Downregulated lincRNA HOTAIR expression in ovarian cancer stem cells decreases its tumorgeniesis and metastasis by inhibiting epithelial-mesenchymal transition
- Jing Wang†1Email author,
- Dengyu Chen†2, 3,
- Xiangfeng He†4,
- Yuxia Zhang1, 2,
- Fangfang Shi2, 5,
- Di Wu1, 2,
- Junsong Chen2,
- Ying Zhang2,
- Fengsu Zhao2 and
- Jun Dou2Email author
© Wang et al.; licensee BioMed Central. 2015
Received: 6 September 2014
Accepted: 10 February 2015
Published: 25 February 2015
Emerging evidence indicates that dysregulated long intervening non-coding RNA (lincRNA) HOTAIR correlates highly with tumor invasion and metastasis but a link between the high expression of HOTAIR and the metastatic cascade of cancer stem cells (CSCs) needs to be further studied. The purpose of this study was to investigate the effect of down-regulated HOTAIR expression on tumorgeniesis and metastasis of epithelial ovarian cancer (EOC) CSCs. CD117+CD44+CSCs were isolated from human EOC SKOV3 cell line by using a magnetic-activated cell sorting system, and were then transfected with the expression vector-based small hairpin RNA targeting HOTAIR; the stably transfected cells were selected for the study. Colony-forming, wound-healing, cellular metastasis and tumorigenicity assays were performed.
The results demonstrated that the HOTAIR expression in clinical EOC tissues and SKOV3 CD117+CD44+CSCs was higher than in SKOV3 tumor tissues and non-CD117+CD44+CSCs. The CD117+CD44+-shHOTAIR showed an inhibited HOTAIR expression, reduced cell migration and invasion than CD117+CD44+- scramble, suggesting the inhibition of an epithelial-mesenchymal transition. Moreover, the downregulated HOTAIR expression in CD117+CD44+ CSCs significantly decreased the tumor growth and lung metastasis in xenograft mice.
Our findings demonstrated the shHOTAIR-mediated down-regulation of the HOTAIR expression in CD117+CD44+ CSCs can be a promising new opportunity for future clinical trials.
Human epithelial ovarian cancer (EOC) is one of malignant tumors in gynecological cancers and currently remains to be the number one the first leading cause of cancer-related deaths in women due to factors such as failure for its early dectection and diagnosis, its proneness to pelvic and peritoneal metastasis, and its resistance to chemotherapy after recurrence. Although many studies have been published in the past decade and have contributed to the advance of the knowledge in this field, the complex biology of EOC is still insufficiently understood [1,2]. Therefore, more recent studies have focused on the molecular mechanisms underlying the EOC progression and the new strategies for early clinical diagnosis and effective therapy [3,4].
Recent studies have demonstrated that cancer stem cells (CSCs) are responsible for tumour-initiating potential, metastasis and eventual relapse. Thus, the treatments that target CSCs may lead to the discovery of effective methods to eradicate the malignant tumor cells [5-7].
Emerging evidence supports that long intervening non-coding RNAs (lincRNAs) play a critical role in regulating cellular processes such as differentiation, proliferation, and metastasis [8,9]. HOX transcript antisense RNA (termed HOTAIR), one of lincRNAs, functions in epigenetic regulatory processes, interacts with polycomb repressive complex 2 and is required for histone H3 lysine-27 trimethylation of the HOD locus. In addtion, HOTAIR has been strongly associated with the invasion and metastasis of cancer cells . Dysregulation of lncRNA HOTAIR has been considered a primary feature of several human cancers including breast cancer [10,11], hepatocellular carcinoma [12,13], colorectal cancer , pancreatic carcinomas , gastrointestinal stromal tumors , and human EOC [17,18]. Of the many functions of HOTAIR, as tumor regulatory factors, the one for silencing HOTAIR transcription in CSCs has remained insufficiently understood [17,19]. For this reason, we investigated whether the downregulated HOTAIR expression would decrease the human EOC SKOV3 CD117+CD44+CSC metastasis by inhibiting epithelial- mesenchymal transition (EMT) in vitro, as well as cellular tumorigenicity in nude mice. The data from our current study showed that epigenetic silencing of lncRNA HOTAIR in SKOV3 CD117+CD44+CSCs resulted in reduced cellular tumorgeniesis and metastasis in mouse model. This fingings suggested that the streatgy of down-regulating the HOTAIR expression may serve as a potential anti-cancer regimen for inhibiting EOC CSC’s invasiveness and metastasis. Future investigations of this possibility are fully warranted.
Materials and methods
SKOV3 cell line was acquired from an ovarian cancer patient, which is a well-established ovarian cancer model system; the line was purchased from the Cellular Institute in Shanghai, China. Cells were cultured in complete media consisting of RPMI 1640, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum. The medium was refreshed every 3 days to maintain adherent cells. When the SKOV3 cells reached 90% confluence, cells were harvested with 0.25% trypsin −1 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA) treatment for 2 min.
Isolation of CD44+CD117+cells and identification of cell phenotype
CD44+CD117+cells were sorted from the SKOV-3 cell line by using the magnetic-activated cell sorting (MACS, Miltenyi Biotec., Bergisch Gladbach, Germany). First, CD44+subsets were isolated by using the mouse antihuman CD44 antibody coupled to magnetic microbeads (code number:130-095-194, antibody dilution,1:20, Miltenyi Biotec., Bergisch Gladbach, Germany) and followed by the magnetic column selection or depletion. Second, the resulting cells were then depleted of CD117−subsets by using mouse antihuman CD117 antibody coupled to magnetic microbeads (code number:130-091-332, antibody dilution,1:20, Miltenyi Biotec., Bergisch Gladbach, Germany), and we named the CD44+CD117+cells for the EOC cancer stem cells as ‘EOC SKOV-3 CD44+CD117+CSCs’ [20-22]. The isolated cells were placed in stem cell culture medium by resuspension in serum-free DMEM/F12 supplemented with 5 μg/mL insulin (Sigma-Aldrich, Missouri, USA), 20 ng/mL human recombinant epidermal growth factor (Invitrogen, CA, USA), 10 ng/mL basic fibroblast growth factor (Invitrogen, CA, USA) and 0.5% bovine serum albumin (Sigma-Aldrich, Missouri, USA) [23,24]. The isolated CD44+CD117+cells were further identified by using flow cytometer (FCM, BD, USA) .
The short hairpin RNA sequence design
A short hairpin RNA sequence of lncRNA HOTAIR was designed based on the HOTAIR RNA sequence (Gene ID: 100124700) by using the siDESIGN design software (Dharmacon, http://www. thermoscientificbio.com/design-center/) and the Block-iTTM RNAi Designer (Invitrogen, Grand island, NY) as well as BLAST (http:// www. ncbi. nlm.nih.gov/BLAST). The target sequence site for HOTAIR shRNA includes 19 base pairs of the HOTAIR RNA sequence. In addition, one scramble sequence was designed as a negative control. The shRNA sequences are as follows: pSUPER-EGFP1-HOTAIR-shRNA (pSUPER- EGFP1-shHOTAIR), Forward 5′-GATCCCCGAACGGGAGTACAGAGAGATTCAAGAG A TCTCTC TGTACTCCCGTTCTTTTTGGAAA-3′; antisense,5′-AGCTTTTCCAAAAAGAACGGG A GTACAGAGAGATCTCTTGAATCTCTCTGTACTCCCGTTCGGG-3′;scramble-siRNA: sense, 5′- GATCCCCTTC TCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGA ATTTTTGG A AA-3′; antisense, 5′-AGCTTTTCCAAAATTCTCCGAACGTGTCACGT-TCTCTTGAAACGTGAC ACGTTCGGAGAAGGG-3′. All the primers were synthesized by Gene and Technology of China in Shanghai .
Construction of pSUPER-EGFP1-HOTAIR -shRNA and production of stably transfected clones
A pSUPER-EGFP1 (enhanced green fluorescent protein 1) vector was used to construct recombinant. The recombinant pSUPER-EGFP1-HOTAIR-shRNA (shHOTAIR) was developed as previously describled [10,26]. A pSUPER-EGFP1-scrambled shRNA (Scramble-HOTAIR) was used as a negative control. These recombinants were verified by the analysis of endonuclease digestion and sequencing. The shHOTAIR and SCHOTAIR were respectively transfected into CD44+CD117+CSCs and the stably transfected clones were selected with G418 (Clontech, CA). ShHOTAIR-expressing and Scramble-HOTAIR-expressing clones were labeled ‘CD44+CD117+ shHOTAIR’and ‘CD44+CD117+ scramble’, respectively.
Four fresh surgical tissue samples of EOC patients with a median age of 55 years (age range 45–63) were collected at the Department of Gynecology & Obstetrics, Zhongda Hospital, Medical School, Southeast University between March 2012 and October 2012. The four surgical tumor samples used in this study (designated T1-T5) were categorized as malignant Fesddration Internationale des Gynaecologistes et Obstetristes (FIGO) stage II-III serous adenocarcinomas. All samples were stored immediately in liquid nitrogen until analysis .
Quantitative RT-PCR of HOTAIR
Total RNA was extracted from the SKOV-3 CD44+CD117+CSCs or SKOV-3 cells or microdissected tumor tissue samples of EOC patients by using a Qiagen RNeasy Kit (Qiagen, Valencia, CA, USA) following by the manufacturer’s protocol. A single-stranded cDNA was prepared by the using SuperScript III reverse transcriptase (Invitrogen). Quantitative real-time reverse transcription-PCR (qRT-PCR) was carried out on an ABI step one plus real-time system (Applied Biosystems, USA). The cDNAs were amplified by PCR with primers as follows: HOTAIR: sense, 5′-GGTAGAAAAAGCAACCACG AAGC-3′; antisense, 5′-TTGGGGAAGCA TTTT CTGAC-3′; β-actin (sense, 5′-GGACTTCGAG CAAG AGATGG-3′; antisense, 5′-AGCACTGT GTTGGCGTACAG-3′). U6-RT Primer, 5′-GTCGTATCCAG TGCAGGGTCCGAGGTATTCGCACTGGATACGACAAATATGGAAC-3′; sense, 5′-TGCGGGTGCT CGCTTCGG CAGC-3′; URP (Universal Reverse Primer), 5′-CCGGCAGGGTCCGAGGT-3′; E-cadherin: sense, 5′-TACACTGCCCAGGAGCCAGA-3′; antisense, 5′-TGGCACCAGTGTCCGGATTA-3′; Vimentin: sense, GGAACAGCATGTCCAAATCG; antisense, GCACCT GTCTCCGGTACTCA. The mRNA levels of the genes of interest were expressed as the ratio of each gene of interest to β-actin or U6 mRNA for each sample. The comparative Ct (ΔΔCt) method was used to determine the expression fold change [27,28].
Colony forming assay
The colony formation ability of SKOV3 CD117+CD44+-shHOTAIR was investigated. A colony with a diameter larger than 75 μm or having more than 50 cells was counted for 1 positive colony according to our previous report . The plate clone formation efficiency was calculated as (number of colony/number of cells inoculated) × 100%.
Cell migration assay
To determine the role of down-regulated HOTAIR expression on migration, SKOV3 CD117+CD44+- shHOTAIR were used in the wound healing assay. Briefly, SKOV3 CD117+CD44+-shHOTAIR or SKOV3CD117+CD44+-scramble were plated in 6-well plates (5 × 105 cells per well) to form a monolayer one day before the assay; non-adherent cells were removed by PBS washing. On the following day, a uniform scratch was made down in the center of the well using a sterile micropipette tip. The distance travelled by the cells was measured between the two boundaries of the acellular area at 0, 24, 48 and 96 hours respectively, after incubation. Each experiment was performed in triplicate .
Cell invasion assay
The invasion ability of SKOV3 CD117+CD44+-shHOTAIR or SKOV3 CD117+CD44+-scramble was evaluated by using the transwell invasion assay as previously described . Briefly, the transwell inserts with 8 μm pores were coated with Matrigel (20 μg/well; Becton Dickinson, Waltham, MA, USA); cells were seeded in the upper chamber in RPMI1640 medium supplemented with 10% fetal bovine serum. After incubation at 37°C, the cells that invaded to the lower surface of the Matrigel-coated membranes were fixed with 70% ethanol and stained with trypan blue; the cells from five randomly selected fields were then counted under a light microscope.
Western blotting analysis
Approximately 1 × 106 SKOV3 CD117+CD44+-shHOTAIR or SKOV3CD117+CD44+-scramble were collected and lyzed in the protein extraction buffer (Novagen, Madison, WI, USA), and 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed and proteins (10 μg/lane) were loaded by following the pulished papers [6,31]. The rabbit antibody specific to human E-cadherin (code number: 31955) or Vimentin (code number: 57415) was used in the assay (Bioworld Technology, Dublin, OH, USA). The antibody dilution was 1: 1000.
Tumorigenicity of shHOTAIR-SKOV3 CD117+CD44+ CSCs in an xenograft mice
Balb/c nude mice (female, weight: 16-18 g and age between 5 and 6 weeks) were ordered from the Animal Center of Yang Zhou University of China and were raised under sterile conditions at the Experimental Animal Center, Medical School of Southeast University. The experiments were performed in compliance with the guidelines of the Animal Research Ethics Board of Southeast University, China. Twelve nude mice were randomly divided into two groups of equal size (six per group): the SKOV3 CD117+CD44+- shHOTAIR group, and the SKOV3CD117+CD44+-scramble group. The nude mice was subcutaneously injected in the back with 5 × 104 SKOV3 CD117+CD44+-shHOTAIR or SKOV3 CD117+CD44+-scramble. Tumor formations in each mouse were monitored every three days by taking 2-dimensional measurements of individual tumors from each mouse .
Lung tissues were removed from the xenograft mice, fixed in 10% formalin, and then embedded in paraffin. Lung tissue sections of 4μm thin were cut and mounted on SuperFrost Plus glass slides; the tissues were fixed in methanol and stained in hematoxylin and eosin (HE). The slides were viewed under a Zeiss Axioplan light microscope at a magnification of × 200 .
Values of interest were presented as the mean plus or minus two standard deviation. Statistical comparisons were performed using the Student’s t-test method. Results for all analyses with a P value < 0.05 indicate the statistically significant differences.
HOTAIR expression in EOC tissues and SKOV3 CD117+CD44+ CSCs
Effects of down-regulated HOTAIR in SKOV3 cells on the ability of migration and invasion
Effect of down-regulated HOTAIR in CD117+CD44+ CSCs on the ability of colony, migration and invasion
Down-regulated HOTAIR in CD117+CD44+CSCs inhibited the tumor growth and metastasis in the xenograft mouse model
To assess the effect of the down-regulation of HOTAIR in CD117+CD44+-shHOTAIR on tumor metastasis, we used H&E staining to detect if there was tumor cell metastasis in the lung tissues. Compared with the mice injected with CD117+CD44+-scramble, no tumor cell metastasis was found in the lung tissues of the nude mice 64 days after they were injected with the CD117+CD44+-shHOTAIR (Figure 5E). It is thus evident from the results that the tumor cell growth and metastasis were significantly inhibited in the mice injected with CD117+CD44+-shHOTAIR compared with the mice injected with CD117+CD44+-scramble.
EOC is the most lethal gynecological malignancy in the world and approximately 30% of cancer patients have died this disease; this emphasizes the need for improved early detection and effective treatment . Evidence has indicated that CSCs are believed to be the ‘seed cell’ in cancer recurrences and metastases, however, treatments targeted at CSCs have remained to be developed and studied . Increasing evidence supports that CSCs possess mesenchymal characteristics and EMT ability, and that the decreased change in lincRNA HOTAIR was associated with alterations in specific EMT markers concurrent with reduced migratory potential [6,10,36]. In this regard, our current study was designed to investigate whether human EOC tissues and SKOV3 CD117+CD44+CSCs expresse HOTAIR and whether the decreased change of HOTAIR in human EOC SKOV3 CD117+CD44+CSCs are closely associated with the tumorgeniesis and metastasis. Indeed, our data indicated that human EOC patients’ tumor tissues and SKOV3 CD117+CD44+ CSCs had significantly high expressions of HOTAIR compared with the SKOV3 tumor tissues and SKOV3 non-CD117+CD44+CSCs, and that the down-regulated HOTAIR expression in CD117+CD44+-shHOTAIR markedly reduced cellular metastasis and invation in vitro as well as the tumorgeniesis in mice. This efficacy in CD117+CD44+-shHOTAIR was refelected in a lower clonogenic potential, a lower metastatic and invasive potential, and a weeker ability to form tumors in the xenografted mice than in CD138−CD34−-scramble. Importantly, the tumor cell lung metastasis was inhibited in the mice injected with CD117+CD44+-shHOTAIR, whereas the metastatic tumor cells were found in the lungs of the mice injected with CD117+CD44+-scramble.
The evident inhibitory activities in the SKOV3 cells transfected with siHOTAIR were apparently associated with inhibited the HOTAIR expression, which resulted in the increase of the epithelium feature molecule of the E-cadherin expression and reduced mesenchymal feature molecule of the Vimentin expression. The knockdown of the HOTAIR expression in CD117+CD44+CSCs may involve the inhibition of EMT of CD117+CD44+CSCs and the reduction of cellular migration and invasion potential and tumorgeniesis ability in vitro and in vivo. Nevertheless, the knockdown of the HOTAIR expression mechanism in inhibiting the metastasis of the SKOV3 cells and CD117+CD44+CSCs warrants further investigation.
In conclusion, the above-mentioned finding from our study is the first proof for demonstrating the novel role of the dawn-regulated HOTAIR in EOC SKOV3 CD117+CD44+CSCs, for suggesting the inhibitory cellular EMT, and for decreasing decreased the migration and and tumorgeniesis potential of CD117+CD44+CSCs. HOTAIR can be a potential therapeutic target for treatment of EOC patients.
This work was supported in part by the National Natural Science Foundation of China (No. 81071769, No. 81202372), Graduate Research and Innovation Projects in Jiangsu Province of China (CXZZ1-0129), and Scientific Research Foundation of Graduated School of Southeast University (No.3224004901).
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