LncRNA-BC069792 suppresses tumor progression by targeting KCNQ4 in breast cancer

Abstract

Background: Breast cancer is the most common malignant tumor that threatens women's health. Attention has been paid on the study of long- non-coding RNA (lncRNA) in breast cancer. However, the specific mechanism remains not clear.

Methods: In this study, we explored the role of lncRNA BC069792 in breast cancer. In vitro and in vivo functional experiments were carried out in cell culture and mouse models. High-throughput next-generation sequencing technology and real-time fluorescence quantitative PCR technology were used to evaluate differentially expressed genes and mRNA expression, Western blot and immunohistochemical staining were used to detect protein expression. RNA immunoprecipitation assay and dual-luciferase activity assay were used to evaluate the competing endogenous RNAs (ceRNA), and rescue and mutation experiments were used for verification.

Results: We found that lncRNA BC069792 was expressed at a low level in breast cancer tissues, and significantly decreased in breast cancer with high pathological grade, lymph node metastasis and high Ki-67 index groups. Moreover, BC069792 inhibited the proliferation, invasion and metastasis of breast cancer cells in vitro and in vivo. Mechanically, BC069792 acts as a molecular sponge to adsorb hsa-miR-658 and hsa-miR-4739, to up-regulate the protein expression of Potassium Voltage-Gated Channel Q4 (KCNQ4), inhibits the activities of JAK2 and p-AKT, and plays a role in inhibiting breast cancer growth.

Conclusions: LncRNA BC069792 plays the role of tumor suppressor gene in breast cancer and is a new diagnostic index and therapeutic target in breast cancer.

Keywords: Breast cancer; IncRNA BC069792; Invasion and metastasis; KCNQ4; Proliferation.

Fig. 1

BC069792 expression in breast cancer tissue. a The expression of LncRNA BC069792 in breast cancer tissue was significantly lower than that in adjacent normal breast tissue, the difference was statistically significant, P < 0.001. b The results of ROC curve analysis showed that BC069792 distinguished breast cancer tissue from normal breast tissue, the area under the curve was 0.9191, and the P value was < 0.0001. c In the MDA-MB-231 cell line, about 27.6% of BC069792 was expressed in the cytoplasm and 72.4% in the nucleus. In the MDA-MB-468 cell line, about 26.4% of BC069792 was expressed in the cytoplasm and 73.6% in the nucleus. d The results of RNA FISH assay showed that BC069792 was distributed in both nucleus and cytoplasm in MDA-MB-468 cell line. e Stability assay of BC069792 in MDA-MB-231 and MDA-MB-468 (P < 0.05, P < 0.01, P < 0.001,. P < 0.0001, ns P > 0.05)^**********

Fig. 2

BC069792 inhibited the proliferative capacity of breast cancer cells. a The growth curve of CCK-8 assay showed that overexpression of BC069792 significantly inhibited the proliferation of MDA-MB-231 (P = 0.035) and MDA-MB-468 (P = 0.021) cells. b The results of EdU experiment showed that overexpression of BC069792 significantly inhibited the proliferation of MDA-MB-231 (P = 0.047) and MDA-MB-468 (P = 0.049) cells. c Overexpression of BC069792 inhibited the migration of MDA-MB-231 (P = 0.011) and MDA-MB-468 (P = 0.008) cells. It also inhibited the invasive ability of MDA-MB-231 (P = 0.020) and MDA-MB-468 (P = 0.006) cells (P < 0.05, P < 0.01, P < 0.001,. P < 0.0001, ns represent P > 0.05)^**********

Fig. 3

BC069792 inhibits the proliferation and invasion of breast cancer cells in vivo. a In vivo imaging of nude mice showed that the volume of the subcutaneous tumor in the LV-BC069792 group was significantly smaller than that of the LV-NC group. One nude mouse in the LV-BC069792 group had no tumor. b The growth curve showed that the tumor growth rate of the LV-NC group was significantly higher than that of the LV-BC069792 group (P = 0.0081). c The removal of subcutaneous tumors showed that the tumor volume in the LV-BC069792 group was significantly smaller than that of the LV-NC group, and the tumor of a nude mouse in the LV-BC069792 group were suppressed. d The content of BC069792 in tumor tissue in LV-BC069792 group was significantly higher than that in LV-NC group (P = 0.0051). e The Ki-67 index of the LV-BC069792 group was about 25%, and the Ki-67 index of the LV-NC group was about 80% (IHC × 200). The Ki-67 index of the LV-BC069792 group was significantly lower than that of the LV-NC group (P = 0.0059). f In lung metastasis, the expression of BC069792 in LV-BC069792 group was significantly higher than that in LV-NC group (P = 0.0228). g The gross image of the lung metastases in nude mice showed that the tumor growth at the hilum and outside the lung of the nude mice in the LV-NC group was obvious. In LV-BC069792 group, there were no obvious tumors in the lungs of nude mice. HE staining results showed that the lung metastases in the LV-NC group were significantly increased and the volume was significantly increased. The scatter plot showed that the number of metastases in the overexpression BC069792 group was significantly reduced (P = 0.0267). h There was liver-diaphragm metastasis in the LV-NC group, but no cancer metastasis in the liver and diaphragm of the LV-BC069792 group. i The tumor cells in the LV-NC group invaded the skin adnexa, while the tumor cells in the LV-BC069792 group had a clear boundary with the surrounding adipose tissue and mammary tubules, and no obvious invasion was found. (P < 0.05, P < 0.01, P < 0.001,.P < 0.0001, ns represent P > 0.05)^**********

Fig. 4

KCNQ4 is a downstream target gene of BC069792. a RT-qPCR results showed that overexpression of BC069792 affected the expression of multiple genes in MDA-MB-231 cells, among which the increase of KCNQ4 was the most significant. b Western blot showed that the expression of KCNQ4 protein in the BC069792 overexpression group was significantly higher than that in the control group (MDA-MB-231: P = 0.0271, MDA-MB-468: P = 0.0088). c The mRNA expression of KCNQ4 in normal breast tissue was significantly higher than that in breast cancer tissue (P = 0.0145). d The protein expression of KCNQ4 in normal breast tissue was significantly higher than that in breast cancer tissue (P = 0.031). e and f In axillary tumors of mice, the expression of mRNAand protein of KCNQ4 in LV-BC069792 group was significantly higher than that in LV-NC group. g and h In lung metastasesof mice, the expression of mRNAand proteinof KCNQ4 in LV-BC069792 group was significantly higher than that in LV-NC group. i The content of KCNQ4 protein in LV-BC069792 tumor tissues of nude mice was significantly higher than that in LV-NC nude mice (P = 0.0051). j In human tumor tissue and nude mouse tumor tissue, BC069792 and KCNQ4 had a significant positive correlation. k BC069792 can promote the RNA stability of KCNQ4.(P < 0.05, P < 0.01, P < 0.001,.P < 0.0001, ns represent P > 0.05)^**********

Fig. 5

BC069792 upregulates the expression of KCNQ4 by binding to miR-658 and miR-4739. a Compared with the IgG control group, endogenous BC069792 and KCNQ4-3'UTR were specifically enriched by Ago2 antibody (BC069792: P = 0.017; FOS: P = 0.012; KCNQ4-3'UTR:P < 0.0001). b Through website prediction, database screening and literature query, three miRNAs were identified for further research. c After co-transfection of pmirGLO-BC069792 and mimics in MDA-MB-231 and MDA-MB-468 cell lines, miR-658 (MDA-MB-231: P = 0.021, MDA-MB-468: P = 0.037) and miR-658 -4739 (MDA-MB-231: P = 0.019; MDA-MB-468: P = 0.029) bound to pmirGLO-BC069792 and significantly inhibit luciferase activity. d After co-transfection of pmirGLO-KCNQ4 3'UTR and mimics, both miR-658 (MDA-MB- 231:P = 0.032, MDA-MB- 468:P = 0.017) and miR-4739(MDA-MB-231: P = 0.031; MDA-MB-468:P = 0.046) bound to pmirGLO-KCNQ4 3'UTR and reduced dual-luciferase activity. e There was no significant difference in luciferase activity between the experimental group and the control group after co-transfection of pmirGLO-BC069792-mut (miR-658 binding site mutation) and miR-658 mimics (MDA-MB-231: P = 0.253; MDA-MB-468: P = 0.152). After co-transfection of pmirGLO-BC069792-mut (miR-4739 binding site mutation) and miR-4739 mimics, there was no significant difference in luciferase activity between the experimental group and the control group (MDA-MB-231: P = 0.194; MDA- MB-468: P = 0.084). f There was no significant difference in luciferase activity between the experimental group and the control group after co-transfection of pmirGLO-KCNQ4-3'UTR-mut (miR-658 binding site mutation) and miR-658 mimics (MDA-MB-231: P = 0.160; MDA-MB-468: P = 0.699). After co-transfection of pmirGLO-KCNQ4-3'UTR-mut (miR-4739 binding site mutation) and miR-4739 mimics, there was no significant difference in luciferase activity between the experimental group and the control group (MDA-MB-231:P = 0.210; MDA-MB-468: P = 0.229).g-j In axillary tumors and lung metastases of mice, the expression of miR-658 and miR-4739 in LV-BC069792 group was lower than that in LV-NC group

Fig. 6

The downstream effector molecule regulated by KCNQ4 is JAK2/p-AKT. a After overexpression of BC069792, the mRNA expression of KCNQ4 was positively correlated with BC069792 and negatively correlated with JAK2. b After overexpression of BC069792, the protein expression of KCNQ4 was positively correlated with BC069792 and negatively correlated with JAK2. c In MDA-MB-231 (P = 0.0047) cell lines, overexpression of BC069792 reduced the expression of p-AKT protein. d In the BC069792 overexpression MDA-MB-231 cells, the protein content of KCNQ4 was significantly higher than that of p-AKT (P = 0.032), while the content of KCNQ4 and p-AKT in the pcDNA3.1 group was not statistically significant (P = 0.194). e In MDA-MB-231 cells, KCNQ4 was negatively correlated with p-AKT(r = -0.943, P = 0.017). f The expression of p-AKT protein in tumor tissue of nude mice was significantly different between LV-BC06972 group and LV-NC group, and it was highly expressed in LV-NC group(P = 0.001

Fig. 7

Rescue experiment. a Overexpression of BC069792 inhibits the proliferation of breast cancer cells (P < 0.0001), After simultaneously overexpressing miR-658 mimics (P = 0.0002) and miR-4739 mimics (P < 0.0001), the proliferation of breast cancer cells was enhanced. b Overexpression of BC069792 inhibited the migratory ability of breast cancer cells (P < 0.0001). breast cancer cells have enhanced migratory ability after transfection with miR-658mimics (P = 0.0009) or miR-4739mimics (P < 0.0001). c After overexpression of BC069792 and simultaneous transfection of miR-658 mimics (P = 0.005) or miR-4739 mimics (P = 0.026), the elevated KCNQ4 protein expression level was decreased. d The p-AKT protein level was elevated in rescue experiments. e The negative correlation of KCNQ4 with p-AKT was restored. (:P < 0.05, :P < 0.01, :P < 0.001,.:P < 0.0001, ns: P > 0.05)^**********

Fig. 8

The mechanism of lncRNA BC069792 to suppress tumor progression in breast cancer. Inc RNA BC069792 is expressed in both the cytoplasm and nucleus, which acts as a ceRNA sponge adsorbing miR-658 and miR-4739 and upregulates the transmembrane protein KCNQ4 expression, thereby inhibiting AKT phosphorylation and inhibiting the proliferation and metastasis of breast cancer