Activation of FGD5-AS1 Promotes Progression of Cervical Cancer through Regulating BST2 to Inhibit Macrophage M1 Polarization

Accumulating evidence has elucidated the biological function of lncRNAs in various tumors. FGD5 antisense RNA 1 (FGD5-AS1) is identified as a significant tumor regulator in malignancies. Up to now, the detailed function of FGD5-AS1 in cervical cancer and its underlying molecular mechanisms remain uninvestigated. Bone marrow stromal cell antigen 2 (BST2) can play critical roles in immune response, and the roles of BST2 in cervical cancer was explored currently. The level of FGD5-AS1 and BST2 was detected by qRT-PCR in cervical cancer cells. FGD5-AS1 and BST2 expression was significantly upregulated in cervical cancer cells. Then, the decrease of FGD5-AS1 greatly repressed cervical cancer cell growth in vitro. In addition, FGD5-AS1 silencing repressed BST2 expression and suppressed M2 macrophage polarization. Mechanistically, we confirmed that FGD5-AS1 sponged miR-129-5p to reduce its inhibition on BST2. Furthermore, lack of BST2 depressed cervical cancer cell growth, while inducing apoptosis. Loss of BST2 induced M1 macrophage polarization while blocking M2 macrophage polarization. For another, we demonstrated that FGD5-AS1-triggered M2 macrophage polarization was remarkably reversed by miR-129-5p via suppressing BST2. In conclusion, FGD5-AS1 induced M2 macrophage polarization via sponging miR-129-5p and modulating BST2, thus contributing to cervical cancer development. Our findings revealed FGD5-AS1/miR-129-5p/BST2 as a new potential target for cervical cancer.


Introduction
Cervical cancer is the fourth frequent cancer among females, which can account for 7.5% of female cancer deaths worldwide [1]. It has been reported that human papillomavirus (HPV), early sexual behavior, sexual relations with several partners, and smoking can contribute to cervical cancer [2,3]. Up to now, radiotherapy, radical hysterectomy, and platinum-based chemotherapy are indicated as the standard primary treatment for cervical cancer [4]. Consequently, exploring the mechanism of cervical cancer and looking for suitable therapeutic targets might be helpful for treating cervical cancer.
Macrophages exhibit a central role in host defense [5]. In recent years, the importance of the immune microenvironment in cancers has gained much attention. Tumorassociated macrophages are an important component of the tumor microenvironment. Tumor-associated macrophages can differentiate into antitumor classical activation type (M1 type) or protumor alternative activation type (M2 type) [6]. Furthermore, an increased number of M2 tumor-associated macrophages have been highly correlated with tumor metastasis [7]. lncRNAs are transcripts with over 200 bp nucleotides with poor protein-coding potential [8,9]. Aberrant ncRNAs are involved in many biological processes, including DNA methylation, histone modification, and transcription [10,11]. lncRNAs can exert a critical role in various cancers [12]. Recently, abnormal lncRNAs are identified in cervical cancer development [13][14][15]. For example, lncRNA XLOC_006390 can induce cervical cancer through sponging miR-331-3p and miR-338-3p [16]. CASC11 contributes to cervical cancer via activating the Wnt pathway [17]. In addition, lncRNA SNHG7 induces cervical cancer cell growth [18].
BST2 is identified as CD317 [22]. It can play important roles in innate immune response [23]. In recent studies, BST2 is aberrantly expressed in cancers. For instance, BST2 promotes cell proliferation and migration through activating NF-κB in gastric cancer [24]. Aberrant regulation of BST2 enhances breast cancer cell proliferation and apoptosis evasion [25]. However, its functional roles in cervical cancer require more investigation.
Taken these together, we found that FGD5-AS1 was upregulated in cervical cancer cells. Knockdown of FGD5-AS1 repressed the proliferation, migration, and invasion and increased apoptosis in vitro. Overexpressed, FGD5-AS1 promoted M2 tumor-associated macrophage polarization. Further, miR-129-5p was one of the targets of FGD5-AS1, and BST2 acted as a target for miR-129-5p. It was hypothesized that downregulation of FGD5-AS1 inhibited cervical cancer by inducing M2 tumor-associated macrophage polarization via targeting miR-129-5p and regulating BST2.

Cell Proliferation
Assay. Transfected SiHa cells in the same amount were plated in a 96-well plate, and cell proliferation was tested with CCK-8 (Dojindo, Tokyo, Japan) at 24 h and 48 h. A microplate reader (Bio-Rad, USA) was utilized to test the absorbance at 570 nm.
2.4. TUNEL Staining. After cells were transfected, cells were cultured for a whole night. After cells were fixed using 4% paraformaldehyde, cells were permeabilized in 0.25% Triton-X 100. Then, TUNEL assays were conducted. In brief, cells were treated in a terminal deoxynucleotidyl transferase reaction cocktail for 45 min. Then, incubation with the Click-iT reaction cocktail was followed.

Transwell Assay.
To carry out the Transwell migration assay, cells were seeded in the upper chamber of each insert with the noncoated membrane. Lower chambers were added with 600 μL medium added by 1% FBS. After 24 h, the cells on the lower surface were stained with 0.1% crystal violet. To do the invasion assay, matrigel chambers were carried out. Transfected cells were harvested in medium without serum. Then, the bottom chambers were incubated in 500 μL DMEM added with 10% FBS. Afterwards, the invasive cells on the lower surface were stained with 0.1% crystal violet.
2.6. Real-Time PCR. Total RNA was extracted by the TRI-zol® reagent. Next, extracted RNA was reverse transcribed into cDNA by the ReverTra Ace qPCR RT Kit (Toyobo, Japan). Quantitative PCR was performed using THUNDER-BIRD SYBR®qPCR Mix and a LightCycler 480 Real-Time PCR system. Primer sequences are exhibited in Table 1. Gene expression was calculated according to 2 −ΔΔCT .

Western
Blotting. In brief, cells were lysed using RIPA lysis buffer. 30 μg proteins were exposed to 10% SDS-PAGE electrophoresis and then transferred to PVDF membranes. 5% nonfat milk was used to block the membranes for 1 h, and the membranes were incubated with primary antibodies overnight at 4°C. Antibodies against human BST2 and GAPDH were purchased from Cell Signaling Technology (Boston, MA, USA). The membranes were incubated with a secondary antibody for 1 h. Immobilon Western Chemiluminescent HRP substrate was used to visualize the protein bands.
2.10. RIP Assay. The RIP assay was carried out with the RIP™ RNA-Binding Protein Immunoprecipitation Kit from EMD Millipore. Briefly, SiHa cells transfected with miR-129-5p NC or inhibitor were collected. Then, cell lysate was incubated with RIP buffer containing magnetic beads conjugated with an anti-Ago2 antibody or IgG isotype control. Afterwards, RT-qPCR analysis was employed to evaluate the immunoprecipitated RNA.
2.11. Statistical Analysis. Statistical analysis was carried out using Student's t-test between two groups or one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. P < 0:05 was considered to be statistically significant.

FGD5-AS1 and BST2 Expression in Cervical Cancer
Cells. Firstly, quantitative RT-PCR was used to determine FGD5-AS1 and BST2 expression in cervical cancer cells ). Therefore, we speculated that FGD5-AS1 might promote cervical cancer development.

Effects of FGD5-AS1 on Cervical Cancer Cell
Aggressiveness. To validate our hypothesis, we transfected shFGD5-AS1 into SiHa cells to generate FGD5-AS1 9 knockdown cervical cancer cells and carried out real-time PCR to verify the transfection efficiency. As exhibited in Figure 2(a), FGD5-AS1 shRNA-01 displayed the best knockdown effect on SiHa cells. In Figures 2(b) and 2(c), BST2 mRNA and protein expression was greatly reduced by the lack of FGD5-AS1. After knocking down FGD5-AS1 in cervical cancer cells, cell viability, cell apoptosis, migration capacity, and invasive capacity were assessed. In Figure 2(d), we found that silencing FGD5-AS1 repressed SiHa cell viability evidenced by the CCK assay. SiHa cell apoptosis was triggered by the downregulation of FGD5-AS1 as evaluated using the TUNEL assay in Figure 2(e). As demonstrated in Figure 2(f), SiHa migration and invasive capacity were significantly reduced by decreased FGD5-AS1. Thus, knocking down FGD5-AS1 inhibited the aggressiveness of cervical cancer cells.

Effects of FGD5-AS1 on Cervical Cancer Macrophage M2
Polarization. Furthermore, tumor-associated macrophages can exhibit an M2-like phenotype in the tumor microenvironment. In order to investigate whether FGD5-AS1

BST2 Promoted Cervical Cancer Progression via Inducing
M2 Macrophage Polarization. In order to verify whether BST2 participate in cervical cancer progression via regulating M2 macrophage polarization in cervical cancer cells, SiHa cells were transfected with BST2 shRNA. As shown in Figure 5(a), BST2 was significantly reduced by BST2 shRNA in vitro. In Figures 5(b) and 5(c), loss of BST2 inhibited SiHa cell viability while SiHa cell apoptosis was induced after BST2 shRNA transfection. In Figure 5(d), cervical cancer cell migration and invasive capacity were suppressed by BST2 shRNA. In Figures 5(e) and 5(f), M0 cells were transfected with BST2 shRNA. Then, polarization was induced in M0 cells, and the expression of M1 markers and M2 markers was evaluated. It was indicated that M1 markers (CD80 and CD86) were upregulated while M2 markers (CD206 and CD163) were repressed by the silence of BST2.

Discussion
Recently, lncRNAs are reported to demonstrate tumorinhibitory or tumor-promoting roles in cancers [26]. Then, many lncRNAs are shown to be associated with cervical cancer and serve as a promising biomarker for cervical cancer [27,28]. Our present study clarified the biological significance of FGD5-AS1 in cervical cancer. In our research, we found that lncRNA FGD5-AS1, miR-129-5p, and BST2 were associated with macrophage polarization and cervical cancer. We observed that FGD5-AS1 directly targeted miR-129-5p to promote M2 macrophage polarization through inducing BST2 in cervical cancer. Our findings potentially identify novel targets to design therapeutic strategies for cervical cancer. In our current study, FGD5-AS1 expression was overexpressed in cervical cancer cell lines. Decrease of FGD5-AS1 repressed cell proliferation, migration, and invasion and facilitated apoptosis. These findings provide powerful evidence, which supported that FGD5-AS1 may act as a carcinogenic driver in cervical cancer. FGD5-AS1 promotes lung cancer cell proliferation via sponging miR-107 and upregulating FGFRL1 [29]. In addition, FGD5-AS1/miR-5590-3p can facilitate renal cell carcinoma proliferation and metastasis through ERK/AKT [30].
Within the tumor microenvironment, tumor-associated macrophages are the major inflammatory cells [31,32]. In general, tumor-associated macrophages can exhibit M2 phenotypes and induce tumor progression [33]. Hence, identifying the key factors in M2 macrophage polarization is significant for repressing tumor-associated macrophagemediated tumor progression. To study the mechanisms of tumor-associated macrophage polarization in cervical cancer, we found that decreased expression of lncRNA FGD5-AS1 inhibited M2 macrophage polarization via modulating miR-129-5p and BST2. We reported that FGD5-AS1 could regulate M2 macrophage polarization in cervical cancer.
lncRNAs can display crucial functions in regulating biological cell processes through acting as "sponges" for microRNAs [34,35]. To study the downstream genes of FGD5-AS1, we used bioinformatics analysis to predict miR-129-5p as a potential miRNA with complementary binding at the FGD5-AS1 3 ′ UTR. miR-129-5p has been reported to participate in cervical cancer. For example, miR-129-5p inhibits cervical cancer progression by inhibiting ZIC2 via downregulating Hedgehog [36]. LINC01305 is able to repress the development of cervical cancer by regulating miR-129-5p and Sox4 [37]. A close association between FGD5-AS1 and miR-129-5p was verified by RIP and luciferase reporter assay. In our study, we observed that miR-129-5p reduced cervical cancer cell growth, which was induced by FGD5-AS1. It has been reported that BST2 is aberrantly expressed in cancers. Hence, BST2 is attracting much attention in tumors. Upregulation of BST2 indicates nodal metastasis and a bad prognosis in oral cavity cancer [38]. Overexpression of BST2 is associated with poor survival of esophageal, gastric, or colorectal cancer patients [39]. In our study, BST2 expression was significantly elevated in cervical cancer cells. Loss of BST2 obviously repressed cervical cancer cell proliferation, migration, and invasion while triggering cell apoptosis. Moreover, we found that knockdown of BST2 restrained M2 macrophage polarization. BST2 was predicted and confirmed as a target for miR-129-5p in cervical cancer. We implied the effect of BST2 in M2 macrophage polarization in cervical cancer. In our future study, we will collect enough human cervical cancer tissues to detect FGD5-AS1, miR-129-5p, and BST2 expression.
In summary, our data focused on the association between FGD5-AS1, miR-129-5p, and BST2 and demonstrated that FGD5-AS1 promoted M2 macrophage polarization through regulating miR-129-5p-mediated regulation of BST2 in cervical cancer. We suggested that lncRNA FGD5-AS1 could serve as a valuable prognostic indicator for cervical cancer.

Data Availability
The data used to support the findings of this study are included within the article.