STOX2 Is the Target of Mir-30a to Increase Cell Proliferation and Metastasis in Hydatidiform Mole Through ERK, AKT and P38 Signaling Pathways

Hydatidiform mole is a disease caused by abnormal proliferation of trophoblastic cells. MiR-30a acts as a tumor suppressor gene in most tumors and participates in the development of various cancers. But its role in hydatidiform mole is not clear. RT-qPCR was used to verify the expression level of miR-190 and STOX2. Flow cytometry (FCM) assays were performed to detect cell cycle. CCK-8 assay, EDU assay and colony formation assay was used to detect proliferation ability. Transwell assay was used to test invasion ability. Dual-luciferase reporter assay and Western blotting were used to elucidate the potential mechanisms involved. The low expression of miR-30a promoted the ability of proliferation, migration and invasion in trophoblastic cells (JAR and HTR-8). The dual luciferase assay conrmed that STOX2 was a new target of miR-30a and resisted the effect of up-regulated miR-30a in trophoblastic cells. In addition, up-regulation of STOX2 by miR-30a could activate ERK, AKT and P38 signaling pathways. These results revealed that a new mechanism for ERK, AKT and P38 activation by miR-30a/STOX2, which could create excessive proliferation of trophoblast cells in the hydatidiform mole.


Abstract Background
Hydatidiform mole is a disease caused by abnormal proliferation of trophoblastic cells. MiR-30a acts as a tumor suppressor gene in most tumors and participates in the development of various cancers. But its role in hydatidiform mole is not clear.

Methods
RT-qPCR was used to verify the expression level of miR-190 and STOX2. Flow cytometry (FCM) assays were performed to detect cell cycle. CCK-8 assay, EDU assay and colony formation assay was used to detect proliferation ability. Transwell assay was used to test invasion ability. Dual-luciferase reporter assay and Western blotting were used to elucidate the potential mechanisms involved.

Result
The low expression of miR-30a promoted the ability of proliferation, migration and invasion in trophoblastic cells (JAR and HTR-8). The dual luciferase assay con rmed that STOX2 was a new target of miR-30a and resisted the effect of up-regulated miR-30a in trophoblastic cells. In addition, upregulation of STOX2 by miR-30a could activate ERK, AKT and P38 signaling pathways. These results revealed that a new mechanism for ERK, AKT and P38 activation by miR-30a/STOX2, which could create excessive proliferation of trophoblast cells in the hydatidiform mole.

Conclusions
In this study, we found that miR-30a plays an important role in the development of HM. Taken together, these fndings indicate that miR-30a may effect the malignant transformation of human trophoblastic cells by downregulating STOX2, which strengthens our understanding of miR-30a in regulating BC cell transformation.

Background
Gestational trophoblastic disease (GTD) is a group of placental trophoblast diseases characterized by abnormal proliferation [1], including hydatidiform mole, villus cancer, epithelial itch trophoblastic tumor, placental site trophoblastic tumor [2][3][4]. GTD can be divided into two types: benign and malignant. Benign is hydatidiform mole. The others are malignant. Choriocarcinoma can be caused by hydatidiform mole, ectopic pregnancy and abortion [5]. Hydatidiform mole (HM) is an abnormal pregnancy characterized by placental villus edema and abnormal growth of trophoblastic cells [6]. Placental edema can lead to a series of pathological phenomena, such as edema abortion (HA), partial hydatidiform mole (PHM), complete hydatidiform mole (CHM); the occurrence of hydatidiform mole accounts for 80% of GTD [7], and the incidence varies in different regions of the world [8,9]. There are many factors that induce hydatidiform mole, such as age, race, genetic basis, spontaneous abortion and nutritional restriction [10].
The incidence of women between the ages of 21-35 is lower than those over the age of 35 or under the age of 21 [11]. Compared with the average woman, women who have a history of spontaneous abortion are 2-3 times more likely to have hydatidiform mole [12]; women who have had hydatidiform moles are 10-20 times more likely to have hydatidiform moles than normal, and about 20% have the possibility of malignant transformation after resection. However, the pathogenesis of hydatidiform mole is currently unclear.
MicroRNA (miRNAs) regulates gene expression by binding to the 3'UTR region of target gene mRNA, which is a negative regulatory factor [13]. There is increasing evidence that miRNAs play an important role in the pathogenesis and progression of various tumors, regulating cell proliferation [14], cell cycle regulation [15], in ammatory response[16], cell differentiation [17], Apoptosis and metastasis [18]. It has also been reported in the literature that miRNAs also play a regulatory role during embryonic development [19]. The miRNA-518 family is a speci c biomarker of placenta. miR-518b is abnormally expressed in placental tissues of preeclampsia, which not only regulates EGR1-mediated angiogenesis and migration of trophoblast cells, but also regulates the establishment of hypoxia model of early embryonic development [20]. Previous studies have shown that mir-30a-5p is located in the 6q13 region of the chromosome and is deregulated in some tumors [21]. In hepg2 and mhc97l cancer cells, overexpression of mir-30a completely blocked the activation of the k-ras/c-raf/mek/erk pathway. These ndings suggest that mir-30a may play a role in the growth, apoptosis and metastasis of hepatoma cells by regulating the k-ras/c-raf/mek/erk signaling pathway, which may become a targeted biomarker for liver cancer treatment [22]. MiR-30a is overexpressed in the placenta of patients with eclampsia. The possible mechanism is to in uence the invasion and apoptosis of trophoblast cells by targeting IGF-1 [23].
STOX2 is a winged helix domain, on chromosome 4q35, near the chromosomal region associated with preeclampsia, it is a transcription factor involved in trophoblast differentiation and the most important collateral of STOX1 [24,25]. The abnormal expression of STOX2 in neural crest stem cells and lung cells of the offspring of asthmatic in ammatory model mice was analyzed by transcription expression[26]; compared with the mice without pregnancy, the in ammatory response of stox2 in pregnant mice to air pollutants was increased [27]. Melanoma suppressor protein (MIA) affects the expression of STOX2 by a paracrine manner, promotes the proliferation and metastasis of oral squamous cell carcinoma, and nds that STOX2 is combined with anticancer drugs such as paclitaxel, cisplatin or 5-FU, which can reduce the drug resistance of cancer cells, provide a new treatment for diseases [28]. We compared the expression levels of STOX1 and STOX2 in decidual tissue with pregnancy with pre-eclampsia and/or fetal growth restriction (FGR), and found that STOX1 did not observe differential gene expression in any case group, while the expression of STOX2 in decidua of pregnancy with preeclampsia and FGR was signi cantly lower than that in the control group [29].
In the present study, the results of previous experiments showed that miR-30a was lowly expressed in hydatidiform mole tissue. Combined with laboratory database and biological software prediction, STOX2 is a possible target of miR-30a. We found that miR-30a active the AKT, ERK, P38 signaling pathways affects the development of hydatidiform moles by regulating STOX2.

Materials And Methods
Cell culture and transfection Human trophoblast cell line JAR and the human cervical cancer cell line HeLa were purchased from the American Type Culture Collection (Manassas, VA, USA). HTR-8/SVneo cell was obtained from the Animal Institute of the Chinese Academy of Sciences. And cells were maintained in RPMI-1640 medium (Invitrogen; Thermo Fisher Scienti c, USA) containing 10% fetal bovine serum (ScienCell, USA), 1% penicillin-streptomycin solution (Thermo Fisher Scienti c, USA). Cultures were maintained in the cell incubator with a humidi ed atmosphere of 5% CO2 at 37 °C MiR-30a mimics and mimics negative control miR-30a inhibitor and inhibitor negative control were purchased from Guangzhou RiboBio Co.,LTD. r. Small interfering RNA (siRNA) for STOX2 and negative control were produced by Shanghai GenePharma (Shanghai, China). The si-STOX2 sequence was 5′-AUGGGAGACAUACUGAUGGTT -3′ and the si-NC sequence was 5′-ACGUGACACGUUCGGAGAATT -3′.
STOX2 DNA and corresponding negative control were composed by GeneCopoeia Inc. The cells were seeded in 6-well plates after 70%-80% con uence, the transfection was performed by Lipofectamine® 2000 reagent according to the manufacturer's descriptions.

RNA extraction and Quantitative RT-PCR
The total RNAs were extracted from JAR and HTR-8 cells by using TRIzol reagent. cDNA is synthesized using TransScript All-in-one First-Strand Cdna Synthesis SuperMix for qPCR kit ( One-Step gDNA Removal) (TransGen, Beijing, China) according to the manufacturer's speci cations. qPCR was performed by using a TransStart Top Green qPCR SuperMix (TransGen, Beijing, China) with analysis in an ABI 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scienti c, Inc.). The qPCR conditions were 94°C for 30 sec followed by 40 cycles of denaturation at 94°C for 5 sec and annealing/elongation at 60°C for 30 sec. U6 was used as the internal reference for miR-30a, and GAPDH was performed as the control for STOX2 expression. The PCR primer was shown as follows: STOX2 forward: 5'-AGCCTGTCCCTCCTCAAATCTCA-3', reverse: 5'-CTCTGTGTTCTTGTTTGCCCCT-3'; GAPDH forward: 5'-GTGAAGGTCGGAGTCAACG-3', reverse: 5'-TGAGGTCAATGAAGGGGTC -3'; the data was calculated using 2 -ΔΔct method. All experiments were performed triplicate samples.

CCK-8 assay
Transfected Cells were seeded 96-well plates in 200 μl medium at the density of 5000 per well and cultured for 24, 48, 72h, cck-8 (Dojindo, Tokyo, Japan) was added into the each well according the manufacturer's speci cations. After 4h in 37℃ incubation, the absorbance at 450 nm were detected using a microplate reader(BioTek, USA ).

Colony formation assay
After Transfected 48h, 3×10 3 Cells were seeded 6-well plates and incubated for 15 days. The colonies were xed with methanol for 30 min and stained with 1% crystal violet for 15 min. Cells number was counted by using Image J.

EdU assay
For transfected cells, EdU reagent (Beyotime, Shanghai, China) was added into 96-well plates and cultured 2h in 37℃ incubator. Next, Cells were xed with 4% paraformaldehyde for 30 minutes and 0.1% Triton-X 100 for 15 minutes, and incubated with Azide-488 for 30 minutes at room temperature in the dark room. Images were taken under inverted microscope (Olympus, Japan).
Transwell assay 8-μm pore size 24-well Transwell plates (Corning, USA) were used to evaluate the Migratory and invasive potential of JAR and HTR-8 cells. For migration assay, transfected cells were collected and resuspended in 1 ml serum-free RPMI-1640 medium, 5×10 4 cells were added to the the upper chambers, RPMI-1640 medium with 10% FBS was seeded into the lower chambers. After 24h in incubator with 5% CO 2 37℃,the migratory cells were xed with methanol for 30 min and stained with 0.2% crystal violet for 30 min. For invasion assay, the upper chambers of transwell were pre-coated with Matrigel (BD Biosciences, USA), 5s10 4 transfected cell were seeded into the upper chambers with serum-free RPMI-1640 medium, after incubated 30h, the cells which not invasive were wiped off with a cotton swab, and the invasive cells were xed with methanol for 30 min and stained with 0.2% crystal violet for 30 min. All migratory and invasive chambers were counted under a light microscope (Olympus, Japan).

Western blot assay
After the cells were transfected for 48h, total protein was cleaved using ProteinExt® Mammalian Total Protein Extraction Kit (TransGen, Beijing, China ), and the protein was determined by the BCA method (TransGen, Beijing, China ). 30µg or 60µg protein was separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE, 10%) and transferred onto a nitrocellulose membrane (Millipore, Bedford, MA, USA) using cold transfer buffer. The membrane was stained with Ponceau S (Beyotime, Shanghai, China) and washed with TBST. 5 non-fat milk was used to block the membrane for 2h at room temperature. The membrane was incubated with the primary antibody overnight 4℃. Hematoxylin and eosin (HE) staining The Hydatidiform mole and normal placenta tissues were xed in 4% formaldehyde and embedded in para n for Hematoxylin and eosin (HE) staining. The slices were stained with hematoxylin for 20 min and eosin for 30 s−1 min. Finally, the slices were analyzed under a light microscope.

Statistical Analysis
All data were presented as mean ± SD and analysised by using GraphPad Prism 6.0 software. All experiment was repeated three times independently. Signi cance of differences between the two groups was assessed via One-way ANOVA and P value <0.05 was considered statistically signi cant.

Results
Up-regulation of miR-30a inhibited trophoblastic cells of proliferation cell cycle and metastasis In previous studies, we demonstrated that the expression level of miR-30a was lower in hydatidiform moles which can be seen in our earlier article [49] . However, the regulatory mechanism of miR-30a in hydatidiform mole is still unclear. In this article, we will explore the mechanism of miR-30a in hydatidiform mole disease at the cellular level. The transfection e ciency was detected by treating mi-30a mimics/negative control and miR-30a inhibitor/negative control in HTR-8 cells (Figure1 A). CCK-8 assay was performed to discover the effect of miR-30a on the proliferation of HTR-8 cells, the results showed that the cell vitality of HTR-8 cells was weakened by up-regulating miR-30a, Conversely, the suppression of miR-30a was increased cell proliferation (Figure1 B). The colony formation ability was reduced by transfecting miR-30a mimics (Figure1 C and Figure1S A). Compared with transfected negative control, the uorescence activity of cells transfected miR-30a mimics were decreased, suggesting that cell proliferation was slow (Figure1 D). Furthermore, we analyzed that down-regulated miR-30a could enhanced the cell rate in S stage, promoting the growth of trophoblastic cells (Figure1 E and Figure1SB).
To investigate the effect of migration and invasion of HTR-8 cells, we operated transwell assay, Compared with transfected negative control, miR-30a transfected miR-30a mimics impaired the ability of migration and invasion, and stronger ability of metastasis was observed by down-regulating miR-30a (Figure1 F and G). In conclusion, the data demonstrated that miR-30a restrained cell viability, cell cycle and metastasis in trophoblastic cells.
The effect of different expression levels of stox2 on the proliferation and metastasis of trophoblastic cells To investigate the effect of STOX2 on the proliferation and metastasis of trophoblast cells, rstly, we tested the transfection e ciency by transfecting STOX2 siRNA or STOX2 cDNA using Real-time PCR (Figure2 A and C). We performed CCK-8, colony formation assay and EdU asssy in JAR and HTR-8 cells.
In CCK-8 experiment, the results showed that up-regulation of STOX2 signi cantly promoted the cell proliferation ability; compared with the control group, the proliferation ability of cells was signi cantly weakened following down-regulated STOX2 (Figure2 B and D). In colony formation assay, the ability of cells transfected with stox2 cDNA to form colonies increased, but the ability of cells interfering with STOX2 siRNA group to form colonies decreased signi cantly (Figure2 E and F); Similarly, EdU asssy found that cell replication and the uorescence activity were enhanced after transfecting up-regulated STOX2; otherwise the uorescence activity decreased (Figure2 G and H).
Subsequently, Transwell assay found that overexpression of STOX2 enhanced cell migration and invasion, and the ability of metastasis was impaired by reducing the expression of STOX2 (Figure2 I and J, K and L). These results indicated that up-regulation of stox2 could promote cell proliferation, migration and invasion.
miR-30a targets the 3'-UTR of STOX2 mRNA and is low in hydatidiform mole.
To explore the effects of miR-30a on trophoblast cell proliferation, migration and invasion, STOX2 was predicted to be a candidate target gene for miR-30a through the biological software (miRBase, TargetScan, PicTar). As shown in Fig. 3A, it was found that miR-30a had a binding site with the 3'UTR region of STOX2. Subsequently, we cloned WT and Mut 3 UTR of STOX2 into a luciferase reporter vector it constructs containing the renilla luciferase gene fused to STOX2 3′UTR sequence and the re y luciferase for normalization. These vectors were transfected in HeLa cells with miR-30a mimics or Negative. As expected, compared with co-transfecting WT STOX2 3'UTR PmiR and negative, the luciferase reporter activity in cells was reduced co-transfected WT STOX2 3'UTR PmiR and miR-30a mimcis; in the same way, we found that the luciferase reporter activity was no changed when we cotransfected Mut STOX2 3'UTR PmiR and miR-30a mimics or negative (Figure3 B). Further evidence of the impact of miR-30a on STOX2 was measured by the expression of STOX2 by western blot. The protein level of STOX2 was reduced in JAR and HTR-8 cells by transfecting with miR-30a mimics, when we inhibited the expression of miR-30a, we found that STOX2 protein was increased (Figure3 C and D). In our previous research, we detected that mi3-30a was lower in hydatidiform mole tissues than normal placenta, but now we also found that the expression of STOX2 was high in hydatidiform mole tissues (Figure3 E). These results con rmed our prediction that STOX2 is a new target of miR-30a and miR-30a might be participated in the pathogenesis of hydatidiform mole by targeting STOX2.

STOX2 mitigates miR-30a stimulation of proliferation and metastasis in trophoblastic cells
To investigate the functions of STOX2 and miR-30a in trophoblastic cells, in previous research, we found that increasing miR-30a could inhibit the proliferation and metastasis abilities in JAR and HTR-8 cells. We performed the CCK-8 (Figure4 A and B, Figure5 A

and B) colony formation assay(Figure4 C and D, Figure5
C and D) EdU assay (Figure4 A and B, Figure5 A and B) to explore the proliferation of cells. The results showed that over-expression of miR-30a and STOX2 could reinforce the proliferation of cells compared with miR-30a mimics group. Conversely, when we transfected the down-expression of cells, the results clearly demonstrated the opposite trends. For the migration and invasion of cells, transwell assay revealed that enhanced expression of STOX2 impaired the ability of miR-30a mimics to decrease the trophoblastic cells metastasis (Figure4 G and I, Figure5 G and I). Compared with miR-30a inhibitor group, co-transfection with STOX2 siRNA and miR-30a inhibitor group was also blocked migration and invasion (Figure4 H and J, Figure5 H and J). These data suggest that STOX2 alleviate the proliferation and metastasis of JAR and HTR-8 cells.
MiR-30a Induces STOX2 expression and through ERK 1/2, AKT, and p38 Signaling Pathway in trophoblastic cells We found that miR-30a and STOX2 mediated trophoblastic cells proliferation and metastasis in vitro. To study the molecules mechanisms for miR-30a and STOX2 induced inhibition of growth, invasiveness and migration of JAR and HTR-8. With transfected miR-30a mimcs, the phosphorylation of AKT ERK and P38 was decreased and the expression of AKT ERK and P38 was not changed. Conversely, up-regulation of STOX2 increased the expression of p-AKT p-ERK p-P38. Co-teatment with miR-30a mimics and STOX2 cDNA could further partially strengthened the expression of p-AKT p-ERK p-P38, and the total protein of AKT ERK P38 was not changes (Figure6 A and C). In the same way, similar levels of AKT ERK and P38 protein expression were observed in the miR-30a inhibitor and STOX2 siRNA-transfected group , miR-30a inhibitor group , STOX2 siRNA group and the NC-transfected group. Transfected STOX2 siRNA weaken the protein level of p-AKT p-ERK p-P38 on miR-30a inhibitor in JAR and HTR-8 cells (Figure6 B and D).

Discussion
Hydatidiform mole is one of the most common Gestational trophoblastic diseases, with a prevalence of 1:1000 in North America and Europe, but is more common in South America and Asia. Due to differences in histology, genetics, and clinical features, hydatidiform moles can be divided into complete moles (CHM) and partial moles (PHM) [30,31]. Ultrasound is the main method for clinical detection of hydatidiform mole, but it is also determined by the age of the fetus. In the world, hydatidiform mole is diagnosed earlier, but in developing countries, there are still patients diagnosed in the early pregnancy with complications [32]. Although hydatidiform moles are benign, there is still a high possibility of developing malignant trophoblastic tumors. The clinical treatment is curettage and hysterectomy. In the later stage, the content of hCG should be detected to prevent recurrence. These not only cause irreparable physical damage to patients, but also cause huge psychological burden. Therefore, new treatment methods are urgently needed.
miRNAs regulate many genes and are involved in the development of many tumors [33,34]. Mir-30a plays a key role in many types of human cancers, mainly anti-cancer effects, non-small cell carcinoma [35] , breast tumor[36], renal cell carcinoma [37], colorectal cancer[38]. miR-30a prevents DNA replication and leads to DNA degradation by targeting RPA1, which induces P53 expression, and triggers S-phase checkpoints, prevents cell cycle progression, ultimately leads to cancer cell death [39]. miR-30a regulates Eya2 to mediate the G1/S cell cycle and the expression of related cyclins [40]. In gallbladder cancer, miR-30a could directly target e2f7 to regulate EMT and metastasis, and participated in cancer progression [41]. The expression of miR-30a-3p was signi cantly increased in the preeclamptic placenta and regulated trophoblast invasion and apoptosis by targeting IGF-1 [23]. In the present study, we have found that down-regulated miR-30a was boosted the ability of proliferation, migration, invasion in trophoblastic cells, and was lower expression in hydatidiform mole tissues than normal placenta, which suggests that miR-30a was involved in the development of hydatidiform mole.
Storkhead box 2 (STOX2) is considered to be the only collateral family of STOX1, but as far as we know, its function is still unclear. However, a large number of studies on STOX1 have reported that the polyploidy defect before incomplete invasion of extravillous trophoblast caused by STOX1 dysfunction seems to be the center of pre-eclampsia, activating PI3K/AKT/FOX signaling pathway [24]. It has been reported in the literature that STOX1A regulates cell cycle by binding to CCNB1 to regulate mitosis [42]. An increase in the level of insulin-like growth factor 1 (IGF1) leads to an increase in the expression of STOX1 in the extravillous trophoblast through the MAPK pathway, thereby identifying a new signaling cascade involved in maternal-fetal communication [43]. Homologous genes have similar biological functions. The RNA encoded by intron 3 of STOX2 transcription can affect the alternative splicing of host genes in placental cells. STOX2-IT3-lncRNA affects genes involved in trophoblast differentiation and invasion, thus affecting the pathogenesis of eclampsia [44]. In this study, we found that STOX2 was not only reduced cell proliferation and metastasis, but also was highly in hydatidiform mole tissues, which suggests that stox2 may induce hydatidiform mole.
It was well known that the biological role of miRNAs was achieved by regulating downstream target genes. Our results revealed that STOX2 was the directly target gene of miR-30a. Moreover, we found that STOX2 was reduced by up-regulating miR-30a in trophoblastic cells. And we also detected that downregulated STOX2 could impair the ability of proliferation and metastasis in trophoblastic cells by transfected miR-30a inhibitor. However, the molecular mechanism by miR-30 regulates the biological role of STOX2 is still not very clear. ERK, AKT, and P38 signaling pathways are important for tumor proliferation and metastasis, and involved in the pathogenesis of various cancers. Some studies have shown that miR-30a-5p targeting NeuroD1 could improve in ammatory response and oxidative stress through MAPK / ERK signaling pathway in case of spinal cord injury. [45]. miR-30a released by p53 r273h mutation can inhibit the expression of IGF-1R, which leads to the activation of igf-1r-akt signal cascade in tumor cells [46]. Mir-30a is signi cantly down-regulated in highly metastatic colorectal cancer cell lines and metastatic tissues, and its mechanism mediates Akt/mTOR signaling pathway by targeting PIK3CD expression, regulating the metastasis of cancer cells [47]. In addition, mir-30-5p-tcf21-mapk/p38 signaling pathway may be a potential biomarker or therapeutic target of atherosclerosis [48]. Interestingly, in our study, we demonstrated that overexpression of miR-30a inhibited the phosphorylation of ERK, AKT, P38, whereas up-regulated STOX2 ameliorated this suppressive effect. These data further con rmed that the aggressive proliferation potential of hydatidiform mole was caused by the low expression of miR-30a, at least in this part, by regulating STOX2 through activating ERK, AKT, P38 signaling pathways. Further research of the speci c molecular mechanism was required on this process.
In the summary, we recognized STOX2 high-regulated in hydatidiform mole. miR-30a was able to suppress the proliferation and invasion ability of trophoblastic cells through targeting STOX2-mediated AKT, ERK and P38 signaling pathways. Our ndings may offer insight into a new mechanism of hydatidiform mole. MiR-30a and STOX2 could be a biomarker and therapeutic target for hydatidiform mole in the future time.

Conclusions
In the summary, we recognized STOX2 high-regulated in hydatidiform mole. miR-30a was able to suppress the proliferation and invasion ability of trophoblastic cells through targeting STOX2-mediated AKT, ERK and P38 signaling pathways. Our ndings may offer insight into a new mechanism of hydatidiform mole. MiR-30a and STOX2 could be a biomarker and therapeutic target for hydatidiform mole in the future time.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.
Availability of data and materials The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
Neither this paper nor any similar paper has been or will be submitted to or published in any other scientifc journal. All authors are aware and agree to the content of the paper and to their being listed as an author on the manuscript.
There is no con ct of interest or competing fnancial interests for all authors      Cell migration was found after co-transfected and STOX2 cDNA or miR-30a inhibitor and STOX2 siRNA into HTR-8 cells. (I and J) Cell invasion was showed after co-transfected and STOX2 cDNA or miR-30a inhibitor and STOX2 siRNA into HTR-8cells. Bar=100μm. *P <0 .05 or **P <0 .01, ***P<0.001.