- Email: [email protected]
Long Noncoding RNA Highly Up-regulated in Liver Cancer Predicts Unfavorable Outcome and Regulates Metastasis by MMPs in Triple-negative Breast Cancer
Archives of Medical Research 47 (2016) 446e453
ORIGINAL ARTICLE
Long Noncoding RNA Highly Up-regulated in Liver Cancer Predicts Unfavorable Outcome and Regulates Metastasis by MMPs in Triple-negative Breast Cancer Feng Shi,a,* Fang Xiao,b,* Ping Ding,c Hong Qin,d and Ruixue Huange a
Internal Medicine Department of Thyroid and Radionuclide Therapy, Hunan Cancer Hospital, The Affiliated Cancer Hospital of Xiangya School of Medicine, bDepartment of Health Toxicology, Xiangya School of Public Health, Central South University, Changsha, China c Center of Preventive Medicine Experiment, Xiangya School of Public Health, Central South University, Changsha, China d Department of Nutrition and Food Hygiene, eDepartment of Occupational and Environmental Health, Xiangya School of Public Health, Central South University, China Received for publication April 18, 2016; accepted October 28, 2016 (ARCMED-D-16-00233).
Background and Aims. Long noncoding RNA (lncRNA) highly up-regulated in liver cancer (HULC) is the first identified lncRNA highly expressed in hepatocellular carcinoma and acts as an oncogenic lncRNA. However, the significance of HULC in triple-negative breast cancer (TNBC) is still unclear. Our purpose is to explore the clinical significance and biological function of HULC in TNBC. Methods. Expression of HULC in TNBC tissues and cell lines was measured and the association between HULC expression and clinicopathological factors was analyzed. Cell proliferation, migration, and invasion assays were executed by using TNBC cell lines transfected with si-HULC or si-NC. The potential mechanism of HULC was explored by Western blot. Results. HULC expression was increased in TNBC tissues and cell lines and associated with malignant status and poor prognosis of TNBC patients. Furthermore, silencing TNBC expression effectively suppressed TNBC cells metastasis through MMP-2 and MMP-9. Conclusion. HULC acts as an oncogene lncRNA in TNBC and as an independent poor prognostic factor in TNBC patients. Ó 2016 IMSS. Published by Elsevier Inc. Key Words: HULC, lncRNA, Triple negative breast cancer, Metastasis, MMP.
Introduction Breast cancer remains the most common cancer in females and the second leading cause of cancer-related mortality in women, suggesting a serious problem worldwide to women (1,2). According to 2016 U.S. Cancer Statistics, an estimated total of 249,260 new breast cancer cases and 40,890 breast cancer deaths will occur in the U.S. in 2016 (3). In China, breast cancer alone is expected to account for 15% of all new cancers in women and is the
*
These authors are co-first authors. Address reprint requests to: Ruixue Huang, Department of Occupational and Environmental Health, Xiangya School of Public Health, Central South University, 410078, China; Phone: 86-18975832122; FAX: 86-731-84805460; E-mail: [email protected].
leading cause of cancer death in women !45 years of age (4). Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer defined by negative expression for estrogen receptors (ER) and progesterone receptors (PR) and normal (low) expression levels of human epidermal growth factor receptor 2 (HER2) (5,6). Due to lack of three important therapeutic markers, TNBC shows higher metastases and relapse rates than other types (7,8). Patients with TNBC carry a poor prognosis (9,10). Therefore, it is urgent to identify novel biomarkers for developing targeted molecular therapies and predicting prognosis in TNBC patients. Long noncoding RNAs (lncRNAs) are transcripts with no protein coding function and longer than 200 nucleotides (11). Dysregulation of lncRNAs has been shown to play a critical role in the development of cancer such as
0188-4409/$ - see front matter. Copyright Ó 2016 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2016.11.001
Role of HULC in Triple Negative Breast Cancer
hepatocellular carcinoma (HCC) (12,13), lung cancer (14,15), and gastric cancer (16,17). Nowadays, some oncogene or anti-oncogene lncRNAs are gradually discovered and identified in TNBC such as HOTAIR (18), MALAT1 (19), RoR (20), and LOC554202 (21). Highly up-regulated in liver cancer (HULC) is the first identified lncRNA highly expressed in HCC (22). HULC is located on human chromosome 6p24.3. Some studies suggested that HULC was overexpressed in a variety of human cancers and acted as an oncogene lncRNA in tumor development and progression (23,24). However, the significance of HULC in TNBC is still unknown. In order to explore the clinical significance of HULC in TNBC, we measured HULC expression in TNBC clinical samples and analyzed the association between HULC expression and clinicopathological characteristics. Furthermore, we performed loss-of-function studies of HULC to identify the biological function of HULC in TNBC cell lines.
Materials and Methods Patients and Tissue Sample Collection Ninety-six TNBC tissues and 22 paired normal mammary tissues were collected at The Affiliated Cancer Hospital of Xiangya School of Medicine from 2008e2013. Clinical tissue samples were obtained from surgery or biopsy and were immediately frozen in liquid nitrogen and kept at 80 C. Clinicopathological characteristics included age, clinical stage, histological grade, tumor size, lymph node metastasis, and distant metastasis. None of the patients had received tumor-specific therapy before diagnosis. Overall survival of TNBC patients was also followed up until December 31, 2015. The Research Ethics Committee of The Affiliated Cancer Hospital of Xiangya School of Medicine approved this protocol and written informed consent was obtained from each patient. The entire study was performed based on the Declaration of Helsinki.
447
Cell Culture and Transfection Four TNBC cell lines (MDA-MB-231, MDA-MB-468, BT549, BT483) and a normal breast epithelial cell line (MCF-10A) were purchased from the Chinese Academy of Sciences (Shanghai Cell Bank). These cells were cultured in DMEM containing 10% fetal bovine serum (FBS) in humidified 5% CO2 atmosphere at 37 C. For transfection, cells were cultured up to 70% confluency and transfected with si-HULC-RNA or si-NC (RiboBio, China), using Lipofectamine 2000 (Invitrogen, Grand Island, NY) by incubating with OptiMem-I media for 4 h. The cells were then cultured in fresh DMEM with 10% FBS. The relative level of HULC in transfected cells was examined using qRT-PCR. Cell Proliferation Analysis Cell proliferation was analyzed using 3-(4,5-cimethylthiazol2-yl)-2,5-diphenyl tetra-zolium bromide (MTT, Sigma, St. Louis, MO) assay. Standard MTT assay was used to detect cell proliferation. Briefly, cells were seeded at a density of 2 103 cells per well in 100 ml culture medium in 96-well culture plates. Cell proliferation was assessed after siRNA transient transfection for 24, 48, 72 and 96 h. The optical density of the wells was measured with using a microplate reader at 490 nm. Each experiment was performed in triplicate. Cell Migration and Invasion Assays Cell migration and invasion assays were examined by 24well plate Transwell system (Corning, New York). Briefly, 1 105 cells were seeded on a fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Corning). After the cells were incubated for 12 h, Giemsa-stained cells adhering to the lower surface were counted under a microscope in five predetermined fields (100). For the cell invasion assay, the procedure was similar to the cell migration assay except that the transwell membranes were precoated with 24 mg/mL Matrigel (Corning). Western Blotting
RNA Extraction and Quantitative RT-PCR (qRT-PCR) Total RNA was isolated using RNAiso Plus (TaKaRa, Dalian, China) according to the manufacturer’s protocol. CCAT2 and GAPDH expressions were quantified using real-time PCR with the SYBRÒ Green (TaKaRa) dye detection method on Light Cycler Roche 480 PCR instrument. Primers are as follows: HULC, forward primer: 50 -ATCTGCAAGCCAGGAAGAGTC-30 ; reverse primer: 50 -CTTGCTTGATGCTTTGGTCTGT-30 . GAPDH, forward primer: 50 -TGACAACTTTGGTATCGTGGAAGG30 ; reverse primer: 50 -GCAGGGATG ATGTTCTGGA GAG-30 . GAPDH was used as an internal control for the expression analysis of HULC. Relative quantification of RNA expression was calculated using the 2DDCt method.
For the protein expression analysis, standard Western blotting was carried out. MDA-MB-231 cells and BT549 cells transfected with si-HULC or si-NC after 48 h were lysed in RIPA lysis buffer (Cwbiotech, China). Thirty mg of total protein was separated by SDS-PAGE and transferred to PVDF membrane (Millipore, Bedford, MA). The following antibodies were used: primary antibodies, MMP-2, MMP-9, E-cadherin, vimentin, Snail, Slug, b-actin (1:1000 dilution; CST, Danvers, MA); secondary antibody, horseradish peroxidase-conjugated goat IgG antibody (1:2000 dilution; CST). Band signal was visualized using ECL reagent (Beyotime, Jiangsu, China). Images were obtained and quantified by Quantity One (Bio-Rad, Hercules, CA). The assays were repeated at least three times.
448
Shi et al./ Archives of Medical Research 47 (2016) 446e453
Statistical Analysis Statistical analysis was performed using SPSS v.16.0 software and GraphPad Prism v.5.0 software. Wilcoxon Signed Rank test was applied to test the differential HULC expression in breast cancer tissue compared to paired adjacent normal mammary tissues. Correlations between HULC expression level and clinicopathological factors were analyzed by c2 test. Two-tailed Student t test was used for comparisons of two independent groups. One-way ANOVA was used to determine the differences between groups in in vitro analyses. Kaplan-Meier method and log-rank test were performed for patients’ survival analyses. Multivariate Cox proportional hazards method was used for analyzing the relationship between the variables and patient’s survival time. Data were expressed as mean standard deviation; p !0.05 was considered statistically significant.
Correlation of HULC with TNBC Clinicopathological Features We aimed to analyze the association between HULC expression and clinicopathological factors in TNBC patients. Levels of HULC expression in 96 TNBC tissues were detected. According to a published study (25), TNBC tissues were classified into the low expression group (n 5 48) and the high expression group (n 5 48) according to median expression level of all TNBC samples. The relationships between HULC expression and clinicopathological factors are shown in Table 1. High expression of HULC was obviously correlated with clinical stage (I;eII vs. IIIeIV, p !0.001), N classification (N0eN1 vs. N1eN2, p !0.001), M classification (M0 vs. M1, p 5 0.026), and histological grade (G0 vs. G1eG2, p 5 0.020). However, there were no significant associations between HULC expression and age ( p 5 0.127) and T classification ( p 5 0.059).
Results Up-regulation of HULC Expression in TNBC Tissues and Cell Lines qRT-PCR was performed to detect the levels of HULC expression in the 22 pairs of TNBC tissues and pairmatched adjacent normal mammary tissues. The level of HULC expression was significantly elevated in TNBC tissues in comparison to pair-matched adjacent normal mammary tissues with an average of 2.89 folds ( p !0.001, Figure 1A). HULC expression was measured in human TNBC cell lines (MDA-MB-231, MDA-MB-468, BT549, BT483) and a normal breast epithelial cell line (MCF-10A). HULC expression was obviously increased in TNBC cell lines compared with normal breast epithelial cell line ( p !0.001, Figure 1B).
Prognostic Significance of HULC in TNBC Patients To explore the prognostic significance of HULC in TNBC, we analyzed the correction between HULC expression and patients’ overall survival through Kaplan-Meier analysis and log-rank test. Kaplan-Meier analysis showed that TNBC patients in the HULC-high group have obviously shorter overall survival than those in the HULC-low group ( p !0.001, Figure 2). Cox regression analyses were performed to screen various prognostic factors for overall survival of TNBC patients. Univariate analysis of overall survival identified five prognostic parameters (Table 2): clinical stage ( p 5 0.002), T classification ( p 5 0.031), N classification ( p !0.001), M classification ( p 5 0.003) and HULC expression ( p !0.001). Parameters with a value of p !0.05 were included in multivariate
Figure 1. Highly upregulated in liver cancer (HULC) expression in triple-negative breast cancer (TNBC) tissues and cell lines. (A) HULC expression is increased in TNBC tissues compared with adjacent normal mammary tissues. (B) HULC expression is elevated in TNBC cell lines in comparison to normal breast epithelial cell line.
Role of HULC in Triple Negative Breast Cancer Table 1. Correlations between lncRNA HULC expression and clinicopathological characteristics in triple negative breast cancer
Characteristics Age (years) !50 $50 Clinical stage IeII IIIeIV T classification T1eT2 T3eT4 N classification N0eN1 N2eN3 M classification M0 M1 Histological grade G1 G2eG3
n
High expression (%)
Low expression (%)
31 65
19 (61.3) 29 (44.6)
12 (38.7) 36 (55.4)
0.127
47 49
11 (23.4) 37 (75.5)
36 (76.6) 12 (24.5)
!0.001
59 37
25 (42.4) 23 (62.2)
34 (57.6) 14 (37.8)
0.059
54 42
14 (25.9) 34 (81.0)
40 (74.1) 8 (19.0)
!0.001
87 9
40 (46.0) 8 (88.9)
47 (54.0) 1 (11.1)
0.036
35 61
12 (34.3) 36 (59.0)
23 (65.7) 25 (41.0)
0.020
p
analysis. Multivariate analysis showed that HULC highexpression was an independent unfavorable prognostic factor of overall survival for TNBC patients ( p 5 0.001, Table 2). Suppression of HULC Expression Has No Effect on the Growth of TNBC Cells We observed that HULC expression was relatively higher in MDA-MB-231 and BT549 TNBC cell lines than the normal breast epithelial cell line (MCF-10A). Thus, we chose MDA-MB-231 and BT549 cell lines for the following loss-of-function studies. In order to knock down HULC
449
expression in TNBC cells, siRNA was transfected into two TNBC cell lines, MDA-MB-231 and BT549, respectively. Suppressing efficiencies of two TNBC cell lines were assessed by qRT-PCR (Figure 3A). Results from the MTT assay showed that both MDAMB-231 and BT549 cells transfected with si-HULC have no effect on cell proliferation compared to the cells transfected with si-NC ( p O0.05, Figure 3B). Silencing of HULC Markedly Decreases TNBC Cells Migration and Invasion To analyze the biological function of HULC in TNBC cell migration, transwell assays were performed in MDA-MB231 and BT549 cells, and the result showed that the migration ability of both the MDA-MB-231 and BT549 transfected with si-HULC were markedly decreased compared with si-NC groups, respectively ( p !0.001, Figure 4A). Using a Boyden chamber coated with matrigel, we measured changes in TNBC cell invasion ability. Compared with the si-NC groups, si-HULC MDA-MB231 and BT549 cells both suggested obviously suppressed invasiveness ( p !0.001, Figure 4B). Involvement of MMP-2 and MMP-9 in HULC-mediated Metastasis To explore the molecular mechanisms by which HULC contributes to cell motility of TNBC, we carried out Western blot to detect the protein expression of cell metastasis-associated molecules including MMP-2, MMP-9, E-cadherin, vimentin, Snail, and Slug. Among these, only MMP-2 and MMP-9 were found to be significantly down-regulated in MDA-MB-231 and BT549 cells transfected with si-HULC (Figure 5). However, epithelial-mesenchymal transition (EMT)-associated gene expressions of E-cadherin, vimentin, Snail, and Slug were not affected by suppressing HULC in MDA-MB-231 and BT549 cells (Figure 5).
Discussion
Figure 2. High expression of HULC predicts unfavorable clinical outcome of TNBC patients. (A color figure can be found in the online version of this article.)
Long noncoding RNAs (lncRNAs) highly up-regulated in liver cancer (HULC) is located on chromosome 6p24.3 and is conserved in primates (22). HULC is one of the strongly overexpressed noncoding transcripts to be primarily identified in human HCC (22). HULC is multifunctional and involves in a variety of cellular processes including tumorigenesis (26). Until now, the clinical significance and biological function of HULC in HCC has been reported in several studies. Originally, Panzitt et al. found that HULC was the most upregulated in HCC through cDNA arrays and RT-PCR and showed a role for HULC in post-transcriptional modulation of HCC-associated gene expression (22). Matouk et al. then
450
Shi et al./ Archives of Medical Research 47 (2016) 446e453
Table 2. Uni- and multivariate Cox regression of prognostic factors for overall survival in triple-negative breast cancer patients Univariate analysis Parameter
HR
Age (years) (!50 vs. $50) Clinical stage (IeII vs. IIIeIV) T classification (T1eT2 vs. T3eT4) N classification (N0eN1 vs. N2eN3) M classification (M0 vs. M1) Histological grade (G1 vs.G2eG3) lncRNA HULC expression (low vs. high)
0.608 2.203 1.730 2.588 3.186 1.703 3.280
Multivariate analysis
95% CI
p
0.367e1.007 1.328e3.656 1.050e2.850 1.563e4.286 1.487e6.825 0.984e2.945 1.944e5.533
0.053 0.002 0.031 !0.001 0.003 0.057 !0.001
demonstrated that HULC overexpressed in hepatic colorectal metastasis (27). H€ammerle et al. reported that HULC was overexpressed in HCC and correlated with staging and grading (28). Meanwhile, Xie et al. first indicated that the expression of HULC in plasma can be used as a noninvasive novel biomarker for the diagnosis of HCC (29). Furthermore, Yang et al. showed that HULC expression was associated with overall survival in HCC patients, and HULC overexpression was an independent favorable factor
HR
95% CI
p
0.495 1.289 2.289 2.153
0.116e2.112 0.763e2.178 0.536e9.773 0.943e4.917
0.342 0.343 0.263 0.069
2.842
1.514e5.333
0.001
for overall survival and disease-free survival time in HCC patients (30). In addition, Liu et al. found that the variant genotypes of rs7763881 in HULC might contribute to reduced susceptibility to HCC in HBV-persistent carriers (31). Zhao et al. suggested that circulation of HULC was obviously increased in plasma samples of HBV-related cirrhosis patients (32). The status of HULC expression in TNBC is still unknown. Our study first showed that HULC expression was
Figure 3. Suppressing HULC expression has no effect on the growth of TNBC cells. (A) The level of knockdown efficiency was determined by qRT-PCR in MDA-MB-231 and BT549 cells. (B) Knocking down HULC expression has no effect on the proliferative ability of MDA-MB-231 and BT549 cells.
Role of HULC in Triple Negative Breast Cancer
451
Figure 4. Silencing of HULC markedly decreases TNBC cell migration and invasive. (A) Knockdown of HULC reduced the ability of MDA-MB-231 and BT549 cells. (B) Suppression of HULC significantly decreased invasiveness of MDA-MB-231 and BT549 cells. (A color figure can be found in the online version of this article.)
increased in TNBC tissues and cell lines compared with paired normal mammary tissues and normal breast epithelial cell line, respectively. Similarly, Sun et al. found HULC expression was significantly up-regulated in osteosarcoma tissues and cell lines compared with normal controls (33). In pancreatic cancer, overexpression of HULC was found in tumor tissues and cell lines as compared to normal pancreatic tissues (34). Moreover, Zhao et al. demonstrated that HULC was significantly overexpressed in gastric cancer cell lines and tissues (35). A similar study was also reported in lymphoma patients (36). These results
consistently suggest that HULC is overexpressed in most cancer tissues. Furthermore, we analyzed the relationships between HULC expression and clinicopathological factors and found that overexpression of HULC was positively correlated with clinical stage, N classification, M classification, and histological grade. Similarly, Zhao et al. indicated that HULC overexpression was correlated with lymph node metastasis, distant metastasis and advanced TNM stages in gastric cancer patients (35). In diffuse large B-cell lymphoma patients, HULC was closely associated with patient characteristics such as Ann Arbor stages, B symptoms,
452
Shi et al./ Archives of Medical Research 47 (2016) 446e453
was an independent unfavorable prognostic factor of overall survival for TNBC patients. However, there is evidence suggesting that HULC overexpression was a favorable factor for HCC overall survival and disease-free survival time (30). The discrepancy toward the data of Yang et al. (30) would be most likely due to the different tumor type. In conclusion, HULC expression was increased in TNBC tissues and cell lines and correlated with malignant status and poor prognosis of TNBC patients. Silencing HULC expression obviously suppressed TNBC cell migration and invasion by regulating MMP-2 and MMP-9. Acknowledgments Figure 5. HULC regulates MMP-2 and MMP-9 expression in TNBC. Knocking down HULC decreased the expression of MMP-2 and MMP-9, but with no effect on EMT-associated gene expressions including E-cadherin, vimentin, Snail, and Slug.
CHOP-like treatment, rituximab and international prognostic index (36). In human gliomas, Zhu et al. reported that HULC expression correlated with angiogenesis and tumor grade (37). In order to explore the biological function and mechanism of HULC in TNBC, we performed loss-of-function studies of HULC in TNBC cells and detected the metastasis-associated gene expression. We found that silencing HULC expression obviously suppressed TNBC cells migration and invasion in vitro and down-regulated MMP-2 and MMP-9 expressions. Moreover, Zhu et al. illustrated HULC inhibited angiogenesis through regulating ESM-1 via the PI3K-Ak-mTOR signaling pathway (37). In addition, knockdown of HULC expression could markedly arrest cell growth and induce apoptosis by suppressing Bcl2 and cyclin D1 in lymphoma cells (36). Several studies showed that HULC served as an endogenous ‘‘sponge’’, which down-regulates a series of microRNA activities (38e40). Generally, HULC acts as an oncogene lncRNA in tumor development and progression. In the past few years, HULC overexpression has been shown to be an independent prognostic biomarker in a variety of human cancers, which has a favorable or unfavorable prognostic value depending on cancer types. In diffuse large B-cell lymphoma patients, HULC overexpression was an independent poor factor that forecasts overall survival and progression-free survival (36). Meanwhile, Peng et al. showed that HULC overexpression served as an independent unfavorable predictor for overall survival in pancreatic cancer (34). Sun et al. similarly reported that higher HULC expression was associated with shorter overall survival of osteosarcoma patients (33). In our study, HULC-high group has an obviously shorter overall survival than those in the HULC-low group for TNBC patients. Multivariate analysis showed that HULC high-expression
This study was funded by the Foundation of Science Technology, Department of Hunan Province, China (No. 2014CFB883). Conflicts of Interest: All authors declare that they have no competing interests. Ethics statement: The Research Ethics Committee of The Affiliated Cancer Hospital of Xiangya School of Medicine approved this protocol and written informed consents were obtained from each patient. The entire study was performed based on the Declaration of Helsinki.
References 1. DeSantis CE, Fedewa SA, Goding Sauer A, et al. Breast cancer statistics, 2015: Convergence of incidence rates between black and white women. CA Cancer J Clin 2016;66:31e42. 2. Torre LA, Bray F, Siegel RL, et al. Global cancer statistics, 2012. CA Cancer J Clin 2015;65:87e108. 3. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin 2016;66:7e30. 4. Chen W, Zheng R, Baade PD, et al. Cancer statistics in China, 2015. CA Cancer J Clin 2016;66:115e132. 5. DeSantis C, Ma J, Bryan L, et al. Breast cancer statistics, 2013. CA Cancer J Clin 2014;64:52e62. 6. Munzone E, Colleoni M. Clinical overview of metronomic chemotherapy in breast cancer. Nat Rev Clin Oncol 2015;12:631e644. 7. Dent R, Hanna WM, Trudeau M, et al. Pattern of metastatic spread in triple-negative breast cancer. Breast Cancer Res Treat 2009;115: 423e428. 8. Boyle P. Triple-negative breast cancer: epidemiological considerations and recommendations. Ann Oncol 2012;23(Suppl 6):vi7evi12. 9. Andre F, Zielinski CC. Optimal strategies for the treatment of metastatic triple-negative breast cancer with currently approved agents. Ann Oncol 2012;23(Suppl 6):vi46evi51. 10. Duffy MJ, McGowan PM, Crown J. Targeted therapy for triplenegative breast cancer: where are we? Int J Cancer 2012;131: 2471e2477. 11. Ponting CP, Oliver PL, Reik W. Evolution and functions of long noncoding RNAs. Cell 2009;136:629e641. 12. Peng W, Fan H. Long non-coding RNA PANDAR correlates with poor prognosis and promotes tumorigenesis in hepatocellular carcinoma. Biomed Pharmacother 2015;72:113e118. 13. Quagliata L, Matter MS, Piscuoglio S, et al. Long noncoding RNA HOTTIP/HOXA13 expression is associated with disease progression and predicts outcome in hepatocellular carcinoma patients. Hepatology 2014;59:911e923. 14. Jiang W, Zhang D, Xu B, et al. Long non-coding RNA BANCR promotes proliferation and migration of lung carcinoma via MAPK pathways. Biomed Pharmacother 2015;69:90e95.
Role of HULC in Triple Negative Breast Cancer 15. Deng J, Liang Y, Liu C, et al. The up-regulation of long non-coding RNA AFAP1-AS1 is associated with the poor prognosis of NSCLC patients. Biomed Pharmacother 2015;75:8e11. 16. Xiong G, Yang L, Chen Y, et al. Linc-POU3F3 promotes cell proliferation in gastric cancer via increasing T-reg distribution. Am J Transl Res 2015;7:2262e2269. 17. Zhao W, Dong S, Duan B, et al. HOTAIR is a predictive and prognostic biomarker for patients with advanced gastric adenocarcinoma receiving fluorouracil and platinum combination chemotherapy. Am J Transl Res 2015;7:1295e1302. 18. Wang YL, Overstreet AM, Chen MS, et al. Combined inhibition of EGFR and c-ABL suppresses the growth of triple-negative breast cancer growth through inhibition of HOTAIR. Oncotarget 2015;6: 11150e11161. 19. Jin C, Yan B, Lu Q, et al. Reciprocal regulation of Hsa-miR-1 and long noncoding RNA MALAT1 promotes triple-negative breast cancer development. Tumour Biol 2016;37:7383e7394. 20. Eades G, Wolfson B, Zhang Y, et al. lincRNA-RoR and miR-145 regulate invasion in triple-negative breast cancer via targeting ARF6. Mol Cancer Res 2015;13:330e338. 21. Augoff K, McCue B, Plow EF, et al. miR-31 and its host gene lncRNA LOC554202 are regulated by promoter hypermethylation in triplenegative breast cancer. Mol Cancer 2012;11:5. 22. Panzitt K, Tschernatsch MM, Guelly C, et al. Characterization of HULC, a novel gene with striking up-regulation in hepatocellular carcinoma, as noncoding RNA. Gastroenterology 2007;132: 330e342. 23. Kitagawa M, Kitagawa K, Kotake Y, et al. Cell cycle regulation by long non-coding RNAs. Cell Mol Life Sci 2013;70:4785e4794. 24. Parasramka MA, Maji S, Matsuda A, et al. Long non-coding RNAs as novel targets for therapy in hepatocellular carcinoma. Pharmacol Ther 2016;161:67e78. 25. Kong R, Zhang EB, Yin DD, et al. Long noncoding RNA PVT1 indicates a poor prognosis of gastric cancer and promotes cell proliferation through epigenetically regulating p15 and p16. Mol Cancer 2015;14: 82. 26. Huang JL, Zheng L, Hu YW, et al. Characteristics of long non-coding RNA and its relation to hepatocellular carcinoma. Carcinogenesis 2014;35:507e514. 27. Matouk IJ, Abbasi I, Hochberg A, et al. Highly upregulated in liver cancer noncoding RNA is overexpressed in hepatic colorectal metastasis. Eur J Gastroenterol Hepatol 2009;21:688e692.
453
28. Hammerle M, Gutschner T, Uckelmann H, et al. Posttranscriptional destabilization of the liver-specific long noncoding RNA HULC by the IGF2 mRNA-binding protein 1 (IGF2BP1). Hepatology 2013;58: 1703e1712. 29. Xie H, Ma H, Zhou D. Plasma HULC as a promising novel biomarker for the detection of hepatocellular carcinoma. Biomed Res Int 2013; 2013:136106. 30. Yang Z, Lu Y, Xu Q, et al. HULC and H19 played different roles in overall and disease-free survival from hepatocellular carcinoma after curative hepatectomy: a preliminary analysis from gene expression omnibus. Dis Markers 2015;2015:191029. 31. Liu Y, Pan S, Liu L, et al. A genetic variant in long non-coding RNA HULC contributes to risk of HBV-related hepatocellular carcinoma in a Chinese population. PLoS One 2012;7:e35145. 32. Zhao J, Fan Y, Wang K, et al. LncRNA HULC affects the differentiation of Treg in HBV-related liver cirrhosis. Int Immunopharmacol 2015;28:901e905. 33. Sun XH, Yang LB, Geng XL, et al. Increased expression of lncRNA HULC indicates a poor prognosis and promotes cell metastasis in osteosarcoma. Int J Clin Exp Pathol 2015;8:2994e3000. 34. Peng W, Gao W, Feng J. Long noncoding RNA HULC is a novel biomarker of poor prognosis in patients with pancreatic cancer. Med Oncol 2014;31:346. 35. Zhao Y, Guo Q, Chen J, et al. Role of long non-coding RNA HULC in cell proliferation, apoptosis and tumor metastasis of gastric cancer: a clinical and in vitro investigation. Oncol Rep 2014;31:358e364. 36. Peng W, Wu J, Feng J. Long noncoding RNA HULC predicts poor clinical outcome and represents pro-oncogenic activity in diffuse large B-cell lymphoma. Biomed Pharmacother 2016;79:188e193. 37. Zhu Y, Zhang X, Qi L, et al. HULC long noncoding RNA silencing suppresses angiogenesis by regulating ESM-1 via the PI3K/Akt/ mTOR signaling pathway in human gliomas. Oncotarget 2016;7: 14429e14440. 38. Lu Z, Xiao Z, Liu F, et al. Long non-coding RNA HULC promotes tumor angiogenesis in liver cancer by up-regulating sphingosine kinase 1 (SPHK1). Oncotarget 2016;7:241e254. 39. Wang J, Liu X, Wu H, et al. CREB up-regulates long non-coding RNA, HULC expression through interaction with microRNA-372 in liver cancer. Nucleic Acids Res 2010;38:5366e5383. 40. Cui M, Xiao Z, Wang Y, et al. Long noncoding RNA HULC modulates abnormal lipid metabolism in hepatoma cells through an miR9-mediated RXRA signaling pathway. Cancer Res 2015;75:846e857.
ORIGINAL ARTICLE
Long Noncoding RNA Highly Up-regulated in Liver Cancer Predicts Unfavorable Outcome and Regulates Metastasis by MMPs in Triple-negative Breast Cancer Feng Shi,a,* Fang Xiao,b,* Ping Ding,c Hong Qin,d and Ruixue Huange a
Internal Medicine Department of Thyroid and Radionuclide Therapy, Hunan Cancer Hospital, The Affiliated Cancer Hospital of Xiangya School of Medicine, bDepartment of Health Toxicology, Xiangya School of Public Health, Central South University, Changsha, China c Center of Preventive Medicine Experiment, Xiangya School of Public Health, Central South University, Changsha, China d Department of Nutrition and Food Hygiene, eDepartment of Occupational and Environmental Health, Xiangya School of Public Health, Central South University, China Received for publication April 18, 2016; accepted October 28, 2016 (ARCMED-D-16-00233).
Background and Aims. Long noncoding RNA (lncRNA) highly up-regulated in liver cancer (HULC) is the first identified lncRNA highly expressed in hepatocellular carcinoma and acts as an oncogenic lncRNA. However, the significance of HULC in triple-negative breast cancer (TNBC) is still unclear. Our purpose is to explore the clinical significance and biological function of HULC in TNBC. Methods. Expression of HULC in TNBC tissues and cell lines was measured and the association between HULC expression and clinicopathological factors was analyzed. Cell proliferation, migration, and invasion assays were executed by using TNBC cell lines transfected with si-HULC or si-NC. The potential mechanism of HULC was explored by Western blot. Results. HULC expression was increased in TNBC tissues and cell lines and associated with malignant status and poor prognosis of TNBC patients. Furthermore, silencing TNBC expression effectively suppressed TNBC cells metastasis through MMP-2 and MMP-9. Conclusion. HULC acts as an oncogene lncRNA in TNBC and as an independent poor prognostic factor in TNBC patients. Ó 2016 IMSS. Published by Elsevier Inc. Key Words: HULC, lncRNA, Triple negative breast cancer, Metastasis, MMP.
Introduction Breast cancer remains the most common cancer in females and the second leading cause of cancer-related mortality in women, suggesting a serious problem worldwide to women (1,2). According to 2016 U.S. Cancer Statistics, an estimated total of 249,260 new breast cancer cases and 40,890 breast cancer deaths will occur in the U.S. in 2016 (3). In China, breast cancer alone is expected to account for 15% of all new cancers in women and is the
*
These authors are co-first authors. Address reprint requests to: Ruixue Huang, Department of Occupational and Environmental Health, Xiangya School of Public Health, Central South University, 410078, China; Phone: 86-18975832122; FAX: 86-731-84805460; E-mail: [email protected].
leading cause of cancer death in women !45 years of age (4). Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer defined by negative expression for estrogen receptors (ER) and progesterone receptors (PR) and normal (low) expression levels of human epidermal growth factor receptor 2 (HER2) (5,6). Due to lack of three important therapeutic markers, TNBC shows higher metastases and relapse rates than other types (7,8). Patients with TNBC carry a poor prognosis (9,10). Therefore, it is urgent to identify novel biomarkers for developing targeted molecular therapies and predicting prognosis in TNBC patients. Long noncoding RNAs (lncRNAs) are transcripts with no protein coding function and longer than 200 nucleotides (11). Dysregulation of lncRNAs has been shown to play a critical role in the development of cancer such as
0188-4409/$ - see front matter. Copyright Ó 2016 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2016.11.001
Role of HULC in Triple Negative Breast Cancer
hepatocellular carcinoma (HCC) (12,13), lung cancer (14,15), and gastric cancer (16,17). Nowadays, some oncogene or anti-oncogene lncRNAs are gradually discovered and identified in TNBC such as HOTAIR (18), MALAT1 (19), RoR (20), and LOC554202 (21). Highly up-regulated in liver cancer (HULC) is the first identified lncRNA highly expressed in HCC (22). HULC is located on human chromosome 6p24.3. Some studies suggested that HULC was overexpressed in a variety of human cancers and acted as an oncogene lncRNA in tumor development and progression (23,24). However, the significance of HULC in TNBC is still unknown. In order to explore the clinical significance of HULC in TNBC, we measured HULC expression in TNBC clinical samples and analyzed the association between HULC expression and clinicopathological characteristics. Furthermore, we performed loss-of-function studies of HULC to identify the biological function of HULC in TNBC cell lines.
Materials and Methods Patients and Tissue Sample Collection Ninety-six TNBC tissues and 22 paired normal mammary tissues were collected at The Affiliated Cancer Hospital of Xiangya School of Medicine from 2008e2013. Clinical tissue samples were obtained from surgery or biopsy and were immediately frozen in liquid nitrogen and kept at 80 C. Clinicopathological characteristics included age, clinical stage, histological grade, tumor size, lymph node metastasis, and distant metastasis. None of the patients had received tumor-specific therapy before diagnosis. Overall survival of TNBC patients was also followed up until December 31, 2015. The Research Ethics Committee of The Affiliated Cancer Hospital of Xiangya School of Medicine approved this protocol and written informed consent was obtained from each patient. The entire study was performed based on the Declaration of Helsinki.
447
Cell Culture and Transfection Four TNBC cell lines (MDA-MB-231, MDA-MB-468, BT549, BT483) and a normal breast epithelial cell line (MCF-10A) were purchased from the Chinese Academy of Sciences (Shanghai Cell Bank). These cells were cultured in DMEM containing 10% fetal bovine serum (FBS) in humidified 5% CO2 atmosphere at 37 C. For transfection, cells were cultured up to 70% confluency and transfected with si-HULC-RNA or si-NC (RiboBio, China), using Lipofectamine 2000 (Invitrogen, Grand Island, NY) by incubating with OptiMem-I media for 4 h. The cells were then cultured in fresh DMEM with 10% FBS. The relative level of HULC in transfected cells was examined using qRT-PCR. Cell Proliferation Analysis Cell proliferation was analyzed using 3-(4,5-cimethylthiazol2-yl)-2,5-diphenyl tetra-zolium bromide (MTT, Sigma, St. Louis, MO) assay. Standard MTT assay was used to detect cell proliferation. Briefly, cells were seeded at a density of 2 103 cells per well in 100 ml culture medium in 96-well culture plates. Cell proliferation was assessed after siRNA transient transfection for 24, 48, 72 and 96 h. The optical density of the wells was measured with using a microplate reader at 490 nm. Each experiment was performed in triplicate. Cell Migration and Invasion Assays Cell migration and invasion assays were examined by 24well plate Transwell system (Corning, New York). Briefly, 1 105 cells were seeded on a fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Corning). After the cells were incubated for 12 h, Giemsa-stained cells adhering to the lower surface were counted under a microscope in five predetermined fields (100). For the cell invasion assay, the procedure was similar to the cell migration assay except that the transwell membranes were precoated with 24 mg/mL Matrigel (Corning). Western Blotting
RNA Extraction and Quantitative RT-PCR (qRT-PCR) Total RNA was isolated using RNAiso Plus (TaKaRa, Dalian, China) according to the manufacturer’s protocol. CCAT2 and GAPDH expressions were quantified using real-time PCR with the SYBRÒ Green (TaKaRa) dye detection method on Light Cycler Roche 480 PCR instrument. Primers are as follows: HULC, forward primer: 50 -ATCTGCAAGCCAGGAAGAGTC-30 ; reverse primer: 50 -CTTGCTTGATGCTTTGGTCTGT-30 . GAPDH, forward primer: 50 -TGACAACTTTGGTATCGTGGAAGG30 ; reverse primer: 50 -GCAGGGATG ATGTTCTGGA GAG-30 . GAPDH was used as an internal control for the expression analysis of HULC. Relative quantification of RNA expression was calculated using the 2DDCt method.
For the protein expression analysis, standard Western blotting was carried out. MDA-MB-231 cells and BT549 cells transfected with si-HULC or si-NC after 48 h were lysed in RIPA lysis buffer (Cwbiotech, China). Thirty mg of total protein was separated by SDS-PAGE and transferred to PVDF membrane (Millipore, Bedford, MA). The following antibodies were used: primary antibodies, MMP-2, MMP-9, E-cadherin, vimentin, Snail, Slug, b-actin (1:1000 dilution; CST, Danvers, MA); secondary antibody, horseradish peroxidase-conjugated goat IgG antibody (1:2000 dilution; CST). Band signal was visualized using ECL reagent (Beyotime, Jiangsu, China). Images were obtained and quantified by Quantity One (Bio-Rad, Hercules, CA). The assays were repeated at least three times.
448
Shi et al./ Archives of Medical Research 47 (2016) 446e453
Statistical Analysis Statistical analysis was performed using SPSS v.16.0 software and GraphPad Prism v.5.0 software. Wilcoxon Signed Rank test was applied to test the differential HULC expression in breast cancer tissue compared to paired adjacent normal mammary tissues. Correlations between HULC expression level and clinicopathological factors were analyzed by c2 test. Two-tailed Student t test was used for comparisons of two independent groups. One-way ANOVA was used to determine the differences between groups in in vitro analyses. Kaplan-Meier method and log-rank test were performed for patients’ survival analyses. Multivariate Cox proportional hazards method was used for analyzing the relationship between the variables and patient’s survival time. Data were expressed as mean standard deviation; p !0.05 was considered statistically significant.
Correlation of HULC with TNBC Clinicopathological Features We aimed to analyze the association between HULC expression and clinicopathological factors in TNBC patients. Levels of HULC expression in 96 TNBC tissues were detected. According to a published study (25), TNBC tissues were classified into the low expression group (n 5 48) and the high expression group (n 5 48) according to median expression level of all TNBC samples. The relationships between HULC expression and clinicopathological factors are shown in Table 1. High expression of HULC was obviously correlated with clinical stage (I;eII vs. IIIeIV, p !0.001), N classification (N0eN1 vs. N1eN2, p !0.001), M classification (M0 vs. M1, p 5 0.026), and histological grade (G0 vs. G1eG2, p 5 0.020). However, there were no significant associations between HULC expression and age ( p 5 0.127) and T classification ( p 5 0.059).
Results Up-regulation of HULC Expression in TNBC Tissues and Cell Lines qRT-PCR was performed to detect the levels of HULC expression in the 22 pairs of TNBC tissues and pairmatched adjacent normal mammary tissues. The level of HULC expression was significantly elevated in TNBC tissues in comparison to pair-matched adjacent normal mammary tissues with an average of 2.89 folds ( p !0.001, Figure 1A). HULC expression was measured in human TNBC cell lines (MDA-MB-231, MDA-MB-468, BT549, BT483) and a normal breast epithelial cell line (MCF-10A). HULC expression was obviously increased in TNBC cell lines compared with normal breast epithelial cell line ( p !0.001, Figure 1B).
Prognostic Significance of HULC in TNBC Patients To explore the prognostic significance of HULC in TNBC, we analyzed the correction between HULC expression and patients’ overall survival through Kaplan-Meier analysis and log-rank test. Kaplan-Meier analysis showed that TNBC patients in the HULC-high group have obviously shorter overall survival than those in the HULC-low group ( p !0.001, Figure 2). Cox regression analyses were performed to screen various prognostic factors for overall survival of TNBC patients. Univariate analysis of overall survival identified five prognostic parameters (Table 2): clinical stage ( p 5 0.002), T classification ( p 5 0.031), N classification ( p !0.001), M classification ( p 5 0.003) and HULC expression ( p !0.001). Parameters with a value of p !0.05 were included in multivariate
Figure 1. Highly upregulated in liver cancer (HULC) expression in triple-negative breast cancer (TNBC) tissues and cell lines. (A) HULC expression is increased in TNBC tissues compared with adjacent normal mammary tissues. (B) HULC expression is elevated in TNBC cell lines in comparison to normal breast epithelial cell line.
Role of HULC in Triple Negative Breast Cancer Table 1. Correlations between lncRNA HULC expression and clinicopathological characteristics in triple negative breast cancer
Characteristics Age (years) !50 $50 Clinical stage IeII IIIeIV T classification T1eT2 T3eT4 N classification N0eN1 N2eN3 M classification M0 M1 Histological grade G1 G2eG3
n
High expression (%)
Low expression (%)
31 65
19 (61.3) 29 (44.6)
12 (38.7) 36 (55.4)
0.127
47 49
11 (23.4) 37 (75.5)
36 (76.6) 12 (24.5)
!0.001
59 37
25 (42.4) 23 (62.2)
34 (57.6) 14 (37.8)
0.059
54 42
14 (25.9) 34 (81.0)
40 (74.1) 8 (19.0)
!0.001
87 9
40 (46.0) 8 (88.9)
47 (54.0) 1 (11.1)
0.036
35 61
12 (34.3) 36 (59.0)
23 (65.7) 25 (41.0)
0.020
p
analysis. Multivariate analysis showed that HULC highexpression was an independent unfavorable prognostic factor of overall survival for TNBC patients ( p 5 0.001, Table 2). Suppression of HULC Expression Has No Effect on the Growth of TNBC Cells We observed that HULC expression was relatively higher in MDA-MB-231 and BT549 TNBC cell lines than the normal breast epithelial cell line (MCF-10A). Thus, we chose MDA-MB-231 and BT549 cell lines for the following loss-of-function studies. In order to knock down HULC
449
expression in TNBC cells, siRNA was transfected into two TNBC cell lines, MDA-MB-231 and BT549, respectively. Suppressing efficiencies of two TNBC cell lines were assessed by qRT-PCR (Figure 3A). Results from the MTT assay showed that both MDAMB-231 and BT549 cells transfected with si-HULC have no effect on cell proliferation compared to the cells transfected with si-NC ( p O0.05, Figure 3B). Silencing of HULC Markedly Decreases TNBC Cells Migration and Invasion To analyze the biological function of HULC in TNBC cell migration, transwell assays were performed in MDA-MB231 and BT549 cells, and the result showed that the migration ability of both the MDA-MB-231 and BT549 transfected with si-HULC were markedly decreased compared with si-NC groups, respectively ( p !0.001, Figure 4A). Using a Boyden chamber coated with matrigel, we measured changes in TNBC cell invasion ability. Compared with the si-NC groups, si-HULC MDA-MB231 and BT549 cells both suggested obviously suppressed invasiveness ( p !0.001, Figure 4B). Involvement of MMP-2 and MMP-9 in HULC-mediated Metastasis To explore the molecular mechanisms by which HULC contributes to cell motility of TNBC, we carried out Western blot to detect the protein expression of cell metastasis-associated molecules including MMP-2, MMP-9, E-cadherin, vimentin, Snail, and Slug. Among these, only MMP-2 and MMP-9 were found to be significantly down-regulated in MDA-MB-231 and BT549 cells transfected with si-HULC (Figure 5). However, epithelial-mesenchymal transition (EMT)-associated gene expressions of E-cadherin, vimentin, Snail, and Slug were not affected by suppressing HULC in MDA-MB-231 and BT549 cells (Figure 5).
Discussion
Figure 2. High expression of HULC predicts unfavorable clinical outcome of TNBC patients. (A color figure can be found in the online version of this article.)
Long noncoding RNAs (lncRNAs) highly up-regulated in liver cancer (HULC) is located on chromosome 6p24.3 and is conserved in primates (22). HULC is one of the strongly overexpressed noncoding transcripts to be primarily identified in human HCC (22). HULC is multifunctional and involves in a variety of cellular processes including tumorigenesis (26). Until now, the clinical significance and biological function of HULC in HCC has been reported in several studies. Originally, Panzitt et al. found that HULC was the most upregulated in HCC through cDNA arrays and RT-PCR and showed a role for HULC in post-transcriptional modulation of HCC-associated gene expression (22). Matouk et al. then
450
Shi et al./ Archives of Medical Research 47 (2016) 446e453
Table 2. Uni- and multivariate Cox regression of prognostic factors for overall survival in triple-negative breast cancer patients Univariate analysis Parameter
HR
Age (years) (!50 vs. $50) Clinical stage (IeII vs. IIIeIV) T classification (T1eT2 vs. T3eT4) N classification (N0eN1 vs. N2eN3) M classification (M0 vs. M1) Histological grade (G1 vs.G2eG3) lncRNA HULC expression (low vs. high)
0.608 2.203 1.730 2.588 3.186 1.703 3.280
Multivariate analysis
95% CI
p
0.367e1.007 1.328e3.656 1.050e2.850 1.563e4.286 1.487e6.825 0.984e2.945 1.944e5.533
0.053 0.002 0.031 !0.001 0.003 0.057 !0.001
demonstrated that HULC overexpressed in hepatic colorectal metastasis (27). H€ammerle et al. reported that HULC was overexpressed in HCC and correlated with staging and grading (28). Meanwhile, Xie et al. first indicated that the expression of HULC in plasma can be used as a noninvasive novel biomarker for the diagnosis of HCC (29). Furthermore, Yang et al. showed that HULC expression was associated with overall survival in HCC patients, and HULC overexpression was an independent favorable factor
HR
95% CI
p
0.495 1.289 2.289 2.153
0.116e2.112 0.763e2.178 0.536e9.773 0.943e4.917
0.342 0.343 0.263 0.069
2.842
1.514e5.333
0.001
for overall survival and disease-free survival time in HCC patients (30). In addition, Liu et al. found that the variant genotypes of rs7763881 in HULC might contribute to reduced susceptibility to HCC in HBV-persistent carriers (31). Zhao et al. suggested that circulation of HULC was obviously increased in plasma samples of HBV-related cirrhosis patients (32). The status of HULC expression in TNBC is still unknown. Our study first showed that HULC expression was
Figure 3. Suppressing HULC expression has no effect on the growth of TNBC cells. (A) The level of knockdown efficiency was determined by qRT-PCR in MDA-MB-231 and BT549 cells. (B) Knocking down HULC expression has no effect on the proliferative ability of MDA-MB-231 and BT549 cells.
Role of HULC in Triple Negative Breast Cancer
451
Figure 4. Silencing of HULC markedly decreases TNBC cell migration and invasive. (A) Knockdown of HULC reduced the ability of MDA-MB-231 and BT549 cells. (B) Suppression of HULC significantly decreased invasiveness of MDA-MB-231 and BT549 cells. (A color figure can be found in the online version of this article.)
increased in TNBC tissues and cell lines compared with paired normal mammary tissues and normal breast epithelial cell line, respectively. Similarly, Sun et al. found HULC expression was significantly up-regulated in osteosarcoma tissues and cell lines compared with normal controls (33). In pancreatic cancer, overexpression of HULC was found in tumor tissues and cell lines as compared to normal pancreatic tissues (34). Moreover, Zhao et al. demonstrated that HULC was significantly overexpressed in gastric cancer cell lines and tissues (35). A similar study was also reported in lymphoma patients (36). These results
consistently suggest that HULC is overexpressed in most cancer tissues. Furthermore, we analyzed the relationships between HULC expression and clinicopathological factors and found that overexpression of HULC was positively correlated with clinical stage, N classification, M classification, and histological grade. Similarly, Zhao et al. indicated that HULC overexpression was correlated with lymph node metastasis, distant metastasis and advanced TNM stages in gastric cancer patients (35). In diffuse large B-cell lymphoma patients, HULC was closely associated with patient characteristics such as Ann Arbor stages, B symptoms,
452
Shi et al./ Archives of Medical Research 47 (2016) 446e453
was an independent unfavorable prognostic factor of overall survival for TNBC patients. However, there is evidence suggesting that HULC overexpression was a favorable factor for HCC overall survival and disease-free survival time (30). The discrepancy toward the data of Yang et al. (30) would be most likely due to the different tumor type. In conclusion, HULC expression was increased in TNBC tissues and cell lines and correlated with malignant status and poor prognosis of TNBC patients. Silencing HULC expression obviously suppressed TNBC cell migration and invasion by regulating MMP-2 and MMP-9. Acknowledgments Figure 5. HULC regulates MMP-2 and MMP-9 expression in TNBC. Knocking down HULC decreased the expression of MMP-2 and MMP-9, but with no effect on EMT-associated gene expressions including E-cadherin, vimentin, Snail, and Slug.
CHOP-like treatment, rituximab and international prognostic index (36). In human gliomas, Zhu et al. reported that HULC expression correlated with angiogenesis and tumor grade (37). In order to explore the biological function and mechanism of HULC in TNBC, we performed loss-of-function studies of HULC in TNBC cells and detected the metastasis-associated gene expression. We found that silencing HULC expression obviously suppressed TNBC cells migration and invasion in vitro and down-regulated MMP-2 and MMP-9 expressions. Moreover, Zhu et al. illustrated HULC inhibited angiogenesis through regulating ESM-1 via the PI3K-Ak-mTOR signaling pathway (37). In addition, knockdown of HULC expression could markedly arrest cell growth and induce apoptosis by suppressing Bcl2 and cyclin D1 in lymphoma cells (36). Several studies showed that HULC served as an endogenous ‘‘sponge’’, which down-regulates a series of microRNA activities (38e40). Generally, HULC acts as an oncogene lncRNA in tumor development and progression. In the past few years, HULC overexpression has been shown to be an independent prognostic biomarker in a variety of human cancers, which has a favorable or unfavorable prognostic value depending on cancer types. In diffuse large B-cell lymphoma patients, HULC overexpression was an independent poor factor that forecasts overall survival and progression-free survival (36). Meanwhile, Peng et al. showed that HULC overexpression served as an independent unfavorable predictor for overall survival in pancreatic cancer (34). Sun et al. similarly reported that higher HULC expression was associated with shorter overall survival of osteosarcoma patients (33). In our study, HULC-high group has an obviously shorter overall survival than those in the HULC-low group for TNBC patients. Multivariate analysis showed that HULC high-expression
This study was funded by the Foundation of Science Technology, Department of Hunan Province, China (No. 2014CFB883). Conflicts of Interest: All authors declare that they have no competing interests. Ethics statement: The Research Ethics Committee of The Affiliated Cancer Hospital of Xiangya School of Medicine approved this protocol and written informed consents were obtained from each patient. The entire study was performed based on the Declaration of Helsinki.
References 1. DeSantis CE, Fedewa SA, Goding Sauer A, et al. Breast cancer statistics, 2015: Convergence of incidence rates between black and white women. CA Cancer J Clin 2016;66:31e42. 2. Torre LA, Bray F, Siegel RL, et al. Global cancer statistics, 2012. CA Cancer J Clin 2015;65:87e108. 3. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin 2016;66:7e30. 4. Chen W, Zheng R, Baade PD, et al. Cancer statistics in China, 2015. CA Cancer J Clin 2016;66:115e132. 5. DeSantis C, Ma J, Bryan L, et al. Breast cancer statistics, 2013. CA Cancer J Clin 2014;64:52e62. 6. Munzone E, Colleoni M. Clinical overview of metronomic chemotherapy in breast cancer. Nat Rev Clin Oncol 2015;12:631e644. 7. Dent R, Hanna WM, Trudeau M, et al. Pattern of metastatic spread in triple-negative breast cancer. Breast Cancer Res Treat 2009;115: 423e428. 8. Boyle P. Triple-negative breast cancer: epidemiological considerations and recommendations. Ann Oncol 2012;23(Suppl 6):vi7evi12. 9. Andre F, Zielinski CC. Optimal strategies for the treatment of metastatic triple-negative breast cancer with currently approved agents. Ann Oncol 2012;23(Suppl 6):vi46evi51. 10. Duffy MJ, McGowan PM, Crown J. Targeted therapy for triplenegative breast cancer: where are we? Int J Cancer 2012;131: 2471e2477. 11. Ponting CP, Oliver PL, Reik W. Evolution and functions of long noncoding RNAs. Cell 2009;136:629e641. 12. Peng W, Fan H. Long non-coding RNA PANDAR correlates with poor prognosis and promotes tumorigenesis in hepatocellular carcinoma. Biomed Pharmacother 2015;72:113e118. 13. Quagliata L, Matter MS, Piscuoglio S, et al. Long noncoding RNA HOTTIP/HOXA13 expression is associated with disease progression and predicts outcome in hepatocellular carcinoma patients. Hepatology 2014;59:911e923. 14. Jiang W, Zhang D, Xu B, et al. Long non-coding RNA BANCR promotes proliferation and migration of lung carcinoma via MAPK pathways. Biomed Pharmacother 2015;69:90e95.
Role of HULC in Triple Negative Breast Cancer 15. Deng J, Liang Y, Liu C, et al. The up-regulation of long non-coding RNA AFAP1-AS1 is associated with the poor prognosis of NSCLC patients. Biomed Pharmacother 2015;75:8e11. 16. Xiong G, Yang L, Chen Y, et al. Linc-POU3F3 promotes cell proliferation in gastric cancer via increasing T-reg distribution. Am J Transl Res 2015;7:2262e2269. 17. Zhao W, Dong S, Duan B, et al. HOTAIR is a predictive and prognostic biomarker for patients with advanced gastric adenocarcinoma receiving fluorouracil and platinum combination chemotherapy. Am J Transl Res 2015;7:1295e1302. 18. Wang YL, Overstreet AM, Chen MS, et al. Combined inhibition of EGFR and c-ABL suppresses the growth of triple-negative breast cancer growth through inhibition of HOTAIR. Oncotarget 2015;6: 11150e11161. 19. Jin C, Yan B, Lu Q, et al. Reciprocal regulation of Hsa-miR-1 and long noncoding RNA MALAT1 promotes triple-negative breast cancer development. Tumour Biol 2016;37:7383e7394. 20. Eades G, Wolfson B, Zhang Y, et al. lincRNA-RoR and miR-145 regulate invasion in triple-negative breast cancer via targeting ARF6. Mol Cancer Res 2015;13:330e338. 21. Augoff K, McCue B, Plow EF, et al. miR-31 and its host gene lncRNA LOC554202 are regulated by promoter hypermethylation in triplenegative breast cancer. Mol Cancer 2012;11:5. 22. Panzitt K, Tschernatsch MM, Guelly C, et al. Characterization of HULC, a novel gene with striking up-regulation in hepatocellular carcinoma, as noncoding RNA. Gastroenterology 2007;132: 330e342. 23. Kitagawa M, Kitagawa K, Kotake Y, et al. Cell cycle regulation by long non-coding RNAs. Cell Mol Life Sci 2013;70:4785e4794. 24. Parasramka MA, Maji S, Matsuda A, et al. Long non-coding RNAs as novel targets for therapy in hepatocellular carcinoma. Pharmacol Ther 2016;161:67e78. 25. Kong R, Zhang EB, Yin DD, et al. Long noncoding RNA PVT1 indicates a poor prognosis of gastric cancer and promotes cell proliferation through epigenetically regulating p15 and p16. Mol Cancer 2015;14: 82. 26. Huang JL, Zheng L, Hu YW, et al. Characteristics of long non-coding RNA and its relation to hepatocellular carcinoma. Carcinogenesis 2014;35:507e514. 27. Matouk IJ, Abbasi I, Hochberg A, et al. Highly upregulated in liver cancer noncoding RNA is overexpressed in hepatic colorectal metastasis. Eur J Gastroenterol Hepatol 2009;21:688e692.
453
28. Hammerle M, Gutschner T, Uckelmann H, et al. Posttranscriptional destabilization of the liver-specific long noncoding RNA HULC by the IGF2 mRNA-binding protein 1 (IGF2BP1). Hepatology 2013;58: 1703e1712. 29. Xie H, Ma H, Zhou D. Plasma HULC as a promising novel biomarker for the detection of hepatocellular carcinoma. Biomed Res Int 2013; 2013:136106. 30. Yang Z, Lu Y, Xu Q, et al. HULC and H19 played different roles in overall and disease-free survival from hepatocellular carcinoma after curative hepatectomy: a preliminary analysis from gene expression omnibus. Dis Markers 2015;2015:191029. 31. Liu Y, Pan S, Liu L, et al. A genetic variant in long non-coding RNA HULC contributes to risk of HBV-related hepatocellular carcinoma in a Chinese population. PLoS One 2012;7:e35145. 32. Zhao J, Fan Y, Wang K, et al. LncRNA HULC affects the differentiation of Treg in HBV-related liver cirrhosis. Int Immunopharmacol 2015;28:901e905. 33. Sun XH, Yang LB, Geng XL, et al. Increased expression of lncRNA HULC indicates a poor prognosis and promotes cell metastasis in osteosarcoma. Int J Clin Exp Pathol 2015;8:2994e3000. 34. Peng W, Gao W, Feng J. Long noncoding RNA HULC is a novel biomarker of poor prognosis in patients with pancreatic cancer. Med Oncol 2014;31:346. 35. Zhao Y, Guo Q, Chen J, et al. Role of long non-coding RNA HULC in cell proliferation, apoptosis and tumor metastasis of gastric cancer: a clinical and in vitro investigation. Oncol Rep 2014;31:358e364. 36. Peng W, Wu J, Feng J. Long noncoding RNA HULC predicts poor clinical outcome and represents pro-oncogenic activity in diffuse large B-cell lymphoma. Biomed Pharmacother 2016;79:188e193. 37. Zhu Y, Zhang X, Qi L, et al. HULC long noncoding RNA silencing suppresses angiogenesis by regulating ESM-1 via the PI3K/Akt/ mTOR signaling pathway in human gliomas. Oncotarget 2016;7: 14429e14440. 38. Lu Z, Xiao Z, Liu F, et al. Long non-coding RNA HULC promotes tumor angiogenesis in liver cancer by up-regulating sphingosine kinase 1 (SPHK1). Oncotarget 2016;7:241e254. 39. Wang J, Liu X, Wu H, et al. CREB up-regulates long non-coding RNA, HULC expression through interaction with microRNA-372 in liver cancer. Nucleic Acids Res 2010;38:5366e5383. 40. Cui M, Xiao Z, Wang Y, et al. Long noncoding RNA HULC modulates abnormal lipid metabolism in hepatoma cells through an miR9-mediated RXRA signaling pathway. Cancer Res 2015;75:846e857.