European Review for Medical and Pharmacological Sciences 2020; 24: 6561-6568 Long noncoding RNA PROX1-AS1 promoted ovarian cancer cell proliferation and metastasis by suppressing KLF6 L. ZHAO1, J.-F. LI2, X.-J. TONG3 1Department of Gynecology Center, The First Affiliated Hospital of XinJiang Medical University, Urumqi, China 2Department of Ultrasound, WeiHai Municipal Hospital, WeiHai, China 3Department of Gynecology, Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute, Shenyang, China Abstract. – OBJECTIVE: Recently, the role of Introduction long noncoding RNAs (lncRNAs) in tumor pro- gression has attracted much attention world- Ovarian cancer remains one of the most fa- wide. Numerous studies have identified lncRNA tal and common tumors in women globally, PROX1-AS1 as an oncogene in cancers. There- accounting for 5-6% cancer-related deaths1,2. It fore, the aim of this research was to explore the function of PROX1-AS1 in the development of has been reported that 22,500 patients are newly ovarian cancer. diagnosed of ovarian cancer in America in 2017, PATIENTS AND METHODS: PROX1-AS1 ex- with 14,100 deaths3,4. Due to the vagueness pression in both ovarian cancer patients and of symptoms and the lack of early detection normal subjects was detected by quantitative tests, 70-75% of patients have already been in Real Time-Polymerase Chain Reaction (qRT- advanced stages when first diagnosed5. Where- PCR). Subsequently, PROX1-AS1 shRNA was constructed and transfected in vitro. 3-(4,5-di- as, almost 80% of the patients may develop methylthiazol-2-yl)-2,5-diphenyl tetrazolium bro- resistance to chemotherapy or recurrence after 6,7 mide (MTT) assay, colony formation assay, tran- surgery . Therefore, the severe situation un- swell assay, and Matrigel assay were utilized derscores the urgency of early detection and the to detect the function of PROX1-AS1 in ovari- establishment of new therapeutic interventions an cancer. In addition, the potential mechanism for ovarian cancer patients. was explored using qRT-PCR and Western blot Long non-coding RNAs (lncRNAs) are a sub- assay. RESULTS: PROX1-AS1 was highly expressed group of non-coding RNAs that do not encode in ovarian carcinoma samples and cell lines proteins. Currently, lncRNAs have emerged as an (p<0.05). The proliferation, migration, and inva- important role in tumorigenesis. Multiple studies sion of ovarian cells were significantly inhibit- have found that lncRNAs have the potential to ed after PROX1-AS1 was downregulated in vi- regulate gene expression. By regulating vasculo- tro (p<0.05). Besides, the mRNA and protein ex- genic angiogenesis, lncRNA MALAT1 promotes pressions of KLF6 were significantly promot- 8. ed after PROX1-AS1 knockdown in ovarian can- tumorigenicity and metastasis in gastric cancer cer cells (p<0.05). Further functional assays LncRNA AC132217.4 promotes the metastasis of showed that KLF6 expression was negatively oral squamous cell carcinoma by regulating IGF2 correlated with PROX1-AS1 expression in ovari- expression modulated by KLF89. By sponging an cancer samples. miR-124-3p, lncRNA OGFRP1 has been report- CONCLUSIONS: PROX1-AS1 enhances the ed to take part in the proliferation of non-small metastasis and proliferation of ovarian cancer cell lung cancer cells10. Upregulation of lncRNA cells via suppressing KLF6. Our findings sug- gest that PROX1-AS1 may be applied as a novel LINC01510 has been confirmed negatively as- target for therapy of ovarian cancer. sociated with the prognosis of colorectal cancer (CRC) patients. Meanwhile, lncRNA LINC01510 Key Words: may serve as a potential independent prognostic Long noncoding RNAs, PROX1-AS1, Ovarian can- biomarker for CRC11. LncRNA HCCL5 activated cer, KLF6. RETRACTEDby ZEB1 accelerates cell viability, cell migra- Corresponding Author: Xiaojing Tong, MD; e-mail: [email protected] 6561 L. Zhao, J.-F. Li, X.-J. Tong tion, epithelial-mesenchymal transition, and the using quantitative Real Time-Polymerase Chain malignancy of hepatocellular carcinoma12. How- Reaction (RT-qPCR). ever, few studies have uncovered the exact role of PROX1-AS1 in ovarian cancer, as well as the RNA Extraction and RT-qPCR underlying mechanism. Total RNA in tissues and cells was extracted In our study, we found that PROX1-AS1 was by using TRIzol reagent (Invitrogen, Carlsbad, remarkably upregulated in ovarian cancer tis- CA, USA). Subsequently, extracted total RNA sues. PROX1-AS1 enhanced the proliferation and was reverse-transcribed to complementary de- metastasis of ovarian cancer in vitro. Moreover, oxyribose nucleic acids (cDNAs) through Reverse KLF6, a tumor suppressor gene, was associated Transcription Kit (TaKaRa Biotechnology Co., with the function of PROX1-AS1 in ovarian can- Ltd., Dalian, China). Thermo-cycling conditions cer. were as follows: 30s at 95°C, 5s for 40 cycles at 95°C, and 35 s at 60°C. 2-ΔΔCt method was utilized for calculating relative expression of genes. The Patients and Methods primer sequences used in this study were as fol- lows: PROX1-AS1 forward 5′-CTAGTTAGCAG- Tissue Specimens GGGCAGCAC-3′, PROX1-AS1 reverse 5′-AA- Paired ovarian carcinoma tissues and adjacent CAGAGAGGCGTGGAAGAA-3′; β-actin, for- normal tissues were sequentially collected from ward 5′-GATGGAAATCGTCAGAGGCT-3′ and 50 ovarian carcinoma patients undergoing sur- reverse 5′-TGGCACTTAGTTGGAAATGC-3′. gery from December 2017 to December 2019 in The First Affiliated Hospital of XinJiang Med- Western Blot Analysis ical University. The selection of patients was Total proteins were collected from cells via ra- based on the guideline proposed by the Union dioimmunoprecipitation assay (RIPA) buffer and for International Cancer Control (UICC). This quantified by using a protein assay (bicinchoninic study was approved by The Ethics Committee of acid method; Beyotime, Shanghai, China). Tar- The First Affiliated Hospital of XinJiang Med- get proteins were separated by sodium dodecyl ical University. The protocol of the study was sulphate-polyacrylamide gel electrophoresis performed as Declaration of Helsinki Principles (SDS-PAGE) and transferred onto polyvinylidene required. difluoride (PVDF) membranes. Then, the mem- branes were incubated with primary antibodies Cell Culture overnight. On the next day, the membranes were Human ovarian cancer cell lines (A2780, TO- incubated with rabbit anti-β-actin (Cell Signal- V112D, HO-8910, and SKOV3) and normal ovar- ing Technology, Danvers, MA, USA) and rabbit ian cell line (ISOE80) were cultured in Dulbec- anti-KLF6 (Cell Signaling Technology, Danvers, co’s Modified Eagle’s Medium (DMEM; Thermo MA, USA). The Pierce enhanced chemilumines- Fisher Scientific, Inc., Waltham, MA, USA) con- cence (ECL) was utilized for visualizing Western sisting of 10% fetal bovine serum (FBS; Thermo Blotting Substrate Immunoreactive bands (Santa Fisher Scientific, Inc., Waltham, MA, USA) and Cruz Biotechnology, Inc., Santa Cruz, CA, USA). penicillin, and maintained in a humidified atmo- sphere with 5% CO2 at 37°C. MTT (3-(4,5-Dimethylthiazol-2-yl)- 2,5-Diphenyl Tetrazolium Bromide) Assay Lentivirus Expressing Short-Hairpin RNA MTT (Roche, Basel, Switzerland) assay was and Cell Transfection used to detect cell proliferation. 200 μL of trans- Lentivirus expressing short-hairpin RNA fected cells were first seeded into 96-well plates (shRNA) directed against PROX1-AS1 were pro- at a density of 2×103 cells per well. Following the vided by GenePharma (Shanghai, China). The manufacturer’s protocol, cell proliferation was complementary DNA encoding PROX1-AS1 was assessed every 24 h. amplified and inserted into pcDNA3.1 (Gene- Pharma, Shanghai, China). Subsequently, they Colony Formation Assay were transfected into ovarian cancer cells, ac- To detect the long-term effect of PROX1-AS1 cording to the instructions of Lipofectamine 3000 on cell proliferation, colony formation assay was (Invitrogen, Carlsbad, CA, USA). PROX1-AS1 conducted. 5×10 2 cells/well cells were seeded into expression in transfectedRETRACTED cells was conducted 6-well plate, and culture medium was replaced 6562 Role of PROX1-AS1 in ovarian cancer every day. 7 day later, formed colonies were fixed two groups were compared by Student t-test. with 75% ethanol for 30 min and stained with p<0.05 was considered statistically significant. 0.2% crystal violet. Finally, colonies were photo- graphed and counted. Results Wound Healing Assay After transfection, the cells were seeded into PROX1-AS1 Was Highly Expressed in 6-well plates and incubated in DMEM medi- Ovarian Cancer Tissues and Cells um overnight. On the next day, the cells were PROX1-AS1 expression in 50 pairs of ovarian scratched with a plastic tip and cultured in se- cancer tissues and adjacent normal tissues was de- rum-free DMEM. Each assay was repeated in tected via qRT-PCR. PROX1-AS1 was highly ex- triplicate independently. Relate distance was pressed in ovarian cancer tissues when compared viewed under a light microscope (Olympus Corp., with adjacent normal tissues (p<0.05, Figure 1A). Tokyo, Japan) at 48 h. Moreover, PROX1-AS1 level in ovarian cancer cells was significantly higher than that of normal Transwell Assay and Matrigel Assay ovarian cell line ISOE80 (p<0.05, Figure 1B). After transfection, 1 ×105 cells in 200 µL serum-free DMEM were replanted in top cham- Cell Proliferation Was Repressed Via ber (Corning, Inc., Corning, NY, USA) with Silence of PROX1-AS1 in Ovarian Cancer or without 50 µg Matrigel (BD, Bedford, MA, In our study, HO-8910 cells were chosen for USA). Meanwhile, DMEM and FBS were added the knockdown of PROX1-AS1 in vitro. qRT- to the lower chamber. These chambers were then PCR was utilized for detecting PROX1-AS1 ex- cultured overnight in an incubator supplemented pression (Figure 2A). MTT assay indicated that with 5% CO2 at 37°C. The top surface of cham- the growth ability of HO-8910 cells was signifi- bers was fixed with methanol for 30 min after cantly repressed after PROX1-AS1 was knocked wiped by cotton swab, followed by staining with down (p<0.05, Figure 2B). Colony formation crystal violet for 20 min. Five fields were ran- assay demonstrated that the number of colonies domly selected for each sample.