HOTAIR/miR-203/CAV1 Crosstalk Influences Proliferation, Migration, and Invasion in the Breast Cancer Cell

Abstract

In recent years, malignant breast cancer metastasis has caused a great increase in mortality. Research on the genetic and molecular mechanisms of malignant breast cancer has continued to deepen, and targeted therapy has become the general trend. Among them, competing endogenous RNA (ceRNA)-related molecules have received much attention. Homeobox transcript antisense RNA (HOTAIR) has been reported to function extensively as a ceRNA in breast cancer. Notably, miR-203 and Caveolin 1 (CAV1) have also been found to play a role in breast cancer. However, the relationship between the three remains unclear. In this study, we present a new mechanic through bioinformatics tool and basic experiments: the HOTAIR/miR-203/CAV1 axis, which complemented the role network of HOTAIR as a ceRNA, thus, it will provide a novel potential idea for breast cancer research and therapy.

Keywords: CAV1; HOTAIR; breast cancer; ceRNA; invasion; miR-203; migration.

Figure 1

HOTAIR is highly expressed in breast cancer. Data analysis from GEPIA and UALCAN. (A) Among various cancers, HOTAIR was significantly overexpressed in breast cancer. (B) HOTAIR was significantly overexpressed in luminal breast cancer, HER2-positive breast cancer, and TNBC. (C) Compared with normal tissues, HOTAIR was significantly overexpressed in breast invasive carcinoma. (D) Breast cancer patients with high expression of HOTAIR might have a poorer prognosis. BRCA, breast invasive carcinoma; LUSC, lung squamous cell carcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; TGCT, testicular germ cell tumors; TNBC, triple-negative breast cancer; UCEC, uterine corpus endometrial carcinoma; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; TNBC, triple-negative breast cancer. Values were significantly different compared with the corresponding control value at * p < 0.05.

Figure 2

Up-regulation of HOTAIR promotes the proliferation, invasion, and migration of MDA-MB-231 cells. (A) RT-qPCR results showed that the expression of HOTAIR was higher in MDA-MB-231 than in MCF-7. (B) CCK-8 results showed that the proliferation ability of MDA-MB-231 cells in the siHOTAIR group decreased after 48 h and 72 h compared to the siCON group. Compared with the oeCON group, the proliferation ability of MDA-MB-231 cells in the oeHOTAIR group was increased. (C) Transwell assay showed that after 48 h, the invasion and migration number of MDA-MB-231 cells in the HOTAIR overexpression group was significantly higher, while that in the siHOTAIR group was lower than that in the NC group. (D) The results of the wound healing assay showed that compared with the NC group, the migration ability of MDA-MB-231 cells in the siHOTAIR group was significantly decreased, and the migration ability of the cells in the overexpression HOTAIR group was significantly higher than that in the NC group. Values were significantly different compared with the corresponding control value at * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

miR-203 functions in cancer, and HOTAIR acts as a molecular sponge for miR-203. (A) Data of possible HOTAIR-bound miRNAs were derived by comprehensive analyses of ENCORI and DAVID. The HOTAIR-miRNA network map was made by Cytoscape3.0 software. (B) Compared with normal tissues, miR-203 was overexpressed in breast invasive carcinoma through the UALCAN analysis of the TCGA database. (C) miR-203 was significantly overexpressed in luminal breast cancer, HER2-positive breast cancer, and TNBC. (D) Breast cancer patients with high expression of miR-203 might have a poorer prognosis. (E) Putative complementary sites between HOTAIR and miR-203. Mutations were generated in the HOTAIR nucleotides complementary to miR-203. (F) Fluorescence expression was detected by the dual-luciferase reporter gene. Compared with the NC group, the luciferase activity of the miR-203 mimics/HOTAIR-wt group decreased, while the luciferase activity of the miR-203 mimics/HOTAIR-mu group did not change significantly (p > 0.05). Values were significantly different compared with the corresponding control value at *** p < 0.001. ns > 0.05. K means 1000. IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; TNBC, triple-negative breast cancer.

Figure 4

HOTAIR reverses the effect of miR-203 on MDA-MB-231 cells in proliferation, invasion, and migration. (A) RT-qPCR results showed that compared with the NC group, the expression level of miR-203 was significantly increased after down-regulation of HOTAIR, while the expression level of miR-203 was extremely significantly decreased after up-regulation of HOTAIR. (B) CCK-8 results showed that after 48 h and 72 h, compared with the NC group, the cell proliferation rate in the miR-203 inhibitor group was significantly higher than that in the miR-203 mimics group; compared with the miR-203 inhibitor group, the rate of cell proliferation decreased in the inhibitor + siHOTAIR group. (C) The transwell invasion and migration assay results showed that, compared with the NC group, the number of cell invasions and migrations in the miR-203 inhibitor group was significantly increased. In the inhibitor + siHOTAIR group, the number of cell invasions and migrations significantly decreased. (D) The results of the wound healing assay showed that the migration area in miR-203 inhibitor cells was significantly increased, while in the inhibitor + siHOTAIR group, the migration ability of cells was decreased. Values were significantly different compared with the corresponding control value at * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

miR-203 binds to CAV1 and inhibits the expression of CAV1. (A) Gene Ontology (GO-Biological Process) for miR-203 involved in gene silencing (GO: 0035195). (B) PPI network diagram between miR-203 target gene-encoded proteins and CAV1-encoded proteins derived from STRING. (C) Western blot detection showed that the expression level of caveolin-1 was significantly increased in the miR-203 down-regulated group. (D) Putative complementary sites between miR-203 and CAV1. Mutations were generated in the CAV1 nucleotides complementary to miR-203. (E) Dual-luciferase reporter gene detection of the interaction between miR-203 and CAV1. Compared with NC, the expression of luciferase in mimics + CAV1-wt decreased, and the expression of luciferase in mimics + CAV1-mu was unchanged. Values were significantly different compared with the corresponding control value at * p < 0.05, *** p < 0.001, and **** p < 0.0001. ns > 0.05.

Figure 6

miR-203 inhibits the effect of CAV1 on MDA-MB-231 cells in proliferation, invasion, and migration. (A) CCK-8 results showed that the down-regulation of CAV1 significantly reduced the cell proliferation rate after 48 h and 72 h. Compared with the oeCAV1 group, the proliferation rate of the cells in the co-transfected miR-203 (oeCAV1 + mimics) group decreased. (B) The transwell invasion and migration assay results showed that the number of MDA-MB-231 cells invading and migrating in the oeCAV1 group increased. The number of cell invasions and migrations in the oeCAV1 + mimics group was lower than that in the oeCAV1 group. (C) The wound healing assay showed that the migration area of cells in the oeCAV1 group increased. The migration area of cells in the oeCAV1 + mimics group was smaller than that in the oeCAV1 group. Values were significantly different compared with the corresponding control value at * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 7

HOTAIR/miR-203/CAV1 crosstalk influences proliferation, migration, and invasion in breast cancer cells. In breast cancer cells, the highly expressed HOTAIR might act as a ceRNA to bind with miR-203, which increases the expression of CAV1, thereby promoting the proliferation, invasion, and migration of breast cancer cells. The figure was constructed with BioRender (https://biorender.com/, accessed on 11 May 2022).