LRRC75A‐AS1 delivered by M2 macrophage exosomes promotes cervical cancer progression via enhancing SIX1 expression

Abstract We aimed to investigate potential roles of LRRC75A‐AS1 delivered by M2 macrophage exosomes in inducing cervical cancer progression. We demonstrated LRRC75A‐AS1 was highly expressed in exosomes from M2 macrophages which could be absorbed by Hela cells. M2 macrophage‐derived exosomes promoted Hela cell proliferation, migration, invasion, and EMT process by delivering LRRC75A‐AS1. LRRC75A‐AS1 directly targeted and suppressed miR‐429 in Hela cells. The regulation of cell functions by exosomes from LRRC75A‐AS1‐overexpressing M2 macrophages was abrogated by miR‐429 mimics. miR‐429 directly targeted and repressed SIX1 expression. SIX1 overexpression alleviated the modulation of cellular functions and STAT3/MMP‐9 signaling by miR‐429 mimics. Also, miR‐429 overexpression or SIX1 silence repressed tumor formation and metastasis in nude mice, which was mitigated by exosomes from LRRC75A‐AS1‐overexpressing M2 macrophages. In conclusion, LRRC75A‐AS1 delivered by M2 macrophage exosomes repressed miR‐429 to elevate SIX1 expression and promote cervical cancer progression through activating the STAT3/MMP‐9 axis.

suitable for the common treatments. 6 Therefore, it is of great clinical significance to elucidate the causes of cervical cancer metastasis.
The epithelial to mesenchymal transition (EMT) is critical to metastasis of cervical cancer, 7 but its regulatory mechanisms underlying cervical cancer pathogenesis deserve further elucidation.
Exosomes and their noncoding RNA cargos have been regarded as key players in cervical cancer metastasis. 8,9 Recent research showed that exosomes from M2 macrophages substantially regulated cancer initiation, metastasis, and chemotherapy resistance. [10][11][12][13] For instance, the migration of gastric cancer cells was reported to be driven by M2 macrophage-derived exosomes via delivery of the apolipoprotein E protein. 13 However, little is known about the roles of macrophage-derived exosomes in cervical cancer growth and metastasis. Furthermore, the antisense of leucine-rich repeat-containing protein 75A (LRRC75A-AS1) was characterized as one key lncRNA widely associated with colorectal carcinoma, glioblastoma, and breast cancer. [14][15][16][17] Bioinformatics data from Kaplan-Meier Plotter indicated that high LRRC75A-AS1 expression was associated with poor survival rate of cervical cancer patients. Our preliminary experiments indicated LRRC75A-AS1 was highly expressed in cervical cancer tissues and exosomes from M2 macrophages, but its potential roles delivered by M2 macrophage-derived exosomes in cervical cancer remain largely unknown. miR-429 suppressed the invasion and migration of cervical cancer cells through targeting zinc finger E-box binding homeobox 1 (ZEB1) and Crk-like adapter protein (CRKL) to inhibit the EMT process. 18 Our bioinformatics analysis (Starbase) showed that LRRC75A-AS1 might also target miR-429, and their association with cervical cancer development deserves further investigation. On the other hand, sine oculis homebox 1 (SIX1) is a homeodomain protein of the SIX family that regulates the EMT process during invasion and metastasis, [19][20][21][22] during which SIX1 activated the signal transducer and activator of transcription 3 (STAT3) to promote matrix metalloproteinase 9 (MMP-9) activity. 22,23 In cervical cancer development, high expression of SIX1 also contributed to cell proliferation, EMT, and lymphangiogenesis. [24][25][26] Moreover, the expression and role of SIX1 in cervical cancer cells were suppressed by miRNAs such as miR-362 and miR-23b. 27,28 However, whether miR-429 could also target SIX1 in cervical cancer is still unaddressed.
In this study, we tested our hypothesis that lncRNA LRRC75A-AS1 in M2 macrophage-derived exosomes might sponge miR-429 to upregulate SIX1 expression and activate the STAT3/MMP-9 signaling axis, thus activating the EMT process and cervical cancer growth and metastasis. Totally

| Exosome isolation and characterization
Exosomes from M2 macrophages were extracted using the ExoQuick® ULTRA EV Isolation System Kit (#EQULTRA-20A-1; System Biosciences) following the manufacturer's instructions. Briefly, culture medium was collected when cell confluence was between 80% and 90% by centrifuge. The supernatant containing exosomes was incubated with 100 μL ExoQuick solution on ice for 30 min with frequent inverting or flicking. After centrifugation at 3200g for 12 min at 4°C, the pellets containing exosomes were resuspended in 200 μL buffer B, mixed with 200 uL buffer A, and loaded into a purification column.
Following mixing for 12 min, exosomes were collected by centrifugation at 900g for 30 s. The morphology and diameter of exosomes were then observed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) as previously described. 11,12

| Exosome uptake analysis
The uptake of M2 macrophage-derived exosomes by Hela cells was validated as previously described. 12 Briefly, the isolated exosomes

| In vivo tumorigenesis
The in vivo tumorigenesis of cervical cancer was evaluated through the xenograft model as previously introduced with minor modification. 11 Briefly, female BALB/c nude mice at the age of 6 weeks were mimics or sh-SIX1 followed by treatment with exosomes from M2 macrophages overexpressing LRRC75A-AS1 as introduced above.
More detailed methods are shown in Appendix S1

| High expression of LRRC75A-AS1 detected in exosomes from M2 macrophages
We first induced the differentiation of THP-1 cells into M2 macrophages, and found the expression levels of CD206, CD163, Arg-1, IL-10, and TGF-β1 in M2 macrophages were all greatly elevated compared with the control group ( Figure 1A,B). These results validated the successful induction of M2 macrophages. Next, we isolated exosomes from the culture medium of M2 macrophages, which exhibited the regular elliptical shape in our TEM observation ( Figure 1C).
Both the TEM and NTA results showed that the diameters of the majority of these exosomes were about 100-150 nm ( Figure 1C,D).

| LRRC75A-AS1 in M2 macrophage-derived exosomes induces Hela cell functions and EMT process through STAT3/MMP-9 signaling
We subsequently found that LRRC75A-AS1 expression level in Hela cells was greatly decreased compared with the control cell line Ect1/

| LRRC75A-AS1 is highly expressed in cervical cancer tissues and negatively regulates miR-429 expression
Kaplan-Meier Plotter database showed that higher expression of LRRC75A-AS1 in cervical cancer tissues was significantly correlated with poor survival of cervical cancer patients ( Figure 3A). Also, we showed the LRRC75A-AS1 expression in the cervical cancer tissues was much higher than in adjacent normal cervical tissues ( Figure 3B).
According to the median expression of LRRC75A-AS1, high expression of LRRC75A-AS1 in our clinical samples was positively associated with tumor stages and lymph node metastasis (Table 1). Interestingly, the ratio of M2 macrophages (CD163 + CD206 + ) in cervical cancer tissues collected from 15 patients was significantly higher than in corresponding adjacent normal cervical tissues ( Figure 3C). Also, the CD163 protein level was also upregulated in the abovementioned cervical cancer tissues ( Figure 3D). Importantly, LRRC75A-AS1 was predicted to directly target miR-429 (Starbase database) ( Figure 3E).
We found the lower expression of miR-429 in cervical cancer tissues was significantly correlated with poor survival of cervical cancer patients via the Kaplan-Meier Plotter platform ( Figure 3F). Moreover, miR-429 expression levels in cervical cancer tissues were much lower than in adjacent cervical tissues ( Figure 3G). Furthermore, LRRC75A-AS1 and miR-429 expression in cervical cancer tissues exhibited significantly negative correlation ( Figure 3H). To validate this, we showed the colocalization of LRRC75A-AS1 and miR-429 in Hela cell cytosols ( Figure 3I). We also found that LRRC75A-AS1 precipitated with Ago2 protein was greatly increased by transfecting Hela cells with miR-429 mimics ( Figure 3J). In addition, miR-429 mimics greatly reduced the luciferase activity in the Hela cells expressing wild-type LRRC75A-AS1, but not in those expressing the mutant LRRC75A-AS1 ( Figure 3K). Consistently, miR-429 expression in Hela cells was remarkably promoted by LRRC75A-AS1 knockdown and greatly repressed by LRRC75A-AS1 overexpression ( Figure 3L).
These results showed that LRRC75A-AS1 could directly target and inhibit miR-429 expression in Hela cells.

| LRRC75A-AS1 in M2 macrophage-derived exosomes represses miR-429 to promote Hela cell proliferation, migration, invasion, and EMT
We then isolated exosomes from M2 macrophages with LRRC75A-AS1 overexpression, which were used to incubate with the Hela cells transfected with miR-429 mimics. We found that  Figure 4E). In addition, miR-429 mimics transfection in Hela cells significantly mitigated the above alterations. These assays indicated that the regulation of Hela cell functions and EMT by LRRC75A-AS1 in M2 macrophage-derived exosomes was mediated by its inhibition on miR-429 expression.
By searching the UALCAN database, we found that SIX1 expression levels in cervical cancer tissues were much higher than in normal cervical tissues ( Figure 5B). SIX1 expression in cancerous tissues was greatly elevated compared with adjacent cervical tissues ( Figure 5C). The miR-429 and SIX1 expression in cervical cancer tissues demonstrated significantly negative correlation ( Figure 5D).
The dual luciferase reporter assay showed great decrease of luciferase activities in Hela cells expressing the wild type of SIX1 by miR-429 mimics, other than in those expressing the mutant SIX1 ( Figure 5E). miR-429 expression was elevated or knocked down in Hela cells by miR-429 mimics or inhibitor, respectively ( Figure 5F).
The SIX1 mRNA and protein levels in Hela cells were significantly repressed by miR-429 mimics, but greatly elevated by miR-429 inhibitor ( Figure 5G,H). These results proved that miR-429 repressed SIX1 expression in Hela cells through directly binding with its 3'-UTR region.

| miR-429 represses SIX1 expression to modulate Hela cell functions and EMT process through STAT3/MMP-9 signaling
The miR-429 expression in Hela cells was markedly elevated by miR-429 mimics, which was not affected by SIX1 overexpression ( Figure 6A). However, SIX1 mRNA and protein levels in Hela cells were greatly reduced by miR-429 mimics, which were then remarkably alleviated by SIX1 overexpression (Figure 6B,C). Hela cell proliferation rates were greatly repressed by miR-429 mimics, but SIX1 overexpression effectively abrogated the inhibition of cell proliferation ( Figure 6D). On the contrary, miR-429 mimics significantly accelerated Hela cell apoptosis, which was also significantly mitigated by SIX1 overexpression ( Figure 6E). Hela cell migration and invasion exhibited similar alteration as cell proliferation ( Figure 6F,G). Furthermore, STAT3 phosphorylation, MMP-9, N-cadherin and slug protein levels in Hela cells were substantially reduced by miR-429 mimics, which, however, caused great increase in the E-cadherin protein level ( Figure 6H). These alterations of EMT biomarker proteins and STAT3/MMP-9 signaling in Hela cells by miR-429 mimics were all significantly abrogated by SIX1 overexpression ( Figure 6H). These observations demonstrated that miR-429 repressed the proliferation, migration, invasion, and EMT but promoted apoptosis in Hela cells through suppressing SIX1 expression.

| LRRC75A-AS1 in M2 macrophage-derived exosomes promotes Hela cell tumorigenesis and metastasis in nude mice
Exosomes from the OE-NC-Exos and shNC-Exos groups increased the tumor volumes and weights in nude mice, and the volumes and weights of tumors were further promoted by exosomes derived from LRRC75A-AS1-overexpressing M2 macrophages, but effectively reduced by exosomes derived from LRRC75A-AS1-silencing M2 macrophages ( Figure 7A-C). In tumor tissues of nude mice, SIX1, N-cadherin, and LRRC75A-AS1 expressions were promoted by exosomes from LRRC75A-AS1-overexpressing M2 macrophages, but inhibited by exosomes from LRRC75A-AS1-silencing M2 macrophages ( Figure 7D,E). Completely opposite alterations of miR-429 levels were observed in the above tumor tissues ( Figure 7E).
In addition, the metastasis potential of Hela cells was then evaluated by histological evaluation and lung nodule numbers ( Figure 7G,H). Compared with the corresponding NC groups, the numbers of lung nodules formed in nude mice were remarkably increased by treatment with exosomes purified from M2 macrophages overexpressing LRRC75A-AS1, but significantly decreased by treatment of exosomes from LRRC75A-AS1-silenced M2 macrophages ( Figure 7G,H). These observations proved that LRRC75A-AS1 from M2 macrophage-derived exosomes could effectively promote both the tumorigenesis and metastasis potential of Hela cells in vivo.

| DISCUSS ION
The progression of EMT during cervical cancer was known to be regulated by a number of inducers and inhibitors, 7 but the epigenetic mechanism mediating EMT in cervical cancer still remains poorly understood. In this study, we comprehensively investigated the pathogenic roles of LRRC75A-AS1 in cervical cancer progression.
We proved that exosomes secreted by M2 macrophages promoted the proliferation, migration, invasion, and EMT of cervical cancer through delivery of LRRC75A-AS1, which was mediated by miR-429, SIX1, and the downstream STAT3/MMP-9 pathway.
Exosomes have been regarded as essential signaling platforms in cancer development. 34,35 For instance, recent report showed that the exosomes derived from M2 macrophages effectively promoted the migration and invasion of colon cancer cells. 10 The development of pancreatic ductal adenocarcinoma could also be promoted by M2 macrophage-derived exosomes. 11 However, the roles of M2

ACK N O WLE D G E M ENTS
The authors have nothing to report.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in this article. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

E TH I C S S TATEM ENT
All experimental operations using human cervical cancer tissues were approved in advance by the Medical Ethical Committee of the Hunan Cancer Hospital.

Approval of the research protocol by an Institutional Reviewer
Board.
Informed Consent: Before the surgical operation, written informed consent was signed by all cervical cancer patients.
Registry and the Registration No: N/A.
Animal Studies: All experiments using mice were approved in advance by the Animal Care and Use Committee of the Hunan Cancer Hospital.

CO N S E NT FO R PU B LI C ATI O N
The informed consent was obtained from study participants.