MIR22HG inhibits breast cancer progression by stabilizing LATS2 tumor suppressor

The long noncoding RNA called MIR22 host gene (MIR22HG) was previously identified as a tumor suppressor in several cancers. However, the biological function of MIR22HG in breast cancer remains unknown. In this study, we aimed to determine the function and molecular mechanism of MIR22HG in breast cancer progression using transcriptomics and biotechnological techniques. Our results showed that MIR22HG expression was lower in the cancerous tissues than in the paired adjacent normal breast tissues. Additionally, MIR22HG was found to be mainly located in the cytoplasm and acted as a miR-629-5p sponge. Notably, MIR22HG stabilized the expression of large tumor suppressor 2 (LATS2), which promoted the LATS2-dependent phosphorylation of YAP1 and suppressed the expression of its downstream target oncogenes, thereby inhibiting the proliferation and migration of breast cancer cells. Therefore, our findings reveal the MIR22HG-dependent inhibition of breast cancer cell proliferation and migration via the miR-629-5p/LATS2 pathway, providing new insights and identifying novel therapeutic targets for breast cancer treatment.


INTRODUCTION
As the most commonly diagnosed cancer worldwide, female breast cancer had an estimated incidence rate of 11.7% in 2020 and was known to greatly influence women's health [1]. Previous studies have successfully identified several biomarkers and therapeutic targets for breast cancer treatment. In addition, increasing evidences show that long noncoding RNAs (lncRNAs) play important roles in breast cancer progression [2].
LncRNAs are defined as noncoding RNA molecules with more than 200 nucleotides [3]. Several lncRNAs, such as MALAT1 [4], RAB11B-AS1 [5], TROJAN [6], and HOST2 [7], have been found to facilitate the proliferation and metastasis of breast cancer cells. MALAT1 binds to the transcription factor TEAD and inactivates the transcriptional activity of the YAP/TEAD complex, subsequently inhibiting the metastatic ability of breast cancer cells [4]. The HIF2induced RAB11B-AS1 recruits RNA polymerase II to upregulate VEGFA and ANGPTL4 expression, consequently promoting tumor angiogenesis and distant metastasis of breast cancer [5]. In triplenegative breast cancer, TROJAN interacts with metastasisrepressing factor ZMYND8, subsequently increasing ZMYND8 degradation via the ubiquitin-proteasome pathway by repelling ZNF592, and thus enhancing cancer progression [6]. In our previous research, HOST2 was revealed to act as a competitive endogenous RNA via decoying of let-7b, thereby promoting STAT3-mediated cell proliferation and migration in triple-negative breast cancer [7].
Notably, the lncRNA called MIR22 host gene (MIR22HG) has also been observed in several tumors. The tumor suppression function of MIR22HG has been described in hepatocellular carcinoma [8], colorectal cancer [9], and glioblastoma [10]. However, the function of MIR22HG in breast cancer remains unknown. Hence, this study aimed to determine the function and molecular mechanism of MIR22HG in breast cancer progression using transcriptomics and biotechnological techniques.

MIR22HG expression is downregulated in breast cancer tissues
To investigate MIR22HG expression in breast cancer tissues, we collected 36 pairs of cancerous tissues and adjacent normal breast tissues for real-time PCR analysis. Our results showed that in most paired tissues (26/36), MIR22HG expression was lower in cancerous tissues than in normal tissues (Fig. 1a). Similarly, the MIR22HG expression was lower in the studied breast cancer cell lines, namely MCF7, MDA-MB-231, BT549, and SKBR3, than in the normal breast epithelial cell line MCF10A (Fig. 1b). In addition, to determine the subcellular localization of MIR22HG in breast cancer cells, we separated the nuclear and cytoplasmic RNA of MDA-MB-231 cells. The real-time PCR results indicated that MIR22HG was mainly localized in the cytoplasm (Fig. 1c). Further analysis via fluorescence in situ hybridization also confirmed that MIR22HG was located in the cytoplasm (Fig. 1d).
Loss-of-function of MIR22HG promotes breast cancer cell proliferation and migration in vitro To determine the biological function of MIR22HG in breast cancer cells, we transfected MDA-MB-231 and BT549 cells with MIR22HG-siRNAs. Considering the off-target effect of siRNAs, the MIR22HG-si1 and MIR22HG-si2 were used. The knockdown efficiency of MIR22HG-si1 and MIR22HG-si2 were detected by real-time PCR (Fig. 2a). After MIR22HG knockdown, the changes in the cell proliferation ability were detected by MTT assays and colony formation assays, while the changes in the cell migration ability were analyzed by Transwell assays. Notably, our results showed that loss-of-function of MIR22HG enhanced the proliferation (Fig.  2b, c) and migration (Fig. 2d) abilities of breast cancer cells.
Gain-of-function of MIR22HG inhibits breast cancer cell proliferation and migration in vitro and in vivo In addition to MIR22HG knockdown, we transfected MDA-MB-231 and BT549 cells with MIR22HG plasmids to achieve overexpression. The overexpression efficiency was detected by real-time PCR (Fig.  3a). Results showed that MIR22HG overexpression suppressed the proliferation (Fig. 3b, c) and migration (Fig. 3d) of breast cancer cells. Furthermore, we constructed MDA-MB-231 cells that stably expressed MIR22HG and a subcutaneous xenograft model to explore the biological function of MIR22HG in vivo. Compared to wild-type MDA-MB-231 cells, the tumorigenicity of MDA-MB-231 cells with stable MIR22HG expression was suppressed (Fig. 3e). Collectively, our results suggest that gain-of-function of MIR22HG inhibited the proliferation of breast cancer cells both in vitro and in vivo.
MIR22HG functions as an miR-629-5p sponge in breast cancer cells Since MIR22HG was mainly located in the cytoplasm, we hypothesized that MIR22HG may function as a miRNA sponge in breast cancer cells. Hence, we searched the starBase database [11] (http://starbase.sysu.edu.cn/) and discovered that miR-629-5p was a potential target of MIR22HG. To validate the relationship between MIR22HG and miR-629-5p, we constructed the pmirGLO-MIR22HG-wild-type and pmirGLO-MIR22HG-mutant vectors for subsequent luciferase reporter assay (Fig. 4a). Our results indicated that miR-629-5p reduced the luciferase activity of the pmirGLO-MIR22HG-wild-type vector and failed to bind with the mutant MIR22HG (Fig. 4b). In contrast, the immunoprecipitation assay demonstrated that AGO2 was able to pull down both MIR22HG and miR-629-5p (Fig. 4c). Taken together, these results suggest that MIR22HG may act as a competing endogenous RNA molecule to suppress miR-629-5p. We then investigated the function of miR-629-5p in breast cancer cells. The MDA-MB-231 and BT549 cells were transfected with miR-629-5p mimics, and the overexpression efficiency of miR-629-5p mimics was detected by real-time PCR (Fig. 4d). We discovered that miR-629-5p overexpression promoted the proliferation and migration of both MDA-MB-231 and BT549 cells. Notably, results of the rescue experiment suggest that MIR22HG impaired the function of miR-629-5p in breast cancer cells (Fig. 4e, f). Furthermore, the colony formation assay and Transwell assay results of breast cancer cells co-transfected with miR-629-5p and wild-type/mutant MIR22HG revealed that only the wild-type MIR22HG impaired the function of miR-629-5p in breast cancer cells (Fig. 5a, b). Collectively, our results suggest that MIR22HG act as a miRNA sponge that can inhibit function of miR-629-5p in breast cancer cells.
potential downstream target of miR-629-5p (Fig. 6b). To confirm the relationship between miR-629-5p and LATS2, we constructed the pmirGLO-LATS2 3′-UTR-wild-type and pmirGLO-LATS2 3′-UTR-mutant vectors (Fig. 6c) and then performed luciferase reporter assay. Our results demonstrated that miR-629-5p can bind with LATS2 3′-UTR (Fig. 6d), which promoted the degradation of LATS2 mRNA (Fig. 6e). We also examined the changes in the expression of LATS2 protein via Western blotting and found that miR-629-5p inhibited LATS2 expression, while MIR22HG slightly counteracted the miR-629-5p-induced decrease in LATS2 expression. As a key member of the Hippo-YAP1 signaling pathway, LATS2 can function as an upstream kinase of the transcriptional cofactor YAP1, which facilitates YAP1 phosphorylation and inactivates the transcriptional activity of the YAP1/TEAD4 complex. Hence, we analyzed the changes in the expression of Hippo pathway signals-CTGF, phosphorylated YAP1, and total YAP1-following MIR22HG knockdown or overexpression. The results showed that MIR22HG promoted YAP1 phosphorylation and slightly counteracted the miR-629-5p-induced decrease in YAP1 phosphorylation, while no changes were observed in total YAP1 expression ( Fig. 6f-h). Finally, investigation of the biological function of LATS2 in breast cancer revealed that the siRNA-induced knockdown of LATS2 enhanced the proliferation and migration of breast cancer cells (Fig. 7a, b), indicating that LATS2 was also a tumor suppressor in breast cancer. Taken together, our results suggest that MIR22HG acts as a miR-629-5p sponge that can inhibit the proliferation and migration of breast cancer cells and stabilize LATS2 expression.  The MDA-MB-231 cells that stably expressed MIR22HG were subcutaneously injected into 6-week-old BALB/c nu/nu female mice. After 30 days, the tumors were collected, and the tumor volume and weight were measured. **P < 0.01, ***P < 0.001, ****P < 0.0001. All experiments were repeated thrice.

DISCUSSION
To determine the function of MIR22HG in breast cancer, we investigated 36 pairs of cancerous tissues and adjacent normal breast tissues via real-time PCR. The results showed that MIR22HG expression was lower in cancerous tissues than in normal tissues. The subcellular distribution of lncRNAs determines their biological function [15]. For example, BCRT1, which is mainly located in the cytoplasm, can competitively bind with miR-1303 [16]. In contrast, BX111, which is primarily located in the nucleus, binds with transcription factor YB1 to regulate the expression of its downstream target genes [17]. In the present study, we discovered that MIR22HG was mainly distributed in the cytoplasm, indicating that MIR22HG may act as a competitive endogenous RNA molecule. Subsequent bioinformatic analysis revealed that MIR22HG can bind with miR-629-5p. Furthermore, the luciferase reporter assay and AGO2 immunoprecipitation assay results confirmed that Fig. 4 MIR22HG functions as an miR-629-5p sponge in breast cancer cells. a Schematic diagram of the interaction between miR-629-5p and wild-type/mutant MIR22HG. b Dual-luciferase assays in HEK293T cells co-transfected with wild-type/mutant MIR22HG and miR-629-5p mimics (25, 50, and 100 nM) or NC. At 48 h after transfection, the 293 T cells were collected for the detection of firefly luciferase activity, with Renilla luciferase activity as internal control. c AGO2 immunoprecipitation assay was performed using HEK293T cells to detect the expression of miR-629-5p associated with AGO2. The AGO2-specific antibody was applied to precipitate the AGO2-RNA complex after MIR22HG overexpression in 293T cells. Total RNA was extracted, and real-time PCR was performed to determine the expression of miR-629-5p associated with AGO2, using IgG-specific antibody as negative control. d Overexpression efficiency of miR-629-5p was analyzed by real-time PCR after MDA-MB-231 and BT549 cells were transfected with miR-629-5p mimics or NC. e The effects of MIR22HG and miR-629-5p co-transfection on the proliferation of MDA-MB-231 and BT549 cells were evaluated by colony formation assay (18-day period). f The effects of MIR22HG and miR-629-5p cotransfection on the migration of MDA-MB-231 and BT549 cells were evaluated by Transwell assay (20-h period, 3 × 10 4 cells per well, 100× magnification). **P < 0.01, ***P < 0.001, ****P < 0.0001. All experiments were repeated thrice.
MIR22HG can act as a miRNA sponge and directly bind with miR-629-5p. Functional analysis via siRNA-induced knockdown of MIR22HG resulted in the enhanced proliferation and metastasis of breast cancer cells, while MIR22HG overexpression inhibited cancer cell proliferation and metastasis. In the Hippo-YAP1 signaling pathway, LATS2 plays a critical role in the phosphorylation and inactivation of the transcription cofactor YAP1/TAZ. The phosphorylated YAP1 is degraded in the cytoplasm by the ubiquitin-proteasome system, consequently inhibiting the expression of YAP1 target genes that are needed for cell proliferation and migration [18]. Taking these into consideration, our results suggest that LATS2 also functions as a tumor suppressor in breast cancer and that miR-629-5p can promote the proliferation and migration of breast cancer cells by targeting LATS2 mRNA. Additionally, MIR22HG can stabilize LATS2 expression by counteracting the miR-629-5p-induced degradation of LATS2 mRNA (Fig.  7c). Therefore, our research reveals the biological function and molecular mechanism of MIR22HG in breast cancer cells and provides novel insights and therapeutic targets for future studies on breast cancer treatment.

MATERIALS AND METHODS Tissue samples
The 36 pairs of cancerous tissues and adjacent normal breast tissues used in this study were collected from the Department of Breast and Thyroid Surgery, Shanghai Tenth People's Hospital (Shanghai, China). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013) and approved by the ethics board of Shanghai Tenth People's Hospital (No. 2020-KN174-01). All study participants were contacted by telephone to obtain verbal informed consent. After collection via surgery, the cancerous and normal tissues were cryopreserved in liquid nitrogen for temporary storage.

Cell culture
The human embryonic kidney cell line HEK293T, human breast cancer cell lines MCF7, MDA-MB-231, BT549, and SKBR3, and human normal breast epithelial cell line MCF10A were purchased from the Chinese Academy of Science, Shanghai (China). The MCF7, MDA-MB-231, and 293 T cells were cultured in DMEM medium with high glucose (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), while the BT549 and MCF10A cells were cultured in RPMI-1640 medium (Gibco, USA) and mammary epithelial cell medium (Procell, Wuhan, China), respectively. The growth media were added with 10% fetal bovine serum (Gibco, USA) and 100 U/mL penicillin and 100 μg/mL streptomycin (Servicebio, Wuhan, China). A 0.25% trypsin-EDTA solution (Servicebio, China) was also applied for cell passage cultivation. All cell lines were cultivated in a cell incubator at 37°C and 5% CO 2 .

RNA extraction and cDNA synthesis
Total RNA was extracted from MCF10A, MDA-MB-231, BT549, SKBR3, and 293 T cells and from cancerous tissues and paired adjacent normal breast tissues using TRIzol ® Reagent (Invitrogen, USA). For first strand cDNA synthesis, 1000 ng total RNA was reverse-transcribed using Hifair ® III 1st Strand cDNA Synthesis SuperMix for qPCR (Yeasen, Shanghai, China) and miRNA 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China), following the manufacturers' instructions. Fig. 7 LATS2 knockdown promoted the proliferation and migration of breast cancer cells. a Colony formation assay was used to determine the viability of MDA-MB-231 and BT549 cells after LATS2 knockdown. b Transwell assay was performed to evaluate the migration ability of MDA-MB-231 and BT549 cells after LATS2 knockdown (100× magnification). c A scheme of the proposed molecular mechanisms. MIR22HG stabilizes LATS2 by counteracting the miR-629-5p-induced degradation of LATS2 mRNA, consequently resulting in the promotion of LATS2dependent YAP1 phosphorylation and YAP1 inactivation. ***P < 0.001. All experiments were repeated thrice.

Subcellular fractionation
The nuclear and cytoplasmic separation in cells were performed using PARIS Kit (Ambion, Life Technologies, Grand Island, NY, USA), following the manufacturer's protocols.

Fluorescence in situ hybridization
The subcellular localization of MIR22HG in MDA-MB-231 cells was analyzed using Fluorescent In Situ Hybridization Kit (RiboBio, Guangzhou, China), according to the manufacturer's instructions. The Cy3-labeled MIR22HG probes were designed and synthesized by RiboBio (China). Representative images were obtained using a confocal microscope (Carl Zeiss Jena, Germany).

Real-time PCR
Real-time PCR was performed using Hieff UNICON ® qPCR SYBR Green MasterMix (Yeasen, China), with β-actin as internal control for normalizing MIR22HG and LATS2 expression and U6 as internal control for normalizing miR-629-5p expression. All primers were synthesized by Generay Biotechnology (China). The primers used for qPCR include the following: MIR22HG

MTT assay
The transfected MDA-MB-231 and BT549 cells were placed in 96-well plates at a density of 1 × 10 3 cells/well. Cell viability was assessed at 24, 48, 72, 96, and 120 h of incubation. A 200-μL complete medium was used for cell incubation, with 20 μL MTT (Yeasen, China) added to each well at set time points. After 6 h incubation at 37°C and 5% CO 2 , the supernatants were removed, and 150 μL DMSO (Sangon Biotech, Shanghai, China) was added to each well to dissolve formazan. The absorbance was quantified at a wavelength of 490 nm.

Colony formation assay
First, the transfected MDA-MB-231 and BT549 cells were placed in 6-well plates at a density of 5 × 10 2 cells/well. Second, the cells were incubated for 7-14 days at 37°C and 5% CO 2 , with the medium changed every 3 days. Third, the medium was removed after colonies formed, and cells were washed thrice with phosphate buffered saline (Servicebio, China) and then fixed with 95% ethanol for 10 min. Finally, the clones were stained with 0.1% crystal violet (Yeasen, China). Representative images were obtained using a camera.

Transwell assay
The transfected cells (3 × 10 4 ) were placed in the upper chamber of Transwell ® (Corning Incorporated, Corning, NY, USA) and incubated in serum-free medium, while medium supplemented with 10% fetal bovine serum was added to the lower chamber. After 18 h, the cells that moved to the lower chamber were fixed with 4% paraformaldehyde and stained with 0.05% crystal violet. Representative images were collected using a light microscope (Nikon, Japan).

Dual-luciferase reporter assay
The dual activity of firefly and Renilla luciferase were detected using Dual-Luciferase Reporter Gene Assay Kit (Promega, Madison, WI, USA), following the manufacturer's protocol.

Xenograft mouse model
The MDA-MB-231 cells (1 × 10 7 ) and transfected cells with stable MIR22HG expression were suspended in 150 μL DMEM (without fetal bovine serum and penicillin/streptomycin) and subcutaneously injected into 6-week-old BALB/c nu/nu female mice. Mice were divided into 2 groups randomly with 4 mice in each group, which were not blinded to investigators. After 30 days, the mice were euthanized, and the tumors were collected. The tumor volume and weight were subsequently measured. All animal experiments were approved by the Animal Care and Use Committee of the Shanghai Tenth People's Hospital (No. SHDSYY-2020-0600).

Statistical analysis
The data were presented as means ± standard deviation (SD) and analyzed using GraphPad Prism (version 9.0; GraphPad, San Diego, CA, USA). The variance was similar between the groups that are being statistically compared. Student's t test was used for two-group comparisons, with Pvalues < 0.05 considered as significantly different.

DATA AVAILABILITY
All data included in this study are available upon request by contact with the corresponding author.