LncRNA HAND2-AS1 suppressed the growth of triple negative breast cancer via reducing secretion of MSCs derived exosomal miR-106a-5p

Background: Triple-negative breast cancer (TNBC) is a special type of breast cancer, its tumor cell metastasis rate is much higher than other types, and at the same time has a high rate of postoperative recurrence, which significantly threatens the health of women. Thus, it is urgent to explore a new treatment for TNBC. Results: MiR-106a-5p was up-regulated in TNBC tissues and cells, and was positively correlated with the tumor grade, which indicated poor prognosis in TNBC patients. Mesenchymal stem cells (MSCs) can transport miR-106a-5p into TNBC cells via exosomes. Functional analysis showed exo-miR-106a-5p secreted by MSCs promoted tumor progression in TNBC cells. Furthermore, lncRNA HAND2-AS1 inhibited miR-106a-5p levels, and HAND2-AS1 was decreased in TNBC tissues and cells. Besides, overexpression of HAND2-AS1 reduced the secretion of exo-miR-106a-5p secretion from MSCs, thus suppressed TNBC development. Conclusion: Our study revealed that HAND2-AS1 inhibited the growth of TNBC, which were mediated by the inhibitory effects of MSC-derived exosomal miR-106a-5p.


INTRODUCTION
Triple negative breast cancer (TNBC) refers to breast cancer with negative expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 [1]. TNBC is characterized by a poor prognosis, high recurrence and metastasis rate, and high mortality, and has become the focus of breast cancer research in recent years [2]. Because TNBC patients lack the expression of 3 receptors mentioned above, they cannot benefit from endocrine therapy and targeted therapy against human epidermal growth factor receptor 2 [3]. Thus, it is urgent to seek a new treatment for TNBC.
Exosome is a kind of cystic vesicle with a double-layer membrane structure, with a diameter of 30~100 nm. And exosomes originate from the late intracellular body of the endocytosis system [4]. It was first discovered by PAN et al. [5] in the study of extracellular cytoplasmic fusion of reticulocyte vesicles. Exosomes contain a variety of bioactive substances such as proteins, cytokines, transcription factor receptors, mRNAs, miRNAs and lncRNAs [6]. Exosomes promote intercellular communication by transporting these small molecules and become essential participants in intercellular connection under normal physiological and pathological conditions. Moreover, cells can produce different exosomes containing different genetic information under various physiological and pathological conditions [7]. Although the mechanism by which this genetic information is sorted into exosome contents is still not fully understood, it is inevitable that specific gene sequences are involved in sorting and regulating the positioning of miRNA molecules in exosomes [8]. At the same time, it can be determined that exosomes can transport these components to specific receptor cells and play particular functions, including participating in the occurrence and development of tumors [9]. AGING Mesenchymal stem cells (MSCs) are pluripotent stem cells that can be induced to differentiate into many types of tissue cells [10]. Studies have shown that MSCs can be induced to differentiate into specific cells, such as cardiomyocytes and adipocytes. Since MSCs have the ability of immune regulation and migration [11], many studies have pointed out the role of MSCs in tumor development. MSCs used as carriers for targeted treatment of tumor cells can reach the tumor or inflammation and produce therapeutic factors to play a role in tumor suppression [12], which provides a potential new research has found that in recent years, MSCs role in regulating tumor can also be mediated by paracrine secrete outside body. It has showed that MSCs can secreted exosomes containing microRNA-144, which then inhibited the growth of tumor cells in non-small cell lung cancer [13].
MiRNA is a naturally non-coding RNA a length of about 22 nt [14]. Despite the presence of high concentrations of extracellular RNA enzymes, extracellular miRNAs have high concentrations and structural integrity in body fluids, suggesting that extracellular miRNAs may be encapsulated in a particular structure to prevent digestion by enzymes [15]. Exosomes can transport miRNAs to specific cells for specific functions, including regulating the growth of tumor cells. Studies have found that melanoma cells release small molecules including miR-214-3p and miR-199a-3p into tumor microenvironment for information communication and regulation of the development of melanoma through exosome encapsulation [16]. miR-106a-5p is a conserved miRNA involved in a variety of cancer processes [17]. And miR-106a-5p promoted ovarian cancer progression by targeting ARHGAP24, while lncRNA HOTAIRM1 sponged miR-106a-5p and inhibited ovarian cancer development [18]. However, whether miR-106a-5p can be packaged into exosomes derived from MSCs and participated in the progression of TNBC remains unclear.
Present study aimed to reveal the role of exosomal miR-106a-5p in triple TNBC process, and further investigating the underlying mechanisms.

Elevation of miR-106a-5p indicated a poor prognosis in TNBC tissues and cells
We first performed bioinformatic analysis, and the data showed the differentially expressed lncRNAs in normal and cancer tissues ( Figure 1A). Then, we collected cancerous and adjacent normal tissues from 20 TNBC patients, the characteristics of patients were shown in Table 1. And qRT-PCR analysis showed that miR-106a-5p was up-regulated in cancer tissues ( Figure 1B). We also found that miR-106a-5p was increased in TNBC cell lines than that in normal breast cell MCF10A ( Figure 1C). According to the median level of miR-106a-5p in Figure 1A, 20 TNBC patients was divided into low (n = 10) and high expression group (n = 10). Kaplan-Meier curves indicated 5-year survival rate of TNBC patients was significantly higher in low expression patients than high expression patients ( Figure 1D). Furthermore, we collected TNBC tissues from different grades (grade 0 to grade IV, n = 6) of TNBC, and found a positive correlation between miR-106a-5p level and tumor grade ( Figure 1E).

Overexpression of HAND2-AS1 suppressed TNBC development by inhibiting exo-miR-106a-5p secretion from MSCs
To confirm the effects of HAND2-AS1 on TNBC progression, we forced or silenced HAND2-AS1 The expression of miR-106a-5p in TNBC tissues from patients with tumor grade 0 to grade IV (n = 6) was measured by qRT-PCR (*p<0.05 vs grade 0). The above measurement data were expressed as mean ± standard deviation. Data among multiple groups were analyzed by one-way ANOVA, followed by a Tukey post hoc test. The experiment was repeated in triplicate.  in response to miR-106a-5p mimic/ AMO-miR-106a-5p transfection, and miR-106a-5p expression was determined by qRT-PCR. n = 6, *p<0.05. The above measurement data were expressed as mean ± standard deviation. Data among multiple groups were analyzed by one-way ANOVA, followed by a Tukey post hoc test. The experiment was repeated in triplicate.

AGING
AGING expression in BT549 cells ( Figure 5A). Then, we performed functional analysis and found that overexpression of HAND2-AS1 suppressed cell viability, migration, invasion and proliferation, while deletion of HAND2-AS1 showed a opposite effects ( Figure 5B, 5E).

DISCUSSION
TNBC is a special type of breast cancer, its tumor cell metastasis rate is much higher than other types, and at the same time has a high rate of postoperative recurrence, which greatly threatens the health of women [19].
Although researchers continue to explore the treatment of TNBC and related drugs, so far there is no effective targeted therapy [20]. Thus, many researchers began to actively seek therapeutic targets at the molecular level.
At present, the extracellular miRNAs have been detected in various physiological and pathological conditions. A large number of miRNAs can be released simultaneously and regulate multiple targets, thereby activating a complex network of transduction [21]. These miRNAs are secreted from exosomes into the microenvironment, and after binding with the corresponding receptor cells, they act on the target mRNA to change the protein expression and perform biological functions [22]. Meanwhile, certain expression patterns have been found in a variety of tumor patients, making them a new class of tumor biomarkers and therapeutic targets in recent years. Rabinowits et al. [23] used exosomes as genetic AGING material for the first time, and analyzed the differences between exosomal miRNAs in normal human serum and miRNAs in NSCLC patients' serum.
As well, there are many specific miRNAs in exosomes of patients with triple negative and Her2 positive breast cancer [24]. In present study, we found that miR-106a-5p was increased in TNBC tissues and cells, and the expression of miR-106a-5p was positively correlated with the tumor grade, which indicated a poor prognosis in TNBC patients. This finding was similar with previous researches targeting on miR-106a-5p and tumor, which also showed an elevation of miR-106a-5p in gastric cancer [25] and hepatocellular carcinoma [26].
When a tumor occurs in the body, MSCs can immediately sense the call of abnormal signals and gather in the tumor microenvironment [27]. Exosomes secreted by MSCs can carry a large amount of proteins and genetic information, and regulate the growth of tumor cells by transferring the therapeutic genes to the target cells [28]. Under the comprehensive influence of multiple factors, the regulation effect of MSC-derived exosomes on tumor is different, mainly presenting as promoting or inhibiting effect [29]. And in present study, we speculated that miR-106a-5p may be packaged into exosomes and secreted by MSCs. Thus, we isolated MSCs from bone marrow of TNBC patients, and further isolated exosomes from MSCs. Then TNBC cells were co-cultured with exosomes  F) The expression of miR-106a-5p and HAND2-AS1 in tumors were detected by qRT-PCR. n = 6, *p<0.05. The above measurement data were expressed as mean ± standard deviation. Data among multiple groups were analyzed by one-way ANOVA, followed by a Tukey post hoc test. The experiment was repeated in triplicate.
At present, numerous studies have shown that lncRNA participates in a variety of physiological and pathological processes through the ceRNA mechanism regulating miRNAs [30]. Herein, we predicted that a binding between miR-106a-5p and HAND2-AS1. And it has been reported that HAND2-AS1 was involved in the progression of breast cancer [31,32]. And our results showed HAND2-AS1 acted as a sponge of miR-106a-5p, and HAND2-AS1 was decreased in TNBC tissues and cells. In addition, overexpression of HAND2-AS1 inhibited the secretion of exo-miR-106a-5p secretion from MSCs, thus suppressed TNBC development both in vitro and in vivo. However, present study only focused HAND2-AS1 and exo-miR-106a-5p function in TNBC development, the underlying mechanism will be further explored in our future study. In addition, we will try to make a specific nanoparticle of HAND2-AS1 and to cue TNBC.
Exosomes, as transport carriers of small molecules in vivo, have their unique advantages over traditional transport methods. Firstly, exosomes are derived from endogenous cells and are a natural carrier with good immunogenicity. Exosomes rarely cause toxicity and immune responses when they are used in organism. Secondly, exosomes with a diameter of only 30 ~ 100 nm are an ideal way to transport miRNA without being phagocytized by macrophages. And exosomes can specifically transport anti-tumor or pro-tumor miRNA antisense sequences to the target sites so as to regulate protein expression. Therefore, as the transport carrier of miRNA, exosomes have a certain practical application prospect, which is conducive to the transformation of basic research of miRNA into clinical application.

CONCLUSIONS
In conclusion, our study revealed exo-miR-106a-5p secreted by MSCs promoted TNBC progression, which can be inhibited by lncRNA HAND2-AS1. And our study provided an attractive target in TNBC clinical treatment.

Clinical samples
Cancerous and normal tissues were taken from 20 TNBC patients January 2018 to January 2019 (Table 1). And TNBC tissues were collected from different grades: grade 0 to grade IV (n = 6)). All of the patients or their guardians provided written consent, and the Ethics Committee of Shanghai Eighth People Hospital approved all aspects of this study.

MSCs and exosome isolation and identification
MSCs were isolated from bone marrow of TNBC patients as previous description [13], which identified with flow cytometry. Several centrifugations were performed to purify exosomes in culture medium. Transmission electron microscopy (TEM) was used to identify exosomes structures. MSCs-derived exosomes were analyzed using exosome marker protein CD63, CD81, Tsg101 and Calnexin via Western blot. Exosomes size was calculated using Zetasizer Nano ZS.

qRT-PCR
Total RNA was isolated from serum and culture medium according to a standard protocol. And then, the purity and concentration of RNA was detected and all the samples were converted into cDNA using reverse transcription kit. We used SYBR Green (Thermo Fisher Scientific, USA) system to perform the qRT-PCR

Western blot
Protein samples were blotted depended on standard protocol. And we used Odyssey Infrared Scanning System (Gene Co. Ltd., Hongkong, China) to scan the membranes. At last, we used Image J software to analyze the western bolt results. The primary antibodies are as list: CD63 (CBL553), CD81 (SAB3500454), Tsg101 (SAB2109010) and Calnexin (SAB4503258) were purchased from Sigma-Aldrich. The secondary antibodies IRDye700/800 Mouse or Rabbit were produced by LICOR (Lincoln, NE, USA).

MTT assay
Cells were plated in 96-well plates and we used MTT assay to detect the cell viability. MTT (0.5 mg/mL; Beyotime Biotechnology, China) was added into cells and incubated for 4 h in incubator. We measured the absorbance of cell in 150 μL DMSO by Spectrophotometer (Tecan, Austria) at 450 nm.

Immunohistochemistry assay
Frozen sections of tumors were fixated in 4% paraformaldehyde and washed using PBS. We penetrated sections using 0.5% Triton X-100. After 3 times wash, we blocked sections with 50% goat serum. Then, sections were incubated with Ki67 antibody overnight. Then, we incubated the sections using secondary antibody. Immunofluorescence was analyzed under an IX73 fluorescence microscope (Olympus, Valley, PA, USA).

Wound healing assay
The cells were spread in a 6-well plate, and when they grew to 80%, horizontal lines were drawn in the cells with a ruler at an interval of 0.5cm, with 4 lines drawn in each well. The cells were washed with PBS for 3 times. Serum free culture medium was added and photographed at 0 hours. The cells were continuously cultured for 24 hours before the photo was taken.

Transwell assay
First, the Matrigel was spread over the Transwell, and the starved cells for 12 hours were inoculated into the upper chamber. The culture medium with serum was added to the lower chamber and cultured for 12 hours.
The Transwell was taken out, the culture medium was discarded. And Transwell was washed using PBS, and fixed with methanol for 30 minutes. After the chamber was dried, the cells were stained with crystal violet for 20min, and the upper cells were removed and washed with PBS for 3 times. The cells were photographed and counted under the microscope.

Clone formation assay
Cells were seeded into 6 well plates at a density of 100 cells/well and cultured for 14 days. Cells were fixed with 4% and stained with 0.1% Crystal Violet (Sigma-Aldrich, USA) at RT for 15min. Then cells were rinsed with distilled water, and the colonies were visualized by inverted microscope.

RNA-Binding Protein Immunoprecipitation (RIP)
We performed a RIP assay to determine the binding between HAND2-AS1 and miR-106a-5p using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) as previous study. Briefly, BT549 cells were transfected with biotinylated miR-106a-5p, and the expression of HAND2-AS1 was detected using qRT-PCR.

Animal experiment
BT549 and MDA-MB-231 cells were injected into nude mice (Guangdong provincial experimental animal center). And exosomes were isolated from MSCs transfected with miR-106a-5p or NC, then a dosage of 5 mg exosomes was administered into mice via tail vein injection once every 3 days for 2 weeks. 1 week later, lentivirus packaging HAND2-AS1 was injected into tumors. And tumor size was measured every 5 days. After 30 days of injection, mice were intraperitoneally injected with 3% pentobarbital sodium and were killed by excessive intraperitoneally anesthesia with a dose of 90 mL/kg, and the tumors were removed for follow-up study.

Statistical analysis
All data is presented as a mean ± S.E.M. Statistical analysis was performed using Student's t-test or Wilcoxon test or a one-way ANOVA through Graphpad Prism 7.0.

AUTHOR CONTRIBUTIONS
Li Xing and Xiaolong Tang designed and coordinated the study, Yi Yi and Kaikai Wu conducted the experiment. Xiong Huang prepared the manuscript. Jinliang Huan participated in data collection. All authors have read and approved the content of the manuscript.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest.

FUNDING
This study was supported by Community screening of breast cancer, No. SHXH201803.