lncRNA FEZF1‐AS1 regulates biological behaviors of cervical cancer by targeting miRNA‐1254

Abstract Aim The purpose of this research was to evaluate lncRNA FEZF1‐AS1 in cervical cancer development and clinical significance. Materials and methods Collecting cervical cancer tissues, measuring FEZF1‐AS1 expression, and analysis correlation between FEZF1‐AS1 and prognosis. In cell vitro study, using MTT assay to measure cell proliferation, evaluating cell apoptosis by flow cytometry, measuring cell invasion and migration by Transwell and wound healing assay; lncRNA FEZF1‐AS1 and miR‐1254 gene expressions were evaluated by RT‐qPCR assay; relative protein (Smurf1, E‐cadherin, Vimentin, N‐cadherin, AKT, p‐AKT, c‐Myc, and ZEB1) expressions were measured by Western blot assay. The correlation among FEZF1‐AS1, miR‐1254, and Smurf1 were analysis by dual luciferase reporter gene assay. Results By clinical analysis, lncRNA FEZF1‐AS1 was high expression in cervical cancer tissues and high expression was closely correlated with poor prognosis in cervical cancer patients. In vitro study, the SiHa and HeLa cell biologically including cell proliferation, migration, and invasion of si‐FEZF1‐AS1 group which knockdown lncRNA FEZF1‐AS1 were significantly depressed (p < .001, respectively). However, with miR‐1254 expression inhibiting, the cell biological activities were significantly increased in si‐FEZF1‐AS1+miRNA inhibitor groups (p < .001, respectively). Conclusion lncRNA FEZF1‐AS1 might be an oncological role in cervical cancer; lncRNA FEZF1‐AS1 knockdown had antitumor effects with miR‐1254 activating in cervical cancer by in vitro study.

Moreover, more and more evidence showed that lncRNAs are involved in the tumorigenesis and development of CC: lncRNA-CTS regulates ZEB2 through a competitive endogenous RNA mechanism, affecting the epithelial-mesenchymal transition (EMT) of CC cells (Feng et al., 2019); lncRNA-CCDST promotes the interaction between MDM2 and DHX9 as a "scaffold," inducing angiogenesis and invasive metastasis of CC (Ding et al., 2019). A new study showed that lncRNA FEZ family zinc finger 1 antisense RNA1 (FeZF1-AS1) is a key regulator of human cancers Wang et al., 2018). However, the expression and relevant mechanisms of FEZB1-AS1 in CC are still unclear.
miR-1254 is a cancer-related gene. Some studies have shown that the expression of miR-1254 in gastric cancer is downregulated; the overexpression of miR-1254 can significantly inhibit the proliferation, metastasis, and invasion of gastric cancer cells . LncRNA DCST1 antisense RNA1 (DCST1-AS1) promotes the proliferation and growth of tumor cells by adsorbing miR-1254 and regulating the expression of FAIM2 . Bioinformatics predictions showed that miR-1254 is a potential target gene of FEZF1-AS1, but it is still unclear whether FEZF1-AS1 affects the biological behaviors of CC cells by regulating the expression of miR-1254. The study is aimed at exploring the biological functions and regulatory mechanism of FEZF1-AS1 in CC by analyzing the expression of lncRNA FEZF1-AS1 in CC and its correlations with the prognosis of patients with CC and then by interfering with the expression of FEZF1-AS1 or overexpressing miR-1254 in CC cells.

| Tissue specimens
The study was approved by the Ethics Committee of the hospital. The pathological tissues of a total of 125 patients treated in Chongqing General Hospital from February 2012 to February 2017 were selected. These patients were informed of the study and signed informed consent forms. The patients enrolled were diagnosed with CC and received radical resection in our hospital. The clinical staging was based on the 2009 FIGO clinical staging criteria for CC. All tumor tissues and adjacent normal tissues (ANTs) were directly sampled from patients. ANTs were more than 5cm from tumor edges; and significantly calcified or necrotic parts were avoided; after the specimens were sampled, they were placed in freezing tubes and immediately stored in liquid nitrogen for subsequent experiments.
These patients did not receive preoperative chemoradiotherapy and had no history of other tumors or major organ diseases. Patients were followed up by phone (5 patients were not followed up) to record their prognostic survival data and overall survival (OS): TNM staging, stages I-II: 73 cases and stages III-IV: 47 cases. Patients were followed up since half a year after the operation. The endpoint of follow-up was death of patients or May 31, 2020, the deadline of this study.

| In situ hybridization (ISH)
In order to detect the expression of lncRNA FEZF-1-AS1 in CC tissues and ANTs, ISH was performed according to the kit instructions (the kit was purchased from Boster, Wuhan). The digoxin-labeled lncRNA FEZF1-AS1 probe (1:400) was added to paraffin sections of tissues, after which tissues were incubated at 55℃ for 1h and then washed. Then, the tissues were sealed with a reagent for 1h; after removing the reagent, tissue sections were placed in TBST containing anti-digoxin antibodies (1:200) and incubated at 37℃ for 1h.
Finally, HE staining was performed; and the dyeing effects were observed and photographed under the microscope. The image analysis software ImageJ was used to analyze IOD values in tissues.

| Cell strains and cell culture
CC cell strains (SiHa and HeLa) were purchased from ATCC (USA).
1% double antibodies to penicillin-streptomycin and 10% fetal bovine serum (FBS) were added to the DMEM. The cells were routinely placed in an incubator containing 5% CO 2 at 37℃ and experienced a passage every 2~3d, until the cells reached the logarithmic phase.

| Cell transfection and experimental grouping
si-FEZF1-AS1, si-NC, and miR-1254 were transfected into Siha or Hela cells by lipofection. The transfected cells were labeled as Si-FezF1-AS1, Si-NC, and Mir-1254. 48h after the transfection, the transfection effect was detected by qRT-PCR. Follow-up studies were performed. The normally cultured CC cells were used as a NC group. In order to further verify whether lncRNA FEZF1-AS1 affects the biological behaviors of CC cells by regulating the expression of miR-1254, si-FEZF1-AS1, and miRNA-1254, inhibitors were cotransfected into CC cell strains. The transfected cells were labeled as NC, si-FEZF1-AS1, miR-1254, and si-FEZF1-AS1+miRNA inhibitor.

| Detection of the expression of FEZF1-AS1 and miR-1254 by RT-qPCR
The transfected CC cells in each group were collected. The reagent TRIzol was used to extract the total RNA, which was reversely tran- and 3min, 95℃ and 30s, 58℃ and 30s, and 72℃ and 30s for a total of 35 cycles. The relative expression quantity of the target genes was calculated by 2 -ΔΔCt .

| Detection of cell proliferation rates by MTT testing
The CC cells from all groups were inoculated into 96-well plate according to 2 × 10 3 /well. 48h after the transfection, 20 μL MTT F I G U R E 1 FEZF1-AS1 expression and correlation with prognosis. (a) FEZF1-AS1 expression in tissues by ISH assay. **p <.01, ***p <.001, compared with adjacent. (b) Correlation between FEZF1-AS1 and prognosis solution (5 mg/ml) was added to each well. Cells were incubated in an incubator for 4h. The supernatant was discarded. Then, 150 μL DMSO was added to each well, after which cells were further incubated for 2h. An automatic microplate reader was used to determine the absorbance (A) at the wavelength of 490nm (zero setting was performed in a blank well).

| Flow cytometry (FCM)
48h after the corresponding treatment of cells in each group, after trypsin digestion, cells in each group were collected by centrifugation and washed with PBS. Binding buffer was added. Cells were resuspended to make the cell concentration reach 1*106 / ml. 100μL cell suspension was taken and added to a flow tube.
5 μL Annexin VFITC and 5 μL PI were added. The mixture was mixed well and incubated at a room temperature and away from light for 15min. 400μL binding buffer was added to each tube.
After mixing, cells were placed on the flow cytometer to detect the apoptosis.

| Transwell assay
Chambers coated with Matrigel were placed in a 24-well plate.
300μL preheated serum-free medium was added to the upper chamber. Chambers were allowed to stand at ambient temperature for 15min. When the Matrigel was hydrated, the remaining culture solution was removed. 48h after the transfection, cells were digested, washed with PBS, and resuspended with serum-free medium to adjust the cell density to 3*105 /ml. 200μL cell suspension was taken and added to the Transwell chamber. 500μL medium containing 10% were wiped off with a cotton swab and stained with 0.1% crystal violet. The Transwell chamber was inverted under a microscope for observation and photography. Five fields were randomly selected to count invading cells. The mean was calculated.

| Would healing assay
When cells in all groups were treated accordingly for 48h, 3*10 4 cells were added to each hole. The next day, low-concentration serum-containing medium was used. A scratch tester was used to push the scratch upward from the central part at the lower end of the 6-well plate. Cells were rinsed with the serum-free medium twice, after which the low-concentration serum-containing medium was added for photography for 0h. Cells were placed in an incubator with 5% CO 2 at 37℃ for incubation and photographed at 24h and 48h. The wound healing rate of cells in each group was calculated.

| Western blot (WB) assay
The total protein of cells in each group was extracted. The BCA was used to quantify protein concentration. 30Μg protein was taken for SDS-PAGE electrophoresis and then transferred to PVDF membrane. The corresponding antibodies were added. The protein was incubated at 4℃ overnight. Secondary antibodies (diluted concentration 1:1,000) were added. ECL development, exposure, and photography were performed. The gel imaging analysis system and the software ImageJ were used to analyze the gray value of each strip.

| Statistical analysis
The statistical software SPSS 23.0 was used for statistical analysis.
The measurement data were expressed as mean±SD. The paired t test was applied to analyze the differences in the expression of lncRNA

| Analysis of FEZF1-AS1 expression and correlation with prognosis
The results of ISH detection showed that the expression of FEZF1-AS1 in CC tissues at stages I-II and stages III-IV was significantly higher than that in ANTs (p <.01, respectively, Figure 1a).
After RT-qPCR detection of the 120 cancer tissues included in this study, 120 patients were divided into a low expression group and a high expression group according to the median. The results of analysis by the Kaplan-Meier method indicated that the OS in the low expression group was significantly longer than that in the high expression group (p <.000, Figure 1b)

| Influence of the knockout of FEZF1-AS1 on proliferation of CC cells
The results of MTT testing showed that the proliferation rates of SiHa and HeLa cells in the FEZF1-AS1-knocked out group were significantly lower than those in the NC group (p <.001, respectively, Figure 2a&b), while no significant difference in the proliferation rate was found between Siha cells and Hela cells in the si-NC group (p >.05), indicating that the transfection process did not impair cell proliferation.

| Influence of the knockout of FEZF1-AS1 on apoptosis of CC cells
The results of the FCM showed that the apoptosis rates of SiHa and HeLa cells in the FEZF1-AS1-knocked out group were significantly higher than those in the NC group (p <.001, respectively, Figure 3a&b), while no significant difference in the apoptosis rate was found between Siha cells and Hela cells in the si-NC group (p >.05), indicating that the transfection process did not affect cell apoptosis.

| Influence of the knockout of FEZF1-AS1 on invasion of CC cells
According to the Transwell results, the invasion of SiHa and HeLa cells in the FEZF1-AS1-knocked out group was significantly lower than that in the NC group (p <.001, respectively, Figure 4a&b), while SiHa and HeLa cells in the si-NC group had no significant difference in invasion (p >.05), indicating that the transfection process did not affect the invasion of SiHa and HeLa cells.

| Influence of the knockout of FEZF1-AS1 on migration of CC cells
According to the wound healing assay results, the 24-hr and 48-hr wound healing rates in the FEZF1-AS1-knocked out group were significantly lower than those in the NC group (p <.001, Figure 5a&b), while the wound healing rates of SiHa and HeLa cells in the si-NC group showed no significant difference (p >.05).

| Influence of the knockout of FEZF1-AS1 on relevant genes
According to the RT-qPCR detection, the expression of lncRNA FEZF1-AS1 gene in the si-FEZF1-AS1 group was significantly lower than that in the NC group (p <.001, respectively, Figure 6a&b), while the expression of miR-1254 gene was significantly increased (p <.001, respectively, Figure 6a&b). The result indicated that lncRNA FEZF1-AS1 could negatively regulate miR-1254 in CC SiHa and HeLa cells.

| Influence of the knockout of FEZF1-AS1 on relevant proteins
The results of WB indicated that the expression of Smuf1, Vimentin, N-cadherin, p-AKT, c-Myc, and ZEB1 proteins in the si-FEZF1-AS1 group was significantly lower than that in the NC group, while the expression of the E-cadherin protein increased significantly (p <.001, respectively, Figure 7a&b); however, the expression of E-cadherin, Smuf1, Vimentin, N-cadherin, p-AKT, c-Myc, and ZEB1 proteins in the si-NC group showed no significant difference (p >.05).

| Role of miR-1254 in regulation of CC cell proliferation by si-FEZF1-AS1
The results of MTT testing showed that the cell proliferation rates in the si-FEZF1-AS1 group and the miR-1254 group were significantly lower than that in the NC group (p <.001, respectively, Figure 8a&b); after the cotransfection of si-FEZF1-AS1 and miRNA inhibitors, the cell proliferation rate in the si-FEZF1-AS1+miRNA inhibitor group was much higher than that in the si-FEZF1-AS1 group (p <.001, respectively, Figure 8a&b). It can be inferred that the inhibition of CC cell proliferation activity through the knockdown of FEZF1-AS1 might be realized by upregulating miR-1254.

| Roles of miR-1254 in regulation of CC cell apoptosis by si-FEZF1-AS1
The FCM results showed that the cell apoptosis rates in the si-FEZF1-AS1 group and the miR-1254 group were significantly higher than that in the NC group (p <.001, respectively, Figure 9a&b); after the cotransfection of si-FEZF1-AS1 and miRNA inhibitors, the cell apoptosis rate in the si-FEZF1-AS1+miRNA inhibitor group was much lower than that in the si-FEZF1-AS1 group (p <.001, respectively, Figure 9a&b).

| Roles of miR-1254 in regulation of CC cell invasion by si-FEZF1-AS1
According to the Transwell results, the number of invading inhibitor group was much higher than that in the si-FEZF1-AS1 group (p <.001, respectively, Figure 10a&b).

| Roles of miR-1254 in regulation of CC cell migration by si-FEZF1-AS1
The results of wound healing showed that the wound healing rates in the si-FEZF1-AS1 group and the miR-1254 group were F I G U R E 9 Roles of miR-1254 in regulation of CC cell apoptosis by si-FEZF1-AS1. (a) Apoptosis rate of Siha cell groups. (b) Apoptosis rate of Hela cell groups. ***p <.001, compared with NC; ###p <.001, compared with si-FEZF1-AS1 significantly lower than that in the NC group (p <.001, respectively, Figure 11a&b); after the cotransfection of si-FEZF1-AS1 and miRNA inhibitors, the wound healing rate in the si-FEZF1-AS1+miRNA inhibitor group was much higher than that in the si-FEZF1-AS1 group (p <.001, respectively, Figure 11a&b).

| Expression of FEZF1-AS1 and miR-1254 in each group
The RT-qPCR detection results indicated that compared with the NC group, the expression of FEZF1-AS1 gene in the si-FEZF1-AS1 group and the si-FEZF1-AS1+miRNA inhibitor group was significantly lower (p <.001, respectively, Figure 12a&b); and the expression of miR-1254 gene in the si-FEZF-AS1 group and the miR-1254 group was significantly higher (p <.001, respectively, Figure 12a&b); compared with the si-FEZF1-AS1 group, the expression of FEZF1-AS1 gene in the miR-1254 group was significantly higher (p <.001, respectively, Figure 12a&b); the expression of miR-1254 in the si-FEZF1-AS1+miRNA inhibitor group was significantly lower (p <.001, respectively, Figure 12a&b).

| Relevant proteins by WB assay
Compared with the NC group, the expression of Smuf1, Vimentin, N-cadherin, p-AKT, c-Myc, and ZEB1 proteins in the si-FEZF1-AS1 group and the miR-1554 group was significantly lower, while the expression of E-cadherin protein was significantly higher (p <.001, respectively, Figure 13a&b significantly higher, while that of E-cadherin proteins was significantly lower (p <.001, respectively, Figure 13a&b).

| Targeted regulation of miR-1254 by FEZF1-AS1 and targeted regulation of Smurf1 by miR-1254
The dual luciferase reporter gene assay showed that FEZF1-AS1 could realize targeted regulation of miR-1254 ( Figure 14a); further detection found that miR-1254 could in turn realize targeted regulation of Smurf1 (Figure 14b).

| D ISCUSS I ON
CC is a malignant tumor of the female reproductive system worldwide and seriously threatens women's health. Research on CC has been gradually refined from the cellular level to the molecular level (Small et al., 2017). As reported by multiple studies, lncRNAs can be closely correlated to tumorigenesis and development by binding to proteins or regulating the expression of miRNAs. Some studies also stressed the carcinogenic features of lncRNAs and lncRNAs' contributions to the malignant phenotype of CC: lncRNA PCAT6 regulates ZEB1 through the competitive endogenous RNA mechanism to affect the chemotherapy resistance of CC cells (Ma et al., 2020); LINC00511 acts synergistically with the transcription factor RXRA to regulate the expression of PLD1 and promote the autophagy and apoptosis of CC cells (Shi et al., 2020); while lncRNA ANRIL, lncRNA CCHE1, and MEG3 may be closely related to the abnormal proliferation and escape from apoptosis of CC cells Zhu and Han, 2019;Chen et al., 2017). According to a recent study (Bian et al., 2018), the expression of FEZF1-AS1 in colon cancer tis- can effectively inhibit the bioactivity of CC cells. It can be seen that FEZF1-AS1 can trigger CC, but the molecular mechanism of its roles is still unclear.
In recent years, studies have found that lncRNAs can reduce the Previous studies have shown that the abnormally elevated expression of Smurf1 is an important participant in tumorigenesis (Xie et al., 2014). Nevertheless, other studies have shown that Smurf1 can inhibit tumor activity . It was found in this study that after the up-regulation of miR-1254 due to the knockout of FEZF1-AS1, the bioactivity of CC cells was effectively controlled, realizing the targeted inhibition of Smurf1 expression by miR-1254.
Previous studies have shown that the overactive PI3K/AKT signaling pathway is an important factor that leads to the enhancement of abnormal proliferation and metastasis of tumors (Xu et al., 2015).
Meanwhile, some reports (Ke et al., 2017) have shown that Smurf1 can regulate the activity of the PI3K/AKT signaling pathway as a regulator. In addition, as the downstream proteins of the PI3K/AKT signaling pathway, c-Myc and ZEB1 play an important role in regulating the activity (proliferation, invasion, and migration) of tumor cells (Deng et al., 2018;Yuan et al., 2014). According to the results of the study, the reduced expression of c-Myc and ZEB1 may be an important factor to decrease the bioactivity of CC cells.
The EMT refers to the biological process in which epithelial cells are transformed into specific mesenchymal phenotypic cells by specific procedures (Pastushenko and Blanpain, 2019). During tumorigenesis, the EMT is closely correlated to the invasion, migration, and drug resistance of tumors (Vynckier and Schmidt, 1998). The abnormally expressed E-cadherin, Vimentin, and N-cadherin are symbolic proteins of the EMT (Yamashita et al., 2018). In this study, the knockdown of FEZF1-AS1 could significantly up-regulate the expression of E-cadherin and down-regulate that of Vimentin and N-cadherin.
Further experiments confirmed that the mechanism was closely related to the elevated expression of miR-1254.
In summary, the results of the study indicated that the abnormal overexpression of FEZF1-AS1 may be one of the factors that lead to the tumorigenesis and development of CC and that the knockout of