Research ArticleKnockdown of lncRNA FOXD2-AS1 Inhibits Proliferation,Migration, and Drug Resistance of Breast Cancer Cells

Qiaohong Nong , Shaokang Yu, Hui Hu, and Xue Hu

Department of Oncology, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, China

Correspondence should be addressed to Qiaohong Nong; 13510331825@139.com

Received 15 October 2021; Revised 3 November 2021; Accepted 7 November 2021; Published 27 November 2021

Academic Editor: Osamah Ibrahim Khalaf

Copyright © 2021 Qiaohong Nong et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Objective. In order to investigate the effect of lncRNA FOXD2-AS1 on breast cancer cells proliferation, migration, and drugresistance as well as its molecular mechanism. Methods. Real-time PCR was used to detect the expression of breast cancertissues and cells from patients admitted to our hospital and the expression of lncRNA FOXD2-AS1 in MCF-7/ADR inadriamycin- (ADR-) resistant breast cancer cells. After interfering with or overexpressing lncRNA FOXD2-AS1 in MCF-7/ADR cells, cell proliferation, apoptosis, invasion, and migration were detected using CCK-8, flow cytometry, Transwell assay,and scratch test, respectively. The protein levels of PI3K, p-PI3K, AKT, and p-AKT in the PI3K/AKT signaling pathway weredetected by Western blot. Results. lncRNA FOXD2-AS1 was upregulated in breast cancer tissues and cells and increased celldrug resistance to ADR. Downregulation of lncRNA FOXD2-AS1 inhibited invasion and migration of MCF-7/ADR cells,promoted apoptosis, increased chemosensitivity of MCF-7/ADR cells, and inhibited the activity of PI3K/AKT signalingpathway in MCF-7/ADR cells. Conclusions. lncRNA FOXD2-AS1 can promote the proliferation, invasion, migration, and drugresistance of breast cancer cells, inhibit apoptosis, and accelerate the development of breast cancer by positively regulating thePI3K/AKT signaling pathway.

1. Introduction

Cancer is one of the leading causes of death worldwide [1].Breast cancer is one of the most common cancer diseasesin women and the second most fatal cancer worldwide, witha gradually increasing mortality rate in China [2]. So far, sig-nificant progress has been made in surgical treatment, che-moradiotherapy, targeted drugs, and other treatments. Dueto the lack of early effective biomarkers, patients are primar-ily diagnosed in the late stage of the disease, resulting in apoor prognosis. Therefore, there is a need to find effectivebiomarkers to diagnose early breast cancer and improvepatient survival effectively.

Studies have shown that patients receiving long-termchemotherapy often develop chemoresistance [3]. For exam-ple, adriamycin (ADR) is an effective chemotherapeutic drugcommonly used for breast cancer treatment. Still, its drughas become a significant clinical problem and a fundamentalreason for treatment failure [4]. However, the mechanism of

drug resistance and treatment is still completely unclear.Tumorigenesis is characterized by uncontrolled cell prolifer-ation with a high rate of metastasis [5, 6]. Therefore, deter-mining the mechanism of abnormal regulation ofproliferation and metastasis of drug-resistant cells is thekey to urgent identification.

Long noncoding RNAs (lncRNAs) are noncodingRNAs that are greater than 200 nucleotides in length. Avariety of lncRNAs to date have been found to be closelyrelated to the development and metastasis of breast cancer[7, 8]. It has been shown that the expression level oflncRNA HOTAIR in breast cancer tissues is significantlyhigher than that in paracancerous tissues, and it can pro-mote the metastasis of breast cancer cells [9]. lncRNAMALAT1 has also been demonstrated to be highlyexpressed in breast cancer cells and is closely related tothe development and metastasis of tumors [10]. It has alsobeen reported that lncRNA DILA1 can promote the drugresistance of breast cancer cells to tamoxifen by stabilizing

HindawiComputational and Mathematical Methods in MedicineVolume 2021, Article ID 9674761, 9 pageshttps://doi.org/10.1155/2021/9674761

the CyclinD1 protein [11]. Chen et al. [12] found thatoverexpression of lncRNA GAS5 significantly increasedthe sensitivity of breast cancer cells to ADR. Zhang et al.[13] demonstrated that lncRNA XIST promotes chemo-therapy resistance to ADR in breast cancer cells by upreg-ulating ANLN through sponge miR-200c-3p. These studiessuggest that lncRNAs are involved in breast cancer devel-opment and drug resistance. It has been shown thatlncRNA FOXD2-AS1 is a newly discovered lncRNAinvolved in developing a variety of tumors, which was firstreported in hepatocellular carcinoma studies [14]. Subse-quently, several studies have found that the expressionlevel of lncRNA FOXD2-AS1 is upregulated in ovarian,gastric, and bladder cancer and is closely related to theproliferation, migration, invasion, and tumor prognosis oftumor cells, which can be used as a potential target forcancer treatment and prognosis [15–17]. In addition,lncRNA FOXD2-AS1 is also involved in tumor drug resis-tance. lncRNA FOXD2-AS1 regulates cisplatin resistancein non-small-cell lung cancer through the miR-185-5p-SIX1 axis [18]. Silencing of lncRNA FOXD2-AS1 inhibitsproliferation and drug resistance of temozolomide-resistant glioma cells via the miRNA-98-5p/CPEB4 axis[19]. It has also been stated that lncRNA FOXD2-AS1can regulate breast cancer malignancy and tumorigenesisby regulating the miR-150-5P/PFN2 axis [20]. Therefore,we speculate that lncRNA FOXD2-AS1 may play a rolein drug resistance in breast cancer cells. However, relatedstudies have not been reported. In this study, MCF-7/ADR, an adriamycin-resistant breast cancer cell, was usedas the study target. The expression of lncRNA FOXD2-AS1 in MCF-7 and MCF-7/ADR cells was first deter-mined. Meanwhile, the expression of lncRNA FOXD2-AS1 in MCF-7/ADR cells was intervened to furtherexplore its role in ADR resistance in breast cancer cells.This study may provide some reference for future clinicalprevention of drug resistance in breast cancer.

2. Materials and Methods

2.1. Tissue Specimens. A total of 60 cases of tumor tissue and60 cases of adjacent normal tissue were collected frompatients with breast cancer admitted to our hospital fromMay 2020 to March 2021. Samples were immediately frozenin liquid nitrogen and kept at -80°C. All diagnosis was con-firmed by the Department of Pathology of all tissue samples,and all patients have not received chemotherapy or radiationtherapy. All patients have signed the informed consent form,and the Peking University of Shenzhen Hospital committeehas approved this study (2020-023).

2.2. Cell Culture. Human normal breast cells (MCF-10A),human breast cancer cells (MCF-7), and adriamycin-resistant breast cancer cells (MCF-7/ADR) were purchasedfrom Shanghai Cell Research Institute, China. MCF-10Acells were cultured in DMEM/F-12 medium (Gibco,USA), and MCF-7 cells were cultured in 1640 medium(Gibco, USA), both containing 10% fetal bovine serum(FBS; Gibco, USA) and 1% penicillin-streptomycin; MCF-

7/ADR cells were cultured in 1640 medium supplementarywith 20% FBS and 1% penicillin-streptomycin. Incubationwas done in an incubator containing 5% CO2 and 95%humidity at 37°C.

2.3. Cell Transfection. MCF-7/ADR cells were cultured tologarithmic growth period, digested and passaged, dilutedto 2 × 106 cells/mL, and seeded in 6-well plates. Transfectionwas performed when cells were cultured to a confluence of50% to 600%. lncRNA FOXD2-AS1 overexpression vector(FOXD2-AS1) and unloaded vector, lncRNA FOXD2-AS1siRNA fragment (si-lncRNA FOXD2-AS1), and controlsiRNA NC (siNC) were transfected into cells according tothe instructions of Lipo2000 transfection kit (Thermo FisherScientific, USA). FOXD2-AS1 overexpression vector andunloaded vector, FOXD2-AS1 siRNA fragment, and controlsiRNA NC were designed and synthesized by GuangzhouRuibo Biotechnology Co., Ltd. (China).

2.4. qRT-PCR. Cells were collected, and total cellular RNAwas extracted with Total RNA extraction kit (Takala, Japan)and stored at −80°C. RNA was then reverse transcribed intocDNA using a reverse transcription-PCR kit (Takala, Japan).After the concentration and purity of the synthesized cDNAwere detected, an appropriate amount of cDNA was takenfor reaction according to the instructions of real-time PCRreagents (Takala, Japan). The reaction program was as fol-lows: 95°C for 1min; 95°C for 40 s, 58°C for 40 s, and 72°Cfor 45 s, for 35 cycles; and 72°C for 10min. Data analysiswas performed with the 2−ΔΔCt method. The primersequences used are shown in Table 1.

2.5. CCK-8. Cells after drug treatment or transfection wereseeded in 96-well plates at 5000 cells per well. After 24 h ofculturing, 10μL of CCK-8 solution and 90μL of completefresh medium were added to each well. The 96-well platewas placed in the incubator for another 2 h, and the absor-bance value at the wavelength of 450 nm was detected witha microplate reader.

2.6. Apoptosis. Annexin V-allophycocyanin (APC) apoptosisdetection kit (BD Pharmingen, San Jose, USA) was used todetect apoptosis in transfected MCF-7/ADR cells. The cellswere washed twice with precooled PBS buffer with a 1 ×Binding Buffer used to prepare a 1 × 106 cells/mL suspen-sion. 100μL of cells was added to the flow tube with anappropriate amount of Annexin V and nucleic acid dye,gently mixed well, and placed in a dark place at room tem-perature for 15 minutes. Cells were washed once using 1 ×Binding Buffer, and the supernatant was removed. 100μLof 1 × Binding Buffer was used to dissolve 0.5μg of SAv-FITC reagent before adding to the flow tube and gentlymix well. 5μL of PI was added and left at room temperaturein the dark for 15min. 400μL of 1 × Binding Buffer wasadded to each assay tube, and the results were measured bya FACScan flow cell flow system (Becton Dickinson, SanDiego, CA, USA) within 1 h.

2.7. Transwell Assay. Matrigel was removed from -20°C andput at 4°C overnight to become liquid and diluted at 1 : 6 in

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serum-free medium at 4°C. 100μL of Matrigel was added tothe center of the upper chamber of the Transwell and placedat 37°C for 3-5 h until solid state. 100μL of transfected cellswas added to the upper chamber, and 500μL containingcomplete medium was added to the lower layer for culture.After 24 hours of culture, they were fixed with 4% parafor-maldehyde, stained with crystal violet solution, observedunder a microscope, and photographed for statisticalanalysis.

2.8. Scratch Test. A ruler and a permanent marker were usedto evenly draw a horizontal line at an interval of 0.5-1 cm onthe bottom surface of the 6-hole plate. At least 5 lines wereto pass through each hole. Transfected MCF-7/ADR cells(1 × 106 cells/well) were added to 6-well plates and grownto confluence. A sterile 200μL micropipette tip was used tomake a scratch perpendicular to the horizontal line on theback. After a straight wound is made, the cells were washedthree times with PBS to remove the cells in the scratchedarea, followed by the addition of a serum-free medium. Thiswas then put into a 37°C, 5% CO2 incubator and cultured for24 h. An inverted microscope was used to take pictures andto observe and record the migration conditions of cells.The migration area was calculated [21].

2.9. Western Blot. The cells in each group were lysed withRIPA lysate for 20min, and the cells were disrupted by son-ication in an ice bath to collect proteins and detect proteinconcentrations. After performing SDS-PAGE to separateproteins, they were transferred on to PVDF membranes.After blocking for 1 h at room temperature, the membraneswere incubated with primary antibodies PI3K, p-PI3K, AKT,and p-AKT (all purchased from CST, USA) overnight at 4°C.The membrane was washed twice, then added with dilutedenzyme-labeled secondary antibody, incubated at room tem-perature for 1 h, and developed using an exposure meter. β-Actin was used as an internal reference to analyze proteinlevels.

2.10. Statistical Analysis. The experimental data were statis-tically analyzed using SPSS 21.0 software. The results wereexpressed as the mean ± standard deviation (SD). Compari-sons between two groups were performed using Student’s t-test, and comparisons of multiple groups were performedusing one-way analysis of variance. P < 0:05 was recordedas statistically significant.

3. Results

3.1. lncRNA FOXD2-AS1 Is Highly Expressed in BreastCancer and Enhanced Adriamycin Resistance. To investigatethe relationship between lncRNA FOXD2-AS1 and drugresistance in breast cancer and breast cancer cells, we exam-ined the expression of lncRNA FOXD2-AS1 in breast cancertissues and cells using qRT-PCR. The results showed that theexpression of lncRNA FOXD2-AS1 was significantly higherin breast cancer tissues than in paracancerous tissues(Figure 1(a), P < 0:05). In addition, we found that comparedwith normal breast cells (MCF-10A), the expression oflncRNA FOXD2-AS1 was significantly upregulated in breastcancer cells (MCF-7). In breast cancer drug-resistant cells(MCF-7/ADR), the expression was even higher(Figure 1(b)). Meanwhile, compared with the MCF-7 group,the cell survival rate of MCF-7/ADR cells was significantlyincreased at different concentrations of ADR, with theIC50 also increased considerably (Figures 1(c)–1(d), P <0:05). This shows that MCF-7/ADR cells were extremelymore resistant to ADR than MCF-7 cells, which may berelated to lncRNA FOXD2-AS1 expression.

3.2. Knockdown of lncRNA FOXD2-AS1 Can Inhibit theInvasion and Migration of MCF-7/ADR Cells and PromoteCell Apoptosis. Further studying the effect of lncRNAFOXD2-AS1 expression on the invasion, migration, andapoptosis of the drug-resistant cell line MCF-7/ADR. Wefirst transfected lncRNA FOXD2-AS1 overexpression vectorand siRNA in MCF-7/ADR cells. The qRT-PCR resultsshowed that after interference or overexpression of lncRNAFOXD2-AS1, the expression of lncRNA FOXD2-AS1 inMCF-7/ADR cells was significantly reduced or increased(Figure 2(a), P < 0:05), confirming the successful transfec-tion (Figure 2(a), P < 0:05). The results of Transwell andscratch testing showed that after overexpression of lncRNAFOXD2-AS1, MCF-7/ADR cell invasion and migrationincreased while interference with the lncRNA FOXD2-AS1expression could significantly inhibit cell invasion andmigration (Figures 2(b) and 2(c), P < 0:05). In addition, theflow cytometry results (Figure 2(d), P < 0:05) showed thatcompared with the vector group, the lncRNA FOXD2-AS1group had significantly reduced cell apoptosis. Comparedwith the siNC group, cell apoptosis increased significantlyafter interfering with the expression of lncRNA FOXD2-AS1. These results suggest that interfering with the expres-sion of lncRNA FOXD2-AS1 can inhibit the metastasis ofMCF-7/ADR cells and promote their apoptosis.

3.3. Downregulation of lncRNA FOXD2-AS1 Can Improvethe Chemosensitivity of MCF-7-ADR Cells. To further vali-date the effect of lncRNA FOXD2-AS1 on drug resistancein breast cancer cells. CCK-8 assay was used to detect the cellsurvival rate of each group of cells under the treatment ofcommonly used chemotherapeutic drugs ADR, cisplatin(DDP), and 5-fluorouracil (5-FU). The results showed thatwhen lncRNA FOXD2-AS1 was overexpressed, cells’ sur-vival rate and IC50 increased under ADR, DDP, and 5-FUtreatments, indicating that their drug resistance was

Table 1: qRT-PCR primer sequence.

Gene name Primer sequence

lncRNA FOXD2-AS1

F: 5′-TGGACCTAGCTGCAGCTCCA-3′R: 5′-AGTTGAAGGTGCACACACTG-3′

GAPDH

F: 5′-GCGAGATCGCACTCATCATCT-3′

R: 5′-TCAGTGGTGGACCTGACC-3′

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enhanced. The cell survival rate and IC50 value decreasedwhen knocked down (Figures 3(a)–3(f), P < 0:05). Thisshows that downregulation of lncRNA FOXD2-AS1 couldimprove the sensitivity of drug-resistant cell lines.

3.4. Downregulation of lncRNA FOXD2-AS1 Can InhibitPI3K/AKT Signaling Pathway in MCF-7/ADR Cells. Furtherinvestigation of the mechanism of lncRNA FOXD2-AS1affecting breast cancer cells was done. The results showedthat when lncRNA FOXD2-AS1 was overexpressed, the pro-tein expression levels of p-PI3K and p-AKT increased signif-icantly in cells. The p-PI3K/PI3K and p-AKT/AKT ratio alsoincreased significantly, while the results were opposite afterthe knockdown of lncRNA FOXD2-AS1 (Figure 4, P < 0:05). This indicates that the PI3K/AKT signaling pathway isinvolved in lncRNA FOXD2-AS1 regulating drug resistanceof breast cancer cells.

4. Discussion

Breast cancer is the most common cancer among women. Itsincidence ranks first among female malignant tumors [22].The clinical treatment of breast cancer is mainly throughchemotherapy [23, 24], but chemotherapeutic drugs arenot only prone to multidrug resistance leading to poor ther-

apeutic effects but also accompanied by severe nausea andvomiting, leukopenia, alopecia, phlebitis, and other adversereactions. Therefore, finding new therapeutic targets forbreast cancer is extremely urgent. lncRNA is considered tobe an important regulator of cancer progression. It partici-pates in multiple processes of gene expression regulation,plays a role in promoting or suppressing cancer in tumors,and is related to tumor cell proliferation, apoptosis, infiltra-tion, metastasis, and other closely related malignant biolog-ical processes [25]. lncRNA FOXD2-AS1 is a newlydiscovered lncRNA that regulates the progression of cancer.A large number of studies have shown that lncRNA FOXD2-AS1 is upregulated in tumors and promotes the occurrenceand development of tumors [26]. There are studies reportinglncRNA FOXD2-AS1 in papillary thyroid carcinoma, colo-rectal cancer, and bladder cancer are highly expressed andhave a more significant correlation with poor prognosis, allof which can be used as molecular markers [17, 27, 28]. Thisindicates that lncRNA FOXD2-AS1 may play an essentialrole in cancer treatment. In this study, it was also found thatthe expression of lncRNA FOXD2-AS1 in breast cancer tis-sues, human breast cancer cells MCF-7, andantiadriamycin-resistant strain MCF-7/ADR was signifi-cantly increased; the MCF-7/ADR expression levels wereconsiderably higher than MCF-7 expression levels.

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Figure 1: lncRNA FOXD2-AS1 highly expressed in breast cancer. (a) qRT-PCR detects the expression of lncRNA FOXD2-AS1 in breastcancer tissues and paracancer tissues; ∗∗P < 0:01, n = 60 per group. (b) The expression of lncRNA FOXD2-AS1 in normal breast cells(MCF-10A), breast cancer cells (MCF-7), and adriamycin-resistant strains of breast cancer was detected by qRT-PCR; ∗∗P < 0:01 vs. theMCF-10A group, ##P < 0:01 vs. MCF-7 group; (c) CCK-8 assay was used to detect cell viability. (d) The IC50 values in both cells; ∗∗P <0:01 vs. MCF-7 groups.

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Further research found that interference with lncRNAFOXD2-AS1 can inhibit the proliferation, invasion, andmigration of MCF-7/ADR cells, promote cell apoptosis,and improve the chemical sensitivity of MCF-7/ADR cells.This is consistent with the results of Wang et al. [29].Their experiments confirmed that overexpression of miR-134 in MCF-7/ADR cells could promote adriamycin-induced cell proliferation inhibition and proapoptosis,thereby increasing the sensitivity of breast cancer cells toadriamycin. These findings suggest that lncRNA FOXD2-AS1 can promote the proliferation and migration of breastcancer cells, inhibit apoptosis, affect the drug resistance ofbreast cancer cells, and promote the malignant biologicalbehavior of breast cancer.

The PI3K/AKT signaling pathway is closely related tothe life activities of the body and can regulate cell prolifera-tion, protein synthesis, and its function and various life pro-cesses. PI3K, as a phosphatidylinositol kinase, can increasethe phosphorylation level of the important downstream tar-get gene AKT to promote AKT activation [30]. AKT is aprotein kinase closely related to cell proliferation, apoptosis,and energy metabolism [31]. In addition, AKT is also associ-ated with inflammation, immune regulation, and autophagy[32]. Studies have reported that AKR1B10 promotes breastcancer cell proliferation and migration through the PI3K/AKT/NF-κB signaling pathway [33]. At the same time, itwas found that during the development of tamoxifen resis-tance, the changes of PI3K/AKT molecules before matrix

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Figure 2: The effect of lncRNA FOXD2-AS1 on the invasion, migration, and apoptosis of MCF-7/ADR cells. (a) The expression of lncRNAFOXD2-AS1 in each group of MCF-7/ADR cells was detected by qRT-PCR; ∗P < 0:05 vs. the MCF-7/ADR-vector group; ##P < 0:01 vs. theMCF-7/ADR-siNC group; (b) Transwell assay was used to evaluate the invasion ability of MCF-7/ADR cells in each group. (c) Scratch testwas performed to detect the migration ability of MCF-7/ADR cells in each group; ∗∗P < 0:01 vs. the MCF-7/ADR-NC group. (d) Flowcytometry was conducted to detect apoptosis of MCF-7/ADR cells in each group; ∗∗P < 0:01 vs. the vector group; ##P < 0:01 vs. the siNCgroup.

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Figure 3: The effect of lncRNA FOXD2-AS1 expression on the chemical sensitivity of MCF-7-ADR cells. CCK-8 assay was used to detectthe cell survival rate of each group of MCF-7/ADR cells under ADR (a), DDP (b), and 5-FU (c) treatment; CCK-8 assay was used to detectthe IC50 value of cells of each group of MCF-7/ADR cells under ADR (d), DDP (e), and 5-FU (f) treatment. ∗P < 0:05 vs. the MCF-7/ADR-vector group; ##P < 0:01 vs. the MCF-7/ADR-siNC group.

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metallopeptidase overexpression are related to the poorprognosis of breast cancer patients [34]. This study foundthat interference with lncRNA FOXD2-AS1 can reduce p-PI3K and p-AKT protein expression levels, thereby inhibit-ing the PI3K/AKT signaling pathway, revealing thatlncFOXD2-AS1 can increase protein expression levels of p-PI3K and p-AKT in breast cancer cells. This can further pro-mote the PI3K/AKT signaling pathway and ultimately pro-mote the occurrence and development of breast cancer,providing new clues for the research on the mechanism ofbreast cancer cell development. However, the specific mech-anism in the animal models remains to be further elucidated.

In summary, the expression of lncRNA FOXD2-AS1 isupregulated in breast cancer and drug-resistant strains.Interfering with lncRNA FOXD2-AS1 can inhibit the prolif-eration, invasion, and migration of breast cancer cells, pro-mote cell apoptosis, improve the chemical sensitivity ofMCF-7/ADR cells, and inhibit the PI3K/AKT signalingpathway. Therefore, lncRNA FOXD2-AS1 can be used as apotential target for early diagnosis and targeted breast cancertherapy.

Data Availability

The data used to support the findings of this study are avail-able from the corresponding author upon request.

Ethical Approval

This study was approved by the Medical Ethics Committeeof the Peking University of Shenzhen Hospital (2020-023).

Conflicts of Interest

The authors claim that there is no conflict of interestbetween them.

References

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Figure 4: The effect of lncRNA FOXD2-AS1 on the PI3K/AKT signaling pathway in MCF-7/ADR cells. The protein expression of p-PI3K,PI3K, p-AKT, and AKT in the ADR-resistant cells (MCF-7) was detected using Western blot. ∗∗P < 0:01 vs. the vector group; ##P < 0:01 vs.the siNC group.

8 Computational and Mathematical Methods in Medicine

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9Computational and Mathematical Methods in Medicine