RETRACTED ARTICLE: LncRNA SOX21-AS1 accelerates endometrial carcinoma progression through the miR-7-5p/RAF1 pathway

Endometrial carcinoma (EC) is one of the world’s typical female reproductive tract malignancies, mostly occurring in postmenopausal women. Many reports have confirmed that long non-coding RNA SOX21 antisense RNA1 (lncRNA SOX21-AS1) is associated with the progressions of various cancer. However, the mechanism of SOX21-AS1 in EC remains unclear. Our study is intended to probe the mechanisms of SOX21-AS1 on EC progression. The CCK-8 assay and colony formation detected cell proliferation. Cell migration and invasion were assessed by transwell analysis. Apoptosis was measured by flow cytometry assay. Bioinformatics software predicted target binding and confirmed using a luciferase reporter analysis. SOX21-AS1 expression was upregulated in EC tumor tissues and cells. High expression of SOX21-AS1 was associated with poor overall survival. Silencing of SOX21-AS1 restrained cell proliferation, migration, invasion, and increased apoptosis in HEC-1A and Ishikawa cells. Additionally, bioinformatics analysis demonstrated that SOX21-AS1 modulated RAF1 expression by competitively binding to miR-7-5p. Functionally, silencing of RAF1 reversed the functions of miR-7-5p inhibitor in the proliferation, invasion, and apoptosis of HEC-1A/sh-SOX21-AS1 and Ishikawa/sh-SOX21-AS1 cells. SOX21-AS1 promoted the pathological development of EC by regulating the miR-7-5p/RAF1 pathway. This research may provide a novel target for EC therapy.


Introduction
Endometrial carcinoma (EC) is a common female reproductive system malignancy in Europe and the USA, accounting for 20 ~ 30% of the overall incidence of female reproductive system malignancy [1].In 2020, the death rate of EC in the USA was about 20%, second only to ovarian cancer [2].With the aging of the population, the incidence of EC in China is gradually increasing, second only to cervical cancer [3,4].Most EC patients could be diagnosed early and the overall prognosis is good; however, the prediction of advanced and unique pathological types of EC is still poor [3,5].EC treatment usually includes radiation, systemic chemotherapy, or endocrine therapy.Still no method of diagnosing endometrial cancer is entirely satisfactory [6,7].Therefore, exploring new biomarkers for early diagnosis and prognosis will benefit EC patients.
LncRNA is a functional RNA molecule that does not code proteins with a length exceeding 200 nucleotides [8].Although lncRNA does not have the function of encoding protein, it plays an essential role in various biological processes, such as pre-and post-transcription regulation, epigenetic regulation, and genetic-related gene regulation [9].Recently, the potential of lncRNA as a marker for cancer diagnosis and prognosis has gradually emerged, and its functions in tumorigenesis have received extensive attention [10].There are also imbalances in the expression of lncRNA in EC.LncRNA HAND2-AS1 is a low expression in EC tissue, and the knockdown of HAND2-AS1 promoted EC occurrences by inactivating neuromedin U, for example [11].LncRNA NORAD is abnormally expressed in EC and is related to prognosis [12].SOX21-AS1, in chromosome 13q32.1, is transcribed into a transcript of 2986 nucleotides [13,14].It has been reported that SOX21-AS1 promotes the occurrence of various tumors, including cervical cancer [15], lung adenocarcinoma [16], and colorectal cancer [17].However, the role of SOX21-AS1 in EC has not been reported.Hence, our study focused on probing the potential mechanism of SOX21-AS1 in EC.

Clinical sample
Thirty pairs of EC tissues and adjacent tissues were gained from the Affiliated Hospital of Jiangnan University.All collected samples were frozen in liquid nitrogen for subsequent studies.The research was ratified and supervised by the ethics committee of the Affiliated Hospital of Jiangnan University.All patients were informed and signed written consent.EC patient information and the correlation between SOX21-AS1 and clinical pathological parameters were summarized in Table 1.

RT-qPCR assay
First, RNA was extracted using the Trizol reagent (Solarbio, Beijing, China).Following, RNA was reverse transcribed to cDNA by PrimeScript RT Master Mix (Yeasen, Shanghai, China).Next, RT-qPCR was implemented by SYBR Green PCR Kit (Qiagen, Dusseldorf, Germany) by Stratagene mx3000p real-time PCR system (Stratagene, USA).GAPDH and U6 acted as the endogenous control, and the 2 −ΔΔCT method measured the transcript expression level.RT-PCR primer was shown in Table 2.

Colony formation assay
The treatment cells were seeded in a 6-well plate.After culturing for 14 days, cells were fixed with methanol for 15 min.After washing, cells were treated with 0.1% crystal violet solution.Conclusively, the colonies were observed using the microscope (KEYENCE, Osaka, Japan) and counted in 6 different areas.

Cell apoptosis assay
Transfected cells were collected and passed through a 100-mesh sieve.Annexin V and PI solution were then incubated in darkness for 20 min.The labeled cells were detected by a flow cytometer (Sysmex-Partec, Germany).

Transwell assay
Migration and invasion activity was measured by the Transwell chamber.For invasion analysis, the transwell chamber membranes were pre-coated with matrigel.Simply put the transfected cells that were plated into the top compartment of the chamber with a basal medium.A medium including 10% FBS was added to the bottom of the cavity.After 48 h, cells in the bottom compartment were fixed with methanol and treated with 0.1% crystal violet at 37 ℃ for 25 min.The cells were recorded and photographed in 5 fields using a microscope (KEYENCE).

Western blot
Transfected cell proteins were cracked at low temperatures with RIPA lysis buffer and measured total protein concentration using a BCA assay kit (Qcbio Science Technologies Co., Ltd., Shanghai, China).Prepared protein samples were separated with 10% SDS-PAGE and transferred into SDS-acrylamide gel and passed to polyvinylidene difluoride membrane (PVDF, Merck, NYC, USA), after sealing with 10% defatted milk for 1 h.Subsequently, bands were incubated with primary antibodies (Antibodies against Bax, Bcl-2, cleaved caspase-3, RAF1, and GAPDH) overnight at 4 ℃ following incubation with an HRP-conjugated secondary antibody at 234 ℃ for 2 h.In the end, an efficient chemiluminescence kit (ECL, Enzyme-linked Biotechnology Co.) was used to visualize these membranes.

Luciferase reporter assay
The sequences matched with miR-7-5p (WT, mutant) in 3′-UTR of the SOX21-AS1, and the RAF1 gene was subcloned into the downstream of a luciferase reporter gene to structure plasmids.In the 24-well plate, SOX21-AS1-WT or SOX21-AS1-MUT (RAF1 WT or RAF1 MUT) was co-transfected with miR-7-5p mimic or miR-NC into cells by lipofectamine 3000.After 48 h, a luciferase assay kit (Yeasen) was performed to evaluate the luciferase activity.

RIP assay
The RIP RNA-Binding Protein Immunoprecipitation Kit (Lab-bio, Beijing, China) performed the RIP assay.First, HEC-1A and Ishikawa were lysed with RIP lysis buffer.Then, cell supernatants were moved into EP tubes and incubated with anti-Ago2 or anti-lgG antibodies and magnetic beads.Next, these samples were incubated with protease K, the proteins were digested, and isolated RNA and qRT-PCR were performed.

Statistical analysis
All data were shown as mean ± standard deviation (SD).The Bonferroni post hoc test was used to compare between the untreated control and treated groups, P < 0.05.

SOX21-AS1 expression is upregulated in EC tissues and cells
The EC tissues and para-carcinoma tissues were collected to detect the functions of SOX21-AS1 on the occurrences of EC.. Firstly, SOX21-AS1 expression was augmented in EC tissues compared with tumor-adjacent tissues (Fig. 1A).Similarly, the expression of SOX21-AS1 was upregulated in EC cells (RL95-2, HEC-1A, Ishikawa, and AN3CA) than ESC, especially in HEC-1A and Ishikawa cells (Fig. 1B), which was used in subsequent experiments.Furthermore, to explore the diagnostic value of SOX21-AS1 for patients with EC, a ROC curve was applied and the AUC of the ROC curve was 0.639 (Fig. 1C).We found SOX21-AS1 expression levels were positively correlated with clinical pathological parameters.Tissues expressing higher SOX21-AS1 levels showed higher tumor grade and the presence of lymph node metastasis (Table 1).Moreover, the Kaplan-Meier method discovered that EC patients with low SOX21-AS1 expression had high overall survival (Fig. 1D).

Knockdown of SOX21-AS1 suppresses the proliferation, migration, invasion, and promotes the apoptosis of EC cells
Next, we transfected sh-SOX21-AS1 into HEC-1A and Ishikawa cells to probe the functions of SOX21-AS1 on EC progression.The knockdown efficiency of SOX21-AS1 was measured, demonstrating that sh-SOX21-AS1 dramatically suppressed SOX21-AS1 expression.Functionally, the CCK-8 and colony formation assay showed that the downregulation of SOX21-AS remarkedly inhibited cell viability and cell proliferation (Fig. 2B-C).
Besides, silencing of SOX21-AS1 inhibited the migration and invasion of EC cells by transwell assay (Fig. 2D).After that, flow cytometry data displayed that the apoptosis ratio was increased in the sh-SOX21-AS1 treated group than in the sh-NC group (Fig. 2E).Consistently, the levels of Caspase-3 and Bax were upregulated, whereas Bcl-2 was downregulated in SOX21-AS1-treated group compared with sh-NC group (Fig. 2F).

MiR-7-5p has a binding site in SOX21-AS1
Bioinformatics software predicted miRNAs targeting SOX21-AS1 and discovered that miR-7-5p had potential binding sequences in SOX21-AS1.The possible crucial line is shown in Fig. 3A.The luciferase reporter analysis was carried out to prove the correlation between miR-7-5p and SOX21-AS1.We found that the relative luciferase activity of miR-7-5p mimic and SOX21-AS1 WT co-transfected cells was dramatically reduced compared with NC mimic and SOX21-AS1 WT co-transfected cells; however, there were no significant changes in SOX21-AS1 MUT transfected cells (Fig. 3B).

R E T R A C T E D A R T I C L E
Additionally, similar results were obtained in the RIP test, which found that SOX21-AS1 could form co-immunoprecipitation with miR-7-5p (Fig. 3C).And silencing of SOX21-AS1 markedly promoted the expression of miR-7-5p (Fig. 3D).Furthermore, miR-7-5p expression was decreased in EC tissues and cells (Fig. 3E).And there was a negative relationship between SOX21-AS1 expression and miR-7-5p expression in EC tissues (Fig. 3F).

RAF1 acts as the downstream target of miR-7-5p
Bioinformatics predicted the target of miR-7-5p and determined RAF1.The combining sequence of miR-7-5p and RAF1 was displayed in Fig. 4A.Then, the correlation between miR-7-5p and RAF1 was testified by the luciferase reporter analysis, founding that luciferase activities were remarkedly lessened in miR-7-5p mimic and RAF1 WT transfected cells compared with NC mimic and RAF1 WT transfected cells.In contrast, the luciferase activity had no change in RAF1 MUT transfected cells (Fig. 4B).In the HEC-1A and Ishikawa cells, RAF1 had a poor expression in the miR-7-5p mimic group compared to the control group (Fig. 4C-D).Moreover, RAF1 expression was enhanced in EC tissues and cells compared to normal tissues and cells (Fig. 4E).

R E T R A C T E D A R T I C L E
the functions of miR-7-5p inhibitor on cell viability and proliferation were reversed by knockdown of RAF1 (Fig. 5B-C).The Transwell assay also demonstrated that downregulation of miR-7-5p accelerated cell migration and invasion in HEC-1A/sh-SOX21-AS1 and Ishikawa/ sh-SOX21-AS1 cells, which was reversed by sh-RAF1 (Fig. 5D).Furthermore, flow cytometry assay uncovered that silencing of miR-7-5p inhibited cell apoptosis, which was reversed by sh-RAF1 in HEC-1A/sh-SOX21-AS1 and Ishikawa/sh-SOX21-AS1 cells (Fig. 5E).

Discussion
Increasing pieces of evidence reveal that tumors are closely related to the abnormal expression of lncRNAs [18,19].Previous reports have confirmed the involvement of various lncRNAs in EC, such as lncRNA H19 [20], lncRNA MALAT1 [21], and lncRNA ZXF1 [22].However, the functions of SOX21-AS1 in EC have yet to be explored.Our research discovered that SOX21-AS1 facilitated cell proliferation and suppressed the apoptosis of EC cells by modulating the miR-7-5p/RAF1 pathway.

R E T R A C T E D A R T I C L E
Previous studies have reported that SOX21-AS1 expression is significantly upregulated in tissue samples and cells of colorectal cancer, and colorectal cancer patients with high SOX21-AS1 expression present poor prognosis [17].Our study disclosed that SOX21-AS1 expression was prominently increased in EC tissues and cells compared with normal tissues and cells.Therefore, SOX21-AS1 may be an oncogene of EC.Consistent with this hypothesis, the downregulation of SOX21-AS1 restrained cell viability, proliferation, migration, invasion, and accelerated cell apoptosis in EC.Consistent with our current data, Zhang XY et al. discovered that the expression of SOX21-AS1 was prominently upregulated, and silencing of SOX21-AS1 reduced cell proliferation and migration in cervical cancer [23].
It is reported that lncRNAs regulate biological function at epigenetic, transcriptional, and post-transcriptional levels [24].When lncRNAs are located in the cytoplasm, they often bind to miRNAs as ceRNAs and then regulate the expression of multiple target genes [25].Hence, we predicted the target of SOX21-AS1 and identified miR-7-5p through the bioinformatics online tool, and there was a potential binding sequence between miR-7-5p and SOX21-AS1.A recent study illustrated that miR-7-5p inhibited cancer metastasis in NSCLC and identified that miR-7-5p might function as an antioncogene [26].So, does miR-7-5p have suppression functions on EC development?Further, we verified that miR-7-5p targets binding with SOX21-AS1 by luciferase reporter analysis and RIP analysis, finding that SOX21-AS1 negatively regulated miR-7-5p expression in EC tissues, suggested that miR-7-5p may exert a significant function on EC occurrences.
MiRNAs combine with the 3′UTR of the downstream gene and modulate the gene translation process, thus affecting the occurrence of cancer [27].In the study, RAF1 was predicted to be a target of miR-7-5p by bioinformatics, and a luciferase reporting experiment verified the relationship between the two.RAF1, a serine-threonine protein kinase, is a critical member of the Ras/Raf/ MEK/Erk signal transduction axis.RAF1 is closely connected with the proliferation, survival, and migration of various cells, including tumor and epithelial cells [28].In epithelial cells, the signal transduction of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor receptor (BFGF) activation passes through the downstream RAF1, which is regarded as the signal hub for several growth factors to induce tumor angiogenesis [29].Blocking RAF1 inhibits the survival and angiogenesis of tumor cells [30], so RAF1 is expected to become a new target for tumor therapy.For example, the knockdown of RAF1 kinase activity inhibits tumor growth in human colorectal cancer [31].We discovered that RAF1 expression was increased in EC tissues and cells.Rescue experimental results proved that the influence of sh-SOX21-AS1 on EC cell viability, proliferation, and apoptosis was reversed by silencing of miR-7-5p, and such reversible effects were abolished by the knockdown of RAF1.

Conclusion
In summary, our study revealed that SOX21-AS1 accelerated EC development through miR-7-5p/RAF1, which may provide a new treatment target for EC.

R E T R A C T E D A R T I C L E
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Fig. 1
Fig. 1 SOX21-AS1 expression is increased in EC tissues and cells.A The level of SOX21-AS1 in EC tissues and para-carcinoma tissues assessed by RT-qPCR, n = 30.B The level of MNX1-AS1 in EC cells and ESC.C The ROC curves.D The overall survival rates of EC patients were compared in SOX21-AS1 high-expression and low-expression groups.** P < 0.01, *** P < 0.001

Fig. 2 RFig. 3 Fig. 4
Fig. 2 Silencing of SOX21-AS1 inhibits the proliferation, migration, and invasion and promotes the apoptosis of EC cells.HEC-1A and Ishikawa cells were treated with sh-SOX21-AS1 and sh-NC, respectively.A The expression of SOX21-AS1 measured by RT-qPCR.B CCK8 assay detected cell viability.C Colony formation assay.D Cell migration and invasion measured through transwell assay.E Flow cytometry detected cell apoptosis.F The protein levels of apoptosis-related genes.** P < 0.01, *** P < 0.001 R E T R A C T E D A R T I C L E

Table 1
Association between SOX21-AS1 expression and clinicopathological characteristics in EC patients (n = 30)

Table 2
The primers used for RT-