than that in the LV-NC group (Fig. 3d). Ki-67 index in the LV-BC069792 group was significantly lower than that in the LV-NC group (Fig. 3e). These experimental results demonstrated that overexpression of BC069792 inhibited the proliferation of breast cancer cells.
The lentivirus-infected MDA-MB-231 cells was injected into nude mice through the tail vein to construct a distant metastasis model. The results showed that 3 nude mice in the LV-BC069792 group had metastases in the lungs. The median number of metastases in the lungs of the nude mice was 2, and the size was small. In the LV-NC group, 6 nude mice were found to have metastases in the lungs, and the median number of metastases in the lungs of nude mice was 13, and tumor size was larger. In the LV-NC group, it was found that the tumor invaded the lung, and the liver of one of the nude mice adhered to the diaphragm, and breast cancer metastasis was seen in the diaphragm. In the LV-BC069792 group, no other metastases except the lung were found (Fig. 3f, g). In the subcutaneous xenograft model, the tumor infiltrated the fat, striated muscle and skin appendages in the LV-NC group, while the tumor in the LV-BC069792 group had a clearer boundary and formed a pseudocapsule without obvious invasion (Fig. 3h). The subcutaneous tumorigenesis and distant metastasis models in nude mice confirmed that BC069792 effectively inhibited the migration and invasion of breast cancer cells.
The Rna-seq Reveals That Kcnq4 Is An Important Downstream Target Gene Of Bc069792
To explore the downstream effector molecules regulated by BC069792, we performed transcriptome sequencing (RNA Sequence, RNA-Seq) in BC069792 overexpression group and control group. The results of principal component analysis showed that the two groups of samples submitted for inspection had a certain degree of distinction, and the consistency within each group was high (Supplementary Fig. 2a). A total of 23365 genes were detected by sequencing. Compared with the pcDNA3.1 group, overexpression of BC069792 caused differential expression of 1209 genes (p < 0.05). At the same time, the log2 fold change was 2 times of the standard. A total of 407 genes were up-regulated and 802 genes were down-regulated (Supplementary Fig. 2b). According to the differential genes obtained by sequencing, GO enrichment analysis and KEGG enrichment analysis were performed, and a total of 212 pathways were analyzed. Compared with the control group, 25 pathways were significantly changed in the BC069792 overexpression group (P < 0.05), of which 7 pathways were concentrated on synaptic transmission and signal transduction pathways (Supplementary Fig. 2c).
According to the mRNA expression level, the log2 difference multiple of more than 2 times and p < 0.05 in the sequencing results, 30 genes were selected from the sequencing results to be verified. RT-qPCR showed that among the 30 genes, the expression of KCNQ4 was the most significantly different between BC069792 overexpression group and control group and had highest expression level in MDA-MB-231 cells (Fig. 4a). The expression of KCNQ4 protein also increased after overexpression of BC069792 (Fig. 4b) but decreased in the BC069792 knockdown cells (Supplementary Fig. 3). Moreover, the mRNA (Fig. 4c) and protein (Fig. 4d) expression of KCNQ4 in normal breast tissue was significantly higher than that in breast cancer tissue. The expression level of KCNQ4 protein in the tumor tissue of nude mice in the LV-BC069792 group was significantly higher than that in the LV-NC group (Fig. 4e). And the expression of BC069792 was positively correlated with KCNQ4 (Fig. 4f, g). The above findings indicated that BC069792 regulates the mRNA and protein expression of KCNQ4 in breast cancer, suggesting that BC069792 may inhibit the progression of breast cancer by promoting the expression of KCNQ4.
BC069792, as an endogenous competing RNA (ceRNA), upregulates the expression of target gene KCNQ4 by adsorbing miR-658 and miR-4739
Argonaute2 protein (Ago2) plays an important role in the occurrence and development of human tumors by participating in the formation of RNA-induced silencing complexes [5]. We performed RIP experiments with IgG as a control. The results showed that endogenous BC069792 and KCNQ4-3'UTR were specifically enriched by Ago2 antibody (Fig. 5a), suggesting that BC069792 and KCNQ4 can bind to specific miRNAs in the cytoplasm. The target miRNAs that can bind to BC069792 and KCNQ4 were predicted through miRWalk, RegRNA2.0 and other websites and ENCORI database, and the differentially expressed miRNAs in breast cancer and normal breast tissues were screened through the TCGA database. Three pro-tumor miRNAs, including miR-658, miR-632, and miR-4739 were found in the intersection of the three groups (Fig. 5b). The mimics or mimics NC of pmirGLO-BC069792/pmirGLO-NC and three miRNAs were co-transfected into MDA-MB-231 and MDA-MB-468 cell lines, and the fluorescein in the cells was detected by a microplate reader 48 hours later compared with the control group, miR-658 and miR-4739 significantly inhibited the activity of pmirGLO-BC069792 luciferase (Fig. 5c), but not miR-632. Finally, the mimics of miR-658 and miR-4739 and pmirGLO-KCNQ4 3'UTR plasmids were co-transfected into MDA-MB-231 and MDA-MB-468 cells, and the fluorescence in the cells was detected by a microplate reader 48 hours later. Both miR-658 and miR-4739 were found to reduce pmirGLO-KCNQ4 3'UTR dual-luciferase activity (Fig. 5d). To demonstrate the specificity of miR-658 or miR-4739 binding to BC069792 and KCNQ4, we performed the above experiments again after mutating the binding sites. The results showed that miR-658 and miR-4739 were no longer able to reduce the luciferase activity of pmirGLO-BC069792 and pmirGLO-KCNQ4 3'UTR after the mutation of the binding sites (Fig. 5e-f). The above results indicated that miR-658 and miR-4739 could specifically bind to the 3'UTR of BC069792 and KCNQ4. When BC069792 and KCNQ4 3'UTR mutations deleted the miR-658 and miR-4739 binding sites, they could not bind to miR-658 and miR-4739. These results indicated that BC069792 adsorbed miR-658 and miR-4739 as a sponge, and relieved its inhibition on the target gene KCNQ4, thereby up-regulating the expression of KCNQ4.
Bc069792 Upregulates Kcnq4 And Thus Inhibits Jak2 And Akt Phosphorylation To Suppress Breast Cancer Progression
According to our RNA-Seq data, BC069792 on cholinergic synapses (hsa04725) increased the expression of KCNQ4 (control group 33.37, experimental group 67.12, P = 0.011), PI3KR3 (control group 48.19, experiment group 28.25, P = 0.015), and AKT (control group 930.34, experiment group 711.99, P = 0.0003). It is well known that PI3K/AKT is a classical tumor signaling transduction pathway that plays a crucial role in tumor proliferation, invasion and metastasis. An increasing number of studies have reported that the ion channel function is closely related to the growth of tumors [6, 7]. The human ether-a-go-go-related gene1 (hERG1) potassium channel (aka KV11.1) regulates p-AKT through the formation of hERG1 integrin and phosphatidylinositol-3 kinase p85 subunit macromolecular complex on the plasma membrane, leading to AKT activation, thus promoting the development of human colorectal cancer cells [8, 9]. KCNQ4 is closely related to the non-receptor tyrosine protein kinase JAK 2, which reduces the activity of KCNQ4 in Xenopus oocytes [10], while PI3K/AKT is up-regulated by JAK2 [11]. As reported in literature, hsa04725 pathway was negatively correlated with breast cancer recurrence rate [12]. Our results showed that the mRNA expression of KCNQ4 was positively correlated with BC069792 and negatively correlated with JAK2, and the protein expression of BC069792 was positively correlated with KCNQ4 and negatively correlated with JAK2 (Fig. 6a, b). In MDA-MB-231 cell lines, overexpression of BC069792 significantly decreased the expression of p-AKT (Fig. 6c). The protein expression of KCNQ4 in the same group of samples was significantly higher than that of p-AKT (Fig. 6d). Combined with the results of KCNQ4 and p-AKT Western Blot detection in the BC069792 overexpression group and the control group, the spearman correlation test was performed, and it was found that KCNQ4 was negatively correlated with p-AKT (Fig. 6e). Compared with the LV-NC group, the expression of p-AKT was significantly decreased in the LV-BC069792 group (Fig. 6f). According to this, BC069792 inhibited the expression of JAK2 protein and the phosphorylation of AKT protein by up-regulating KCNQ4, decreased the expression of p-AKT, and then inhibited the progression of breast cancer.
To further verify whether BC069792 exerts biological functions through competitive adsorption of hsa-miR-658 and hsa-miR-4739, we performed rescue experiments. The results showed that breast cancer cells overexpressed BC069792 with reduced proliferation ability, weakened migration ability, and increased KCNQ4 protein expression level. When BC069792 was simultaneously overexpressed with hsa-miR-658 or hsa-miR-4739, the inhibition of breast cancer cell proliferation and migration by BC069792 was reversed (Fig. 7a, b), the elevated KCNQ4 protein expression level was decreased (Fig. 7c), and the level of reduced p-AKT protein was increased (Fig. 7d). Spearman's correlation test showed that the negative correlation of KCNQ4 with p-AKT was restored (Fig. 7e). Based on the above experimental results, it is suggested that BC069792 exerts its biological functions by binding to miRNA, and can act as a molecular sponge to adsorb hsa-miR-658 or hsa-miR-4739, up-regulate the protein expression of the target gene KCNQ4, inhibit the activity of p-AKT, and then play a role in inhibiting breast cancer.