FAM201A Promotes Cervical Cancer Progression and Metastasis through miR-1271-5p/Flotillin-1 Axis Targeting-Induced Wnt/β-Catenin Pathway

This study investigated the role of the family with sequence similarity 201-member A (FAM201A), as previously reported oncogenic, in cervical cancer (CC). FAM201A expression in CC was analyzed through bioinformatics analyses, and its distribution in CC tissues/cells was determined by in situ hybridization. CC cells were transfected/cotransfected with FAM201A/flotillin-1 (FLOT1) overexpression plasmids and miR-1271-5p mimics, followed by functional analysis on viability, migration and invasion. Pearson's correlation tests were performed to analyze the correlation between FAM201A and miR-1271-5p in CC tissues. The targeting relationship between miR-1271-5p and FLOT1 was confirmed by dual-luciferase reporter assay. The expressions of FAM201A, miR-1271-5p, FLOT1, matrix metalloproteinases (MMP)-9, MMP-2, E-cadherin, N-cadherin, and the Wnt/β-catenin pathway-related molecules (Wnt1, β-catenin and p-β-catenin) in CC cells or tissues were assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and/or western blot. The results showed that FAM201A was abundantly expressed and miR-1271-5p expression was downregulated in CC. FAM201A was enriched in CC cell cytoplasm and negatively correlated with miR-1271-5p in CC tissues. FAM201A overexpression enhanced the cell viability, migration, invasion, and tumorigenesis of CC in vivo and increased FLOT1 expression. These trends were all reversed by upregulating miR-1271-5p, which induced opposite effects to FAM201A overexpression. MiR-1271-5p upregulation depleted the levels of MMP-9, MMP-2, N-cadherin, and the Wnt/β-catenin pathway-related molecules and upregulated E-cadherin expression. FLOT1 was a direct target of miR-1271-5p. FLOT1 overexpression induced effects contrary to the upregulation of miR-1271-5p and abolished miR-1271-5p upregulation-induced effects in CC cells. Overall, this study showed that FAM201A promoted cervical cancer progression and metastasis by targeting the miR-1271-5p/FLOT1 axis-induced Wnt/β-catenin pathway.


Introduction
Cervical cancer (CC) is one of the most common malignant tumors affecting women worldwide, second to breast cancer [1], and is the leading cause of cancer-related mortality in some developing countries [2]. The progression of CC is featured as a multistage and multistep process involving the activation of proto-oncogenes and (or) inhibiting tumorsuppressive genes [3]. Currently, the antitumor treatment for CC remains less effective owing to its late-appearing symptom, leading to unsuccessful disease diagnoses and advancedstage disease by the time of diagnosis [4]. It is reported that the five-year survival rate for metastatic CC patients is 16.5%, compared to 91.5% for localized CC patients [5]. Therefore, metastasis is accountable for most unfavorable prognoses, recurrence and high morbidity of CC [6].
Long noncoding RNAs (lncRNAs), a type of transcripts constituted by over 200 nucleotides with no translation ability, have emerged as pivotal regulators for the carcinogenesis and progression of cancers, including CC [6]. Epithelialmesenchymal transition (EMT), a highly conserved transdifferentiation program considered the major driver of cancer progression, is reported to facilitate metastasis of cancer cells by promoting migration and invasion and conferring an apoptosis-resistant property [7]. By directly or indirectly reversing EMT, lncRNAs can repress tumorigenesis, cancer progression, and metastasis, demonstrating their therapeutic potential [8]. The family with sequence similarity 201member A (FAM201A) is a long nonprotein coding RNA derived from an open reading frame (ORF)-lacking RNA transcripts transcribed from a 2.9 Kbp-long gene that is located in genomic 9p13.1 [9]. Several studies exploring anticancer strategies have revealed the involvement of FAM201A in inducing carcinogenesis and promoting the progression of triple-negative breast cancer (TNBC) [10], lung squamous cell cancer (LSCC) [11], and lung adenocarcinoma (LUAD) [12]. Additionally, highly expressed FAM201A was reported to provoke short-term radio-resistance, leading to inferior survival in patients with esophageal squamous cell cancer [13] and nonsmall-cell lung cancer [14]. However, little is known about the biological roles and clinical significance of FAM201A in CC.
Interactive analyses have identified FAM201A as a key regulator in cancer progression in a lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network, via which FAM201A was found to indirectly regulate the expression of messenger RNA (mRNA) by sponging its targeted microRNAs (miRNAs) [10,14,15]. Without lncRNAdirected sponging effects, miRNAs, a class of small noncoding RNAs with 18-24 nucleotides in length that are endogenous and evolutionarily conserved, can destabilize mRNAs or inhibit translation, thereby repressing mRNA expression by complementarily binding to the 3'-untranslated regions of the mRNAs [16,17].
A large number of miRNAs have been implicated in CCassociated ceRNA networks. For instance, MiR-1271-5p expression was previously reported to be aberrantly downregulated in acute myeloid leukemia [18], colon cancer [19], multiple myeloma [20], and LUAD [21], indicating that it played a tumor-suppressive role in the progression of these cancers through related ceRNA networks. Meanwhile, upregulated miR-1271-5p expression was shown to induce oncogenic effects and associated with unfavorable prognoses in hepatocellular carcinoma (HCC) [22]. However, whether FAM201A regulates miR-1271-5p through the ceRNA network and thus participates in CC progression remains unconfirmed.
In this study, we investigated the effects of FAM201A in CC progression using bioinformatics tools and determined the potential miR-1271-5p-targeted mRNA for identifying a FAM201A-miR-1271-5p-mRNA ceRNA regulatory network in CC, with the hope to propose an original molecular therapy for CC.

Materials and Methods
2.1. Ethics Statement. Written informed consent was obtained from all human participants. All animal experiments were performed following the guidelines of the China Council on Animal Care and Use [23]. The human and animal studies Journal of Oncology        7 Journal of Oncology set as negative control (NC). MiR-1271-5p mimic/mimic control (MC) (miR10005796-1-5/miR1N0000001-1-5) was purchased from RIBOBIO (Guangzhou, China). C33a or ME180 cells (4 × 10 4 ) were seeded in 96 well plates until 80% confluence was reached. Transfection working solutions (0.15 μL) were prepared by mixing Lipofectamine 3000 transfection reagents (L3000015, ThermoFisher, USA) and Opti-MEM media (31985062, ThermoFisher, USA). Subsequently, the above plasmids were (2 μg) added into Opti-MEM media (10 μL) together with a P3000 reagent (0.4 μL). Next, the processed plasmids were mixed with the transfection working solution at a ratio of 1 : 1 to obtain an RNAlipid complex, of which 10 μL of the complex mixture was incubated with the cells at 37°C for 24 h or 48 h.
2.10. Murine Xenograft Assay. BALB/c nude mice (Male, 5-6-week-old) were purchased from the Vital River Laboratories (Beijing, China). The mice were maintained under a specific condition (22~24°C, 50% humidity, a 12 h:12 h circadian cycle), with free access to a standard mice chow and water. Then, the mice were randomized into four groups (n = 6 per group): NC+MC group, FAM201A+MC group, NC+miR-1271-5p M group, and FAM201A+miR-1271-5p M group. After transfection, SiHa cells (5 × 10 6 ) with stable expressions of FAM201A, miR-1271-5p or both were subcutaneously injected into the posterior flank of the mice. The size of subcutaneous xenografts (length and width) was measured by a caliper every 7 days, with 5 times in total, and the volume of the xenografts was calculated according to the formula: 0:5 × length × width 2 . Five weeks after the injection, the mice were sacrificed via spinal dislocation under anesthe-tization using pentobarbital sodium (P010, Sigma-Aldrich, USA), following which the subcutaneous xenografts were resected and weighed.

Quantitative Reverse Transcription Polymerase Chain
Reaction (qRT-PCR). Total mRNAs and miRNAs from CC cell lines and HCerEpiC, as well as CC tissues and the adjacent normal tissues, were extracted by TRIzol lysis buffer (15596018, ThermoFisher) and Small RNA kits (9753Q, TaKaRa, Liaoning, China), respectively. Chloroform (48520-U, Sigma-Aldrich, USA) was used to extract the lysate of mRNA and miRNA. The extracted lysate was centrifuged (12000 × g) at 4°C for 15 min. Then, isopropanol (W292907, Sigma-Aldrich, USA) was applied to precipitate the lysate from water layers via centrifugation (12000 × g) at 4°C for 10 min, which was then washed with 75% ethanol (32205, Sigma-Aldrich, USA) and then isolated from the supernatant. Next, it was resuspended and centrifugated (7500 × g) at 4°C for 10 min and dissolved in 20 μL diethyl pyrocarbonate (DEPC; 40718, Sigma-Aldrich, USA). Firststrand cDNAs of the isolated mRNA and miRNA were synthesized using a Synthesis Kit (K1621, ThermoFisher, USA    13 Journal of Oncology as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. The expressions of relative genes normalized to U6 or GAPDH were calculated using the 2 −ΔΔCT method [24]. 2.13. Statistical Analysis. Measurement data with normal distribution were expressed as mean ± standard deviation (SD). All the experiments were conducted in triplicate. SPSS software (version 21.0, SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The differences between CC tissues and the adjacent normal tissues were analyzed by paired t-test. Comparison between the other two groups was performed by independent t-test, and those between multiple      Journal of Oncology groups were conducted by one-way analysis of variance (ANOVA) followed by Dunnett's or Turkey's post-hoc test. Pearson's correlation tests were used to analyze the correlation between FAM201A and miR-1271-5p in CC tissues. Statistics with P < 0:05 were considered statistically significant.

FAM201A Was Highly Expressed in CC, Enriched in CC Cell Cytoplasm, and Its Expression Was Negatively
Correlated with miR-1271-5p. According to Gene Expression Profiling Interactive Analysis (GEPIA) based on Cancer Genome Atlas Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (TCGA-CESC) database, FAM201A expression level was higher in CC tissues than that in normal tissues (P < 0:05; Figure 1(a)). Then, we harvested 33 pairs of clinical samples including CC tissues and the adjacent normal tissues for the examination of FAM201A expression. The results showed that FAM201A was highly expressed in CC tissues compared with adjacent normal tissues (Figure 1(b)). Considering that miR-1271-5p has been widely reported as a regulator of tumor growth, in this present study, miR-1271-5p expression was downregulated CC clinical samples and compared with adjacent normal tissues (Figure 1(c)), which showed a negative correlation between miR-1271-5p and lncRNA FAM201A via Pearson's correlation analysis (Figure 1(d)). Meanwhile, fluorescence in situ hybridization assay confirmed that FAM201A was highly expressed in CC tissues compared with adjacent normal tissues (Figure 1(e)). Moreover, in comparison with HCerEpiC, FAM201A expression level was also highly expressed in CC (HeLa, C33a, SiHa, and ME180) cells, while miR-1271-5p expression was decreased (P < 0:01; Figures 1(f) and 1(g)). A relatively higher expression level of FAM201A was seen in SiHa cells and ME180 cells than in other cells above. Therefore, to investigate the role of FAM201A during the progression of CC, SiHa cells    Taken together, the above results suggested that FAM201A-directed sponging of miR-1271-5p was associated with CC progression.

FLOT1 Overexpression Resisted miR-1271-5p
Upregulation-Induced Decrease in Viability and Inhibition in Migration, Invasion and EMT in CC Cells. The miRNA-mRNA networks are widely known to regulate CC progression [25]. Here, as FLOT1 was identified as a target mRNA of miR-1271-5p, we investigated FLOT1-miR-1271-5p network-delivered regulation on CC cell phenotypes. The results showed that CC cells transfected with plasmid overexpressing EMT, a biological process, displays distinctive cellular phenotypes and plays vital roles in both cell growth and cancer progression [26]. Thus, the protein and mRNA levels of EMT-related markers were assessed by western blot and qRT-PCR. Both the protein and mRNA levels of MMP-9, MMP-2, and N-cadherin were upregulated by FLOT1 overexpression in CC cells compared with those in the NC+MC group (P < 0:05), while compared with those in the NC+MC group, miR-1271-5p upregulation depleted the expressions of these markers (P < 0:01) and abolished the FLOT1 overexpression-induced effects on the expressions of these markers (P < 0:05) (Figures 7(a)-7(e)). Additionally, the effects of miR-1271-5p upregulation on these EMT-related markers were counteracted by FLOT1 overexpression (P < 0:05) (Figures 1(f) and 7(a)). Conversely, E-cadherin, an EMT-related marker, and its protein and mRNA levels were downregulated by FLOT1 overexpression but elevated by miR-1271-5p upregulation (P < 0.05), compared with those in the NC + MC group (Figures 8(a)-8(f)). Furthermore, FLOT1 overexpression counteracted the effects of miR-1271-5p upregulation on E-cadherin expression, and miR-1271-5p upregulation also reversed the effects of FLOT1 overexpression (P < 0:05) (Figures 8(a)-8(f)).

Discussion
In 2003, the World Health Organization considered CC preventable in women [20]. However, due to metastasis, the median survival time of CC patients remains mediocre [30]. Metastasis is a distinctive malignant sign that can be subdivided into two types, hematogenous metastasis and lymphatic metastasis [6,31], of which lymph metastasis is the leading factor for CC-associated poor prognosis and death [32]. Metastasis in most human cancers implicates both cellular and molecular alterations [33], identifying that the molecular mechanism in CC is very important for hindering the development of metastasis and other malignant phenotypes.
LncRNA-mediated mechanisms have been widely unveiled in the carcinogenesis, progression, and therapy resistance of CC [34]. Numerous lncRNAs, including FAM201A, have been confirmed to function as an oncogene in multiple types of human cancers by suppressing malignant phenotypes such as cancer cell proliferation, migration, invasion, and in vivo tumorigenesis [10][11][12]. In line with these studies, our study newly identified FAM201A as a key player in promoting CC carcinogenesis and progression. Analysis of the TCGA-CESC database showed that FAM201A was highly expressed in CC. To increase the credibility of this study's results, CC tissues and cell lines (HeLa, C33a, SiHa, and ME180) were used to assess FAM201A expression. We detected a unanimous increase in FAM201A expression in all CC in vitro and in vivo samples, which was consistent with the FAM201A expression patterns in TNBC 22 Journal of Oncology and lung cancer, where FAM201A played oncogenic roles [10][11][12]. The specific tumor-promoting role of FAM201A was illustrated in previous studies, which reported that knocking down FAM201A led to significantly suppressed proliferation, migration, and invasion of TNBC or LSCC cells [10][11][12]. In line with the role of FAM201A in TNBC and LSCC, our study discovered a positive association between FAM201A overexpression and the biological behaviors of CC, including cell viability, migration, invasion, and in vivo tumorigenesis, which suggested that this oncogenic role of FAM201A also existed in CC. Functional analyses of FAM201A-miRNA-mRNA ceRNA networks indicated that FAM201A could sponge miRNAs and unleash mRNAs from the binding of FAM201A, with miRNAs critical for the promotion of cancer progression [10]. Our study found that miR-1271-5p was negatively correlated with FAM201A in CC tissues, implying that FAM201A sponged miR-1271-5p in CC. Previous studies reported that miR-1271-5p expression was significantly downregulated and miR-1271-5p exerted a tumor-suppressive effect in several cancers [18,35]. Preventing oncogene-directed sponging of miR-1271-5p led to the inhibition of cancer progression, as evidenced by Zhang et al. [35], who found that miR-1271-5p upregulation from the knockdown of lncRNA-ZFAS1 constrained in vitro development of glioma. In light of Zhang et al.'s evidence, our findings demonstrated that miR-1271-5p upregulation reversed the promotive effect of FAM201A overexpression on the progression of CC, suggesting that FAM201A facilitated the progression of CC by sponging miR-1271-5p.
Furthermore, it was reported that miRNA-mRNA interaction emerged following the interaction between lncRNA and miRNA in ceRNA networks associated with the pathological conditions in cancer [36]. Wang et al. showed that the upregulation of miR-1271-5p by MALAT1 knockdown inhibited the growth and migration of ovarian cancer cells and simultaneously silenced its target mRNA E2F5 [37]. In our study, bioinformatics prediction theoretically identified FLOT1 as the target of miR-1271-5p, which was subsequently validated by our dualluciferase reporter assay results. Similarly, our findings showed that FAM201A overexpression downregulated miR-1271-5p expression to elevate FLOT1 expression and concomitantly promoted in vitro CC progression, indicating that the overexpressed FAM201A-directed ceRNA network with the miR-1271-5p/FLOT1 axis promoted CC progression.
FLOT1, a pivotal marker of lipid rafts that modulates membrane receptor signaling, has been reported to participate in membrane trafficking and affect cell adhesion and invasion, thereby displaying a role in tumorigenesis [38,39]. The overexpression of FLOT1 has been previously discovered to promote migration and invasion and induce recurrence of bladder transitional cell carcinoma [38], activate oncogenic ALK signaling to drive malignant phenotypes of neuroblastoma [40], and sustain inflammatory signaling to facilitate the growth and invasion of esophageal squamous cell carcinoma cells [40,41]. For CC, FLOT1 was shown to serve as the downstream target of miR-1294 to form a miR-1294/FLOT1 axis, and its expression can be repressed by the upregulation of miR-1294, thereby inhibiting the progression of CC malignant phenotypes [42,43]. Likewise, in our in vitro experiments, miR-1271-5p upregulation decreased FLOT1 expression and offset FLOT1 overexpression-induced promotion. Meanwhile, FLOT1 overexpression could also counteract the inhibitory effects of miR-1271-5p upregulation on cell viability, migration and invasion. According to these findings, we concluded that targeting the miR-1271-5p/FLOT1 axis could be the underlying mechanism via which FAM201A induced CC progression.
Accumulating evidence indicated that EMT, a hallmark of carcinogenesis, functionally contributed to tumor invasion, migration and metastatic dissemination [44]. The phenotype of EMT mainly involves the transformation of epithelial cells to mesenchymal-like cells, allowing them to invade surrounding tissues [45,46]. Induction of EMT is accompanied by the loss of epithelial adhesion molecule Ecadherin [47] and an increase in mesenchymal marker Ncadherin [48]. Moreover, during EMT, MMPs, a family of zinc-dependent endoproteases, degrade the extracellular matrix to facilitate EMT [49]. Secretion of MMP-2 and MMP-9 was shown to break down the basement membrane and promote lymph node invasion and cancer metastasis, thus leading to poor prognoses [50,51]. In this study, we found that E-cadherin levels in CC cells were decreased by FLOT1 overexpression but increased by miR-1271-5p upregulation, and an opposite trend was seen on the levels of N-cadherin, MMP-2, and MMP-9 when FLOT1 was overexpressed or miR-1271-5p expression was upregulated. Besides, we discovered that FLOT1 overexpression counteracted miR-1271-5p upregulation-induced effects on the expressions of these EMT-related markers and vice versa. Collectively, these findings indicated that FAM201A facilitated EMT and promoted CC progression by targeting the miR-1271-5p/FLOT1 axis.
The Wnt/β-catenin pathway, which plays an essential role in embryogenesis, homeostasis, and stem cell regeneration and pluripotency, is activated in CC as a promoter of cancer progression [52,53]. Likewise, our results demonstrated that the levels of Wnt1, β-catenin and p-β-catenin in CC cells were positively correlated with FLOT1 overexpression, while the levels of these markers were negatively correlated with miR-1271-5p upregulation. Besides, our study revealed that FLOT1 overexpression could also restore the expressions of Wnt1, βcatenin, and p-β-catenin in CC cells after the upregulation of miR-1271-5p, which indicated that FLOT1 overexpression counteracted the inhibitory effects induced by miR-1271-5p upregulation on the Wnt/β-catenin pathway, thus promoting the progression of CC.
Considering that FAM201A was overexpressed in CC cells and acted through the FAM201A-miR-1271-5p-FLOT1 ceRNA network, they might be targeted and used to develop novel potential molecular target to improve CC treatment outcomes, with FAM201A as a potential diagnostic biomarker for CC and possible indicator of FAM201Atargeted treatment for individualized treatment of patients 23 Journal of Oncology expressing high levels of FAM201A. However, considering limitations such as lack of survival analysis, no assessment to determine the association of FAM201A with pharmacological treatment, and others, these findings should be further verified in translational and clinical studies.

Conclusion
In conclusion, the current study revealed that FAM201A, which was highly expressed in CC, promoted CC progression via sponging miR-1271-5p to upregulate FLOT1 expression. Moreover, CC progression was also promoted via regulating the miR-1271-5p/FLOT1 axis by activating the Wnt/β-catenin pathway. Thus, this study proposed the FAM201A-miR-1271-5p-FLOT1 ceRNA network as an original molecular target for prevention against CC.

Data Availability
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.