LncRNA MIR4435-2HG Promotes Proliferation, Migration, Invasion and EMT Via Targeting miR-22-3p/TMEM9B in Breast Cancer

Background: Breast cancer, as the malignancy with the highest incidence rate and mortality rate in women, seriously threatens human life and health. Pieces of evidence have suggested that long noncoding RNAs (lncRNAs) possess important effects on regulating the occurrence and development of breast cancer. Results: In the present study, MIR4435-2HG was highly expressed in breast cancer tissues and cells. Down-regulation of MIR4435-2HG inhibited the viability, proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of breast cancer cell lines by Cell Counting Kit-8 (CCK-8), colony formation, transwell migration and invasion, immunouorescence and western blot assays. Dual-luciferase reporter assay and RNA pull-down analysis conrmed that miR-22-3p was a target of MIR4435-2HG. Over-expression of MicroRNA-22-3p (miR-22-3p) obviously inhibited the viability, proliferation, migration, invasion and EMT of breast cancer cell lines. Transmembrane protein 9 domain family member B (TMEM9B) was up-regulated in breast cancer tissues and cell lines. Dual-luciferase reporter assay conrmed that TMEM9B was a target of miR-22-3p. TMEM9B inhibition partially restored the effects of MIR4435-2HG/miR-22-3p on the viability, proliferation, migration, invasion and EMT of breast cancer cell lines. Conclusions: MIR4435-2HG potentially played a tumor-promoting role in the occurrence and development of breast cancer, which might be achieved by regulating the miR-22-3p/TMEM9B axis. by acting as a ceRNA to down-regulate TMEM9B through competitively binding to miR-22-3p. These results indicated that MIR4435-2HG/miR-22-3p/TMEM9B axis might be a potential therapeutic basis for the treatment of breast cancer. The protein expression of α-SMA in MCF-7 and ZR-75-30 cells transfected with miR-22-3p mimic was assessed by immunouorescence assay. levels of proteins, including E-cadherin, vimentin and α-SMA, in MCF-7 and cells transfected with miR-22-3p mimic were evaluated by western blot assay. 0.01,


Introduction
Breast cancer is a kind of malignant tumor originated from breast epithelial tissues and is also the most common malignant disease in female patients [1]. Recent reports show that the incidence rate of breast cancer is increasing annually [2]. There are more than 1.3 million newly diagnosed cases of breast cancer in the world every year, and in China, breast cancer has accounted for 15% of new female tumors [3]. With the development of early detection, early diagnosis, early treatment, and prognosis monitoring of breast cancer, the overall mortality of breast cancer patients has decreased signi cantly [4]. However, malignant proliferation, recurrence, metastasis and other factors may compensate these bene ts. There are more than 45, 000 deaths of breast cancer patients in the world every year [5]. China accounts for 9.2% of the global deaths and is the greatest contributor [6]. Therefore, breast cancer is still a serious threat to women's life and health in China. Further understanding of the molecular mechanisms of the occurrence and development of breast cancer and nding the key molecules that can effectively inhibit the proliferation, invasion and metastasis of breast cancer cells will be conducive to the targeted treatment of clinical breast cancer [7,8].
Long non-coding RNAs (LncRNA) are a kind of non-coding RNAs with a length from 200 bp to 100 kbp.
LncRNAs account for 4%-9% of the transcripts in the mammalian genome and gained increasing interest [9]. With the rapid development of RNA sequencing, epigenome technology and computational prediction technology, an increasing number of lncRNAs have been found. Previous studies have shown that lncRNAs are abnormally expressed in breast cancer. Yang et al. identify more than 1300 abnormal lncRNAs in breast cancer by sequencing [10]. In addition, Shen et al. identify more than 1750 lncRNAs differentially expressed in triple-negative breast cancer [11]. These results suggest that abnormal expression of lncRNAs may play an important role in the carcinogenesis of breast cancer. Lv et al. nd that the expressions of lncRNAs in triple-negative breast cancer are different from those in non-triple negative breast cancer, which can be used as biomarkers for individual diagnosis, and may also be potential targets for evaluating individual therapeutic e cacy [12]. Moreover, lncRNA H19, SRA, lsinct5 and UCA1 promote the proliferation and/or metastasis of breast cancer. On the contrary, lncRNA GAS5 and XIST inhibit the proliferation and/or metastasis of breast cancer [13].
LncRNA MIR4435-2HG, known as AK001796 and LINC00978, is encoded on human chromosome 2q13 and has been regarded as a new oncogenic lncRNA in many types of cancer [14]. For example, a high level of MIR4435-2HG is detected in colorectal cancer tissues [15]. Increased level of MIR4435-2HG is signi cantly correlated with TNM stage and carcinoembryonic antigen level before treatment. High MIR4435-2HG expression has a poorer progression-free survival and overall survival (OS) rate [15]. In addition, MIR4435-2HG is up-regulated in hepatocellular carcinoma tissues, and the expression of MIR4435-2HG is signi cantly affected by tumor size instead of tumor metastasis [16]. Additionally, MIR4435-2HG is over-expressed in ovarian cancer tissues and cells. It is reported that MIR4435-2HG expression is negatively related to the health conditions of ovarian cancer patients. MIR4435-2HG knockdown inhibits proliferation, invasion and migration but induces apoptosis of ovarian cancer cells via the miR-128-3p/CDK14 axis [17]. However, the exact functions and corresponding mechanisms of MIR4435-2HG in the occurrence and development of breast cancer have been rarely reported [18][19][20]. Therefore, the present study was designed to determine whether MIR4435-2HG exerted its functional role in the growth and metastasis of breast cancer and investigate the possible mechanisms.

Clinical tissue collection
A total of 60 tissues, including 15 tissues in stage I + II and 15 tissues in stage III + IV, and 30 corresponding adjacent tissues were collected from A liated Hospital of Nantong University. All patients did not receive any chemotherapy and signed written informed consent. Moreover, the experimental procedures were approved by the Ethics Committee of A liated Hospital of Nantong University.

Cell Counting Kit-8 (CCK-8) assay
Transfected MCF-7 and ZR-75-30 cells at the density of 1 × 10 4 cells/well were maintained in 96-well plates and cultured for different time points (0, 24, 48 and 72 h, respectively). Then, cell viability was determined using a CCK-8 kit (Beyotime, Shanghai, China) according to the protocols of the manufacturer. The optical density was measured by the OD value at 450 nm by using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA).

Colony formation assay
Transfected MCF-7 and ZR-75-30 cells (1 × 10 3 cells/well) were seeded in six-well plates. The media was replaced with a fresh culture medium every 2-3 days for 2 weeks. Subsequently, MCF-7 and ZR-75-30 cells were stained with 10% crystal violet for 30 min and observed by using a microscope (Olympus, Tokyo, Japan).

Wound healing assay
Transfected MCF-7 and ZR-75-30 cells (5 × 10 5 cells/well) were seeded into a six-well plate. Once the cells were 100% con uent, a wound was made on the surface of the cell using a 200-µL tip, and a serum-free medium was used. After 0 and 48 h, MCF-7 and ZR-75-30 cells were observed under an inverted microscope (Tokyo, Japan), and the distance between the wounds was recorded.

Transwell migration and invasion assays
For migration and invasion assays, transfected MCF-7 and ZR-75-30 cells (1 × 10 6 cells/well) were seeded in the transwell chambers containing an 8 µm size porous membrane (Corning, NY, USA). The upper chamber was inserted without or with matrigel, while the lower chamber was added by 20% FBS.
After 48 h incubation at 37 o C, the non-migrating or invading cells in the upper chamber were removed by cotton wool. The migrated or invaded cells in the upper chamber were stained with 0.1% crystal violet solution (Sangon Biotech, Shanghai, China) and counted using a microscope (Olympus, Tokyo, Japan).
2.8 RNA extraction and qRT-PCR analysis RNA from clinical tissues and cell lines was extracted according to the instructions of the TRIzol reagent, and the OD 260 /OD 280 of the RNA solution was also detected. The reverse transcription reaction was conducted according to the instructions of the cDNA synthesis kit, and the PCR reaction solution was con gured according to the instructions of the Taq enzyme mixture. The ampli cation program was set up for primer sequence as listed below, and a PCR ampli cation reaction was conducted. PCR reaction

Subcellular fractionation analysis
Cytoplasmic and nuclear fractions were extracted from MCF-7 and ZR-75-30 cells using the NE-PER Nuclear and Cytoplasmic Extraction reagent (Thermo Fisher Scienti c, Inc. USA). RNA from each fraction was measured by qRT-PCR. U6 was used as internal references for MIR4435-2HG in cytoplasmic and nuclear fractions, respectively.
2.12 RNA pull-down analysis MCF-7 and ZR-75-30 cells were incubated with biotin-labeled miR-22-3p-wild-type (WT) or -mutant (Mut) for 48 h and lysed in the speci c buffer. The lysate was incubated with magnetic beads and washed three times with precooled lysis buffer and salt buffer solution. Finally, the bound RNA was puri ed with TRIzol®. MIR4435-2HG or miR-22-3p enrichment was analyzed using qRT-PCR.

Statistical analysis
GraphPad Prism 6.0 software was used for graphing. All data were expressed as mean ± standard deviation (SD). SPSS 17.0 software was used for statistical analysis. Data comparison between groups was performed by analysis of variance. A pairwise comparison between means was performed by t-test. P < 0.05 was considered statistically signi cant.

MIR4435-2HG is highly expressed in breast cancer tissues and cell lines
To investigate the possible role and underlying mechanisms of MIR4435-2H in breast cancer, the expression of MIR4435-2H in breast cancer tissues was rstly evaluated by qRT-PCR. The data of Fig. 1A showed that MIR4435-2H was signi cantly over-expressed in breast cancer tissues compared with nontumor tissues. In addition, the expression of MIR4435-2H in breast cancer patients at different TNM stages was also analyzed. The data of Fig. 1B revealed that MIR4435-2H expression was higher in patients at advanced stage III + IV than that in patients at stage I + II. Moreover, the clinical signi cance of aberrant MIR4435-2H expression in the prognosis of patients was further investigated. The results in Fig. 1C indicated that patients with low MIR4435-2H expression exhibited improved percent survival compared with patients with high MIR4435-2H expression. Furthermore, qRT-PCR was performed to detect the expression of MIR4435-2H in breast cancer cells. As expected, the data of Fig. 1D showed that compared with MCF-10A cells, the expression of MIR4435-2H was obviously higher in breast cancer cells, especially in MCF-7 and ZR-75-30 cells. These data suggested that MIR4435-2H was up-regulated in breast cancer tissues and cell lines and might function as an oncogene in the occurrence and development of breast cancer.
3.2 Down-regulation of MIR4435-2H inhibits the viability, proliferation, migration, invasion and EMT of breast cancer cells To determine the potential role of MIR4435-2H in breast cancer, MCF-7 and ZR-75-30 cells were transfected with sh-MIR4435-2H, and transfection e ciency was detected by the qRT-PCR assay. As shown in Fig. 2A, the expression of MIR4435-2H was obviously decreased in MCF-7 and ZR-75-30 cells transfected with sh-MIR4435-2H. Then, a CCK-8 assay was used to evaluate the effects of MIR4435-2H on the viability of MCF-7 and ZR-75-30 cells, and the data of Fig. 2B showed that down-regulation of MIR4435-2H signi cantly inhibited the viability of MCF-7 and ZR-75-30 cells in a time-dependent manner.
Besides, a colony formation assay was performed to assess the effects of MIR4435-2H on the proliferation of MCF-7 and ZR-75-30 cells. The data in Fig. 2C showed that MIR4435-2H inhibition signi cantly suppressed the proliferation of MCF-7 and ZR-75-30 cells. Moreover, the role of MIR4435-2H in metastasis of MCF-7 and ZR-75-30 cells was also investigated. The data of wound healing and transwell results showed that down-regulation of MIR4435-2H signi cantly inhibited migration and invasion of MCF-7 and ZR-75-30 cells displayed in Fig. 2D and 2E. Furthermore, the effects of MIR4435-2H on the EMT progress of MCF-7 and ZR-75-30 cells were explored. The data of immuno uorescence assay showed that sh-MIR4435-2H signi cantly inhibited α-SMA expression (Fig. 2F). Western blotting was performed to evaluate the effects of MIR4435-2H on the expression levels of EMT-related proteins, including E-cadherin, vimentin and α-SMA. The data of Fig. 2G revealed that sh-MIR4435-2H signi cantly decreased the protein expressions of Vimentin and α-SMA and increased the level of E-cadherin protein in MCF-7 and ZR-75-30 cells. These data suggested that down-regulation of MIR4435-2H inhibited the viability, proliferation, migration, invasion and EMT of breast cancer cells.

MIR4435-2H acts as a competing endogenous RNA (ceRNA) via sponging miR-22-3p
To determine the possible mechanisms of MIR4435-2H in breast cancer, the distribution of MIR4435-2H was initially investigated by subcellular fraction analysis. As shown in Fig. 3A, MIR4435-2H was primarily found in the cytoplasm. In addition, to verify whether MIR4435-2H acted as a ceRNA, bioinformatics tools were utilized to acquire miRNAs that potentially bound to MIR4435-2H. As indicated in Fig. 3B, miR-22-3p was predicted to possess the target domain for MIR4435-2H. Then miR-22-3p mimic and mimic NC were transfected into MCF-7 and ZR-75-30 cells, and qRT-PCR was performed to detect transfection e ciency (Fig. 3C). Dual-luciferase reporter analysis was performed to validate the interaction between MIR4435-2H and miR-22-3p. As expected, the data of Fig. 3D showed that exogenous expression of miR-22-3p signi cantly decreased the luciferase intensity of MCF-7 and ZR-75-30 cells transfected with MIR4435-2H-WT. Similarly, RNA pull-down data showed that MIR4435-2H was highly enriched in MCF-7 and ZR-75-30 cells treated with miR-22-3p-WT (Fig. 3E). Furthermore, the expression of miR-22-3p was downregulated in breast cancer tissues and cell lines (Fig. 3F and 3G) and was signi cantly up-regulated in MCF-7 and ZR-75-30 cells transfected with sh-MIR4435-2H (Fig. 3H). Finally, a negative association was also discovered between MIR4435-2H and miR-22-3p levels in breast cancer tissues (Fig. 3I). These data suggested that MIR4435-2H served as a ceRNA for miR-22-3p in breast cancer.  Fig. 4C and 4D. Additionally, a immuno uorescence assay was performed to evaluate the effects of miR-22-3p on the expression of α-SMA. The results showed that miR-22-3p mimic signi cantly inhibited α-SMA expression (Fig. 4E). Furthermore, western blotting was performed to evaluate the effects of miR-22-3p on the expression levels of EMT-related proteins. The data in Fig. 4F revealed that miR-22-3p mimic signi cantly decreased the protein expressions of Vimentin and α-SMA and increased the level of E-cadherin protein in MCF-7 and ZR-75-30 cells. These data suggested that up-regulation of miR-22-3p inhibited the viability, proliferation, migration, invasion and EMT of breast cancer cells.

TMEM9B acts as a direct target of miR-22-3p
To investigate the downstream targets of miR-22-3p, bioinformatics tools were utilized to acquire mRNAs potentially bound to miR-22-3p. As indicated in Fig. 5A, TMEM9B was predicted to possess the target domain for miR-22-3p. Dual-luciferase assay results further con rmed the target relationship between miR-22-3p and TMEM9B (Fig. 5B). In addition, qRT-PCR was performed to detect TMEM9B expression in breast cancer tissues and cell lines. As shown in Fig. 5C and 5D, TMEM9B was highly expressed in breast cancer tissues and cell lines. Furthermore, qRT-PCR and western blot assays were performed to evaluate the mRNA and protein levels of TMEM9B in MCF-7 and ZR-75-30 cells transfected with miR-22-3p mimic or NC mimic. The data in Fig. 5E and 5F revealed that the mRNA and protein expressions of TMEM9B were signi cantly decreased in MCF-7 and ZR-75-30 cells transfected with miR-22-3p mimic. Finally, a negative association was discovered between miR-22-3p and TMEM9B levels in breast cancer tissues (Fig. 5G). These data suggested that TMEM9B was a direct target of miR-22-3p and was negatively associated with miR-22-3p in breast cancer.

Discussion
A large number of studies have con rmed that abnormal expression of MIR4435-2HG plays an essential role in multiple tumors, including gastric cancer, prostate carcinoma, and oral squamous cell carcinoma [21][22][23][24]. In this manuscript, MIR4435-2HG was signi cantly up-regulated in breast cancer tissues and cell lines. In addition, breast cancer patients with low MIR4435-2HG expression exhibited improved percent survival compared with those with high MIR4435-2HG expression, which is consistent with previous studies [15][16][17], suggesting that MIR4435-2HG may act as an oncogene in the occurrence and progression of breast cancer. Functionally, our data showed that down-regulation of MIR4435-2HG notably inhibited the viability, proliferation, migration, invasion and EMT progress of breast cancer cells. Similarly, MIR4435-2HG exhibited its role in promoting the proliferation and metastasis of colorectal cancer and ovarian cancer [25][26][27]. These data suggested that MIR4435-2HG might act as an oncogene in the occurrence and progression of breast cancer.

LncRNAs affect the occurrence and development of tumors via post-transcriptional regulation [28].
Multiple lncRNAs may regulate gene expression, thus reducing the number of available miRNAs in cells [29]. Therefore, lncRNAs act as competing ceRNAs to modulate the expression of the target gene via sponging miRNAs [30]. MIR4435-2HG has been reported to exert ceRNA function in osteoarthritis, glioma, hepatocellular carcinoma and lung cancer [31,32]. In this manuscript, bioinformatic analysis of MIR4435-2HG-miRNA prediction was performed through online software, and the target relationship was veri ed by a dual-luciferase reporter and pull-down assays. All the data indicated that miR-22-3p was a direct target of MIR4435-2HG. Previous studies have illustrated that miR-22-3p exerted essential effects on the development of many tumors, including bladder cancer, gastric cancer, and lung cancer [33][34][35][36]. The data showed that the level of miR-22-3p was dramatically reduced in breast cancer tissues and cell lines, which was inversely correlated with MIR4435-2HG expression in breast cancer tissues. Moreover, up-regulation of miR-22-3p inhibited the viability, proliferation, migration, invasion and EMT progress of breast cancer cells. These results indicated that miR-22-3p acted as a tumor suppressor in breast cancer.
MiRNAs act as tumor suppressors through the restraint of their target genes to participate in breast cancer progression [37]. In this study, TMEM9B was con rmed as a target of miR-22-3p containing the putative miRNA response sequences within its 3'-UTR by starBase v2.0. Tmem9B, as a glycosylated protein located on the lysosomal membrane, has been previously discovered as an NF-κB inducer in large-scale cDNA over-expression screens [38]. Furthermore, TMEM9B is a key component of in ammatory signaling pathways [39]. A remarkable negative correlation between the expression levels of TMEM9B and miR-22-3p in breast cancer tissues was found in this study. TMEM9B was a direct target of miR-22-3p in breast cancer cells, which indicated that the ceRNA system existed among MIR4435-2HG, miR-22-3p and TMEM9B in breast cancer. Next, whether MIR4435-2HG/miR-22-3p/TMEM9B axis conduced to the progression of breast cancer in vitro was further explored. The relationship between MIR4435-2HG/miR-22-3P/TMEM9B axis and the progression of breast cancer was investigated in vitro. The data presented that the effects of MIR4435-2HG knockdown reduced TMEM9B expression level by the promotion of miR-22-3p. TMEM9B inhibition partly restored the effects of the miR-22-3p inhibitor on the viability, proliferation, migration, invasion and EMT progression of breast cancer cells transfected with sh-MIR4435-2HG.
To conclude, MIR4435-2HG was regarded as a vital mediator of cell growth and metastasis in breast cancer. MIR4435-2HG was obviously up-regulated in breast cancer tissues and cell lines, and MIR4435-2HG knockdown inhibited breast cancer progression by acting as a ceRNA to down-regulate TMEM9B through competitively binding to miR-22-3p. These results indicated that MIR4435-2HG/miR-22-3p/TMEM9B axis might be a potential therapeutic basis for the treatment of breast cancer.

Declarations Con icts of interest
The authors state that there are no con icts of interest.

Ethics approval and consent to participate
The analysis was performed according to the principles of Helsinki Declaration. Written informed consent was obtained from all patients. The experimental procedures were approved by the Ethics Committee of A liated Hospital of Nantong University.

Consent for publication
The authors give consent to the publication in the journal.