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Research paper
Long non-coding RNA UCA1 promoted the growth of adrenocortical cancer cells via modulating the miR-298-CDK6 axis
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Ningning Guoª,*, Qingfeng Sunb, Dongxia Fuª, Yunna Zhangª
ª The Central Hospital of Cangzhou, Second Department of Endocrine Diabetes, Cangzhou City 061000, Hebei Province, China
b The People Hospital of Cangzhou, Department of Urology, Cangzhou City 061000, Hebei Province, China
| ARTICLE INFO | ABSTRACT |
|---|---|
| Keywords: Adrenocortical cancer UCA1 miR-298 CDK6 Cell cycle | Adrenocortical cancer (ACC) is an aggressive malignancy with no available effective treatments; therefore, ex- ploring the molecular mechanisms involved in the initiation and progression of ACC is quite important. Here, we found that the long noncoding RNA urothelial carcinoma-associated 1 (UCA1) was highly expressed in ACC tissues and closely associated with the TNM stage and metastasis of ACC patients. Overexpression of UCA1 significantly promoted the proliferation and suppressed the apoptosis of ACC cells. Mechanism study showed that UCA1 acted as sponge of miR-298 and decreased the expression abundance of miR-298 in ACC cells. Further investigation identified that miR-298 bound the 3'-UTR of the cyclin-dependent kinase 6 (CDK6) and inhibited the expression of CDK6. Consistently, ectopic expressed UCA1 suppressed miR-298 and up-regulated the ex- pression of CDK6, which promoted the cell cycle progression of ACC cells. Taken together, our results identified the potential oncogenic function of UCA1 in ACC by regulating the miR-298-CDK6 axis. |
1. Introduction
Adrenocortical cancer (ACC) is a rare malignancy occurring in the adrenal cortex (Guida et al., 2015; Wong et al., 2016). Approximately one or two persons per million per year was diagnosed as ACC around the world (Wooten and King, 1993). Due to the aggressive character- istics, most patients have developed into metastatic stage at the time of diagnosis, and the five-year survival of these patients remains < 10%. Currently, there were no effective therapies for the treatment of pa- tients with locally advanced and metastatic stages. Therefore, it is cri- tical to identify novel factors involved in the initiation and pathophy- siology of ACC.
Aberrant expression of long noncoding RNAs (lncRNAs) have been found in the pathogenesis of ACC (Glover et al., 2015). LncRNAs are defined as RNA transcripts longer than 200 nucleotides without pro- tein-coding capacity (Wang and Chang, 2011; Fu et al., 2016; Zhang et al., 2016; Hu et al., 2018). LncRNAs regulated the gene expression via epigenetic silencing, splicing regulation or inducing the degradation of miRNAs (Fu et al., 2016). Interestingly, the dysfunction of lncRNAs has been recognized as a critical phenotype in the initiation and de- velopment of cancers (Bach and Lee, 2018; James de Bony et al., 2018;
Shi et al., 2018; Sun et al., 2018; Xia et al., 2018). The lncRNA ur- othelial carcinoma-associated 1 (UCA1) is located in chromosome 19p13.12 (Wang et al., 2017). Previous studies have suggested the important roles of UCA1 in the progression of cancers, drug resistance and glucose metabolism (Pan et al., 2016; Li et al., 2017b). Highly expressed UCA1 was found in bladder cancer, colon cancer and breast cancer, which might serve as biomarker for the diagnosis of cancers (Tao et al., 2015; Tuo et al., 2015; Li et al., 2016; Pan et al., 2016; Cui et al., 2017; Li et al., 2017a; Li et al., 2017b; Luo et al., 2017; Gou et al., 2018). However, the expression and the function of UCA1 in ACC has not been characterized.
MiRNAs are a class of non-coding RNAs with the length of 20-22 nt (Bartel, 2004). miRNAs negatively modulated the gene expression via binding to the 3’-untranslated region (UTR) of the target mRNA tran- scripts, which subsequently triggers mRNA degradation or protein translation inhibition (Bartel, 2004; Fabian et al., 2010; Mohr and Mott, 2015). Aberrantly expressed miRNAs acted as oncogenes or tumor suppressors to regulate the tumor progression (Kwak et al., 2010; Farazi et al., 2011; Gentilin et al., 2016). Interestingly, the competing en- dogenous (ceRNA) hypothesis was proposed that lncRNAs function as molecular sponges of miRNAs, which reduces the stability of miRNAs
Abbreviations: UCA1, urothelial carcinoma-associated 1; ACC, adrenocortical cancer; CDK6, cyclin-dependent kinase 6; IncRNAs, long noncoding RNAs; 3’-UTR, 3’- untranslated region; ceRNA, competing endogenous; CCK-8, cell counting kit 8; PVDF, polyvinylidene fluoride; HRP, horseradish peroxidase
* Corresponding author at: The Central Hospital of Cangzhou, Second Department of Endocrine Diabetes, No. 16 of Xinhua West Road, Cangzhou City 061000, Hebei Province, China.
E-mail address: ningning458@126.com (N. Guo).
https://doi.org/10.1016/j.gene.2019.03.066
and modulated the downstream targets of miRNAs (Yao et al., 2017; Gao et al., 2018). It has been found that UCA1 regulated the expression of GLS2 via interfering the miR-16 in bladder cancer (Li et al., 2015). Recent publication showed that UCA1 was highly expressed in breast cancer, which directly interacted with miR-143 and decreased the ex- pression level of miR-143 (Tuo et al., 2015). We confirmed this data by transfecting UCA1 in both SW-13 and NCI-H295R cells. The data in- dicated that overexpression of UCA1 decreased the abundance of miR- 143 in ACC cells (Supplementary Fig. 1A). These results demonstrated that UCA1 acted as sponges of miRNA and played oncogenic roles in regulation the growth of cancer cells.
In this study, we investigated the expression of UCA1 in ACC tissues and corresponding normal tissues. The results indicated that UCA1 was highly expressed in ACC tissues and correlated with the worse prognosis of cancer patients. The further molecular studies demonstrated that UCA1 sponged miR-298 and up-regulated the downstream target CDK6. Our results shed light on the potential involvement of UCA1 in the progression of ACC.
2. Materials and methods
2.1. Cell lines and tissues
The SW-13 cell line was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured with the Leibovitz- 15 (L-15) medium at 37 ℃ with 10% fetal bovine serum (FBS). Cell transfection was performed using the Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After transfection for 48 h, cells were harvested for the western blot or RT-qPCR analysis. The fresh-frozen adrenocortical carcinoma tissues and corresponding ad- jacent non-tumorous adrenocortical carcinoma. Tissues were obtained from the patients who were not received radiotherapy or chemotherapy before surgery at The Central Hospital of Cangzhou between July 2011 and October 2013. All the tissue samples were confirmed by three in- dependent pathologists. Written informed consents were obtained from all the patients and this study was approved by the Ethical committee of The Central Hospital of Cangzhou (No. CZH20160108).
2.2. RNA extraction and RT-qPCR analysis
Total RNA was extracted from the tissues with the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The concentration of RNA was de- tected with the NanoDrop-2000 (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). RNA was reversed tran- scribed into cDNA using the Reverse Transcription Kit (Takara, Dalian, China). The real-time PCR assay was performed with the SYBR green mix with the ABI7500 quantitative PCR instrument (Applied Biosystems). The results were normalized to the expression of U6 RNA. Amplification conditions were as follows: 95 ℃ for 10 min, followed by 40 cycles of 95 ℃ for 10 s and 58 ℃ for 60 s. The relative abundance of genes was determined with the 2-44Cq method. The primers used in this study were presented as Table 1.
2.3. Cell proliferation assay
Cells transfected with the control lncRNA or UCA1 were seeded into the 96-well plate with the density of 1000 cells per well. The cell proliferation rate was detected at the interval of 24h with the cell counting kit 8 (CCK-8 kit, Dojindo, Japan) according to the manufac- turer’s instructions. 10 ul of CCK-8 was added into the cells and in- cubated for 3 h in the CO2 incubator. The cell viability was determined by detecting the absorbance of each well at the wavelength of 450 nm. The experiment was performed in triplicate.
| Primer | Sequence (5'-3') |
|---|---|
| UCA1 Forward | CCCAAGGAACATCTCACCAATT |
| UCA1 Reverse | TGAGGGGTCAGACTTTTGACAAG |
| MiR-298 Forward | CTAGCCTGCAGGTTCCCAGCTACGTGCGCTCAG |
| MiR-298 Reverse | ATCCGGCCGGCCTACAGGATACTTGCCACACCAT |
| U6 Forward | GCTTCGGCAGCACATATACTAAAAT |
| U6 Reverse | CGCTTCACGAATTTGCGTGTCAT |
| CDK6 Forward | TCTTCATTCACACCGAGTAGTGC |
| CDK6 Reverse | TGAGGTTAGAGCCATCTGGAAA |
| GAPDH Forward | CCATGTTCGTCATGGGTGTG-3 |
| GAPDH Reverse | GGTGCTAAGCAGTTGGTGGTG |
| STAT3 Forward | GGAGGAGGCATTCGGAAAG |
| STAT3 Reverse | TCGTTGGTGTCACACAGAT |
2.4. Western blot
SW-13 cells were lyzed with the RIPA buffer (Beyotime, Shanghai, China) containing PMSF. The protein concentration was detected with the BCA protein assay kit (Beyotime, Shanghai, China). Proteins were separated with the SDS-PAGE by loading 20 µg of protein. The proteins were then transferred onto the polyvinylidene fluoride membrane (PVDF, Millipore, Billerica, MA, USA) followed by blocking with 5% non-fat milk. The membrane was incubated with the primary antibody of anti-CDK6 (#3136, Cell Signaling Technology, Danvers, MA, USA) for 2h at room temperature. After washing twice with TBST, the membrane was then incubated with horseradish peroxidase (HRP)- conjugated secondary antibodies for 1 h at RT. The signals were de- tected with KeyGEN Enhanced ECL detection kit according to the manufacturer’s instructions (KeyGEN, Nanjing, China). The expression of GAPDH was detected with anti-GAPDH antibody (#G8795; Sigma- Aldrich, St. Louis, MO, USA) for normalization.
2.5. Luciferase reporter assay
Oligonucleotides of the wild-type or mutant sequence of UCA1 containing the putative binding sites of miR-298 were constructed into the pMIR-REPORT luciferase reporter vector, respectively. The SW-13 and NCI-H295R cells were seeded into the 96-well plate and cultured overnight. The firefly and renilla luciferase plasmids, as well as the miRNA control or miR-298 mimics were transfected into the cells with the Lipofectamine 2000 reagent (Invitrogen). After transfection for 48 h, cells were harvested and the luciferase activity was measured with the Dual Luciferase Reporter Assay Kit (Promega, Corporation, Madison, WI, USA) according to the manufacturer’s protocol. The ex- periment was performed in triplicate.
2.6. Cell cycle analysis
ACC cells transfected with the corresponding expression vector were washed twice with pre-cold PBS and fixed in 70% ethanol overnight at 4 ℃. Cells were incubated with 40 µg/ml RNase A for 30 min at 37 ℃. After washed twice with PBS, cells were resuspended with PBS con- taining 50 µg/ml propidium iodide (PI). The cell cycle progression was determined immediately with the BD FACScan Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The cell cycle distribution was also detected using the BD BrdU FITC Assay on the BD FACSVerse™ system (559,619, BD Franklin Lakes, NJ, USA) according to the man- ufacturer’s instruction.
2.7. Statistical analysis
Data were presented as mean + SD. The statistical analysis was performed with the GraphPad Prism 5 Software (GraphPad Software, San Diego, California, USA). The comparisons between two groups were
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carried out with the Student’s t-test. P < 0.05 was considered as sta- tistically significant.
3. Results
3.1. The expression of UCA1 was up-regulated in ACC
The relative expression of UCA1 was detected by RT-qPCR assay with 40 paired ACC tissues and adjacent normal tissues. As shown in Fig. 1A, UCA1 was significantly overexpressed in ACC tissues in com- parison with that of the normal tissues. To detect the relationship be- tween the expressions of UCA1 with the progression of ACC, the ex- pression of UCA1 in patients with or without metastasis was compared. The data indicated that the abundance of UCA1 was higher in ACC tissues from patients with metastasis than that of patients without metastasis (Fig. 1B). This result suggested that UCA1 might be involved in the progression of ACC.
3.2. Overexpression of UCA1 promoted the growth of ACC cells
To investigate the function of UCA1 in regulating the growth of ACC cells, UCA1 was transfected into SW-13 and NCI-H295R cells and the expression of UCA1 was confirmed by the RT-qPCR analysis. As pre- sented in Fig. 2A, the level of UCA1 was significantly up-regulated in ACC cells with the transfection of UCA1. CCK-8 assay was performed to evaluate the influence of UCA1 on the proliferation of both SW-13 and NCI-H295R cells. The data showed that overexpression of UCA1 sig- nificantly promoted the proliferation of SW-13 and NCI-H295R cells (Fig. 2B and C). To further confirm the promotion effect of UCA1 on the growth of ACC cells, in vitro colony formation was performed with SW- 13 and NCI-H295R cells expressing lncRNA control vector or UCA1. The result showed that ectopic expression of UCA1 remarkably increased the number of colonies compared with the control group (Fig. 2D). Additionally, the regulation of UCA1 on the migration of SW-13 and NCI-H295R cells was also detected with the wound-healing assay. The data indicated that transfection of UCA1 significantly promoted the migration of ACC cells (Fig. 2E). To further characterize whether the promoting effect of UCA1 on the growth of ACC cells was associated with the apoptosis of cells, SW-13 and NCI-H295R cells were treated with cisplatin to jump the apoptosis of cells. FACS analysis showed that exposure of cisplatin enhanced the apoptosis of cells, while over- expression of UCA1 significantly decreased cisplatin-induced apoptosis of both SW-13 and NCI-H295R cells (Fig. 2F). Collectively, these results demonstrated that overexpression of UCA1 positively regulated the
growth of ACC cells.
3.3. UCA1 targeted miR-298 in SW-13 cells
Previous studies have suggested that lncRNAs function as sponge to sequester miRNAs and modulate the expression of downstream targets. To characterize the molecular mechanism by which UCA1 regulated the growth of ACC cells, the potential miRNAs that might be regulated by UCA1 were predicted by the miRDB database (http://mirdb.org/ miRDB/custom.html). The results suggested that miR-298 was a can- didate-binding partner of UCA1. The predicted binding sites of miR-298 in UCA1 were shown in Fig. 3A. To confirm this observation, the wild- type or mutant sequence of UCA1 that contained the putative binding sites of miR-298 was constructed into the luciferase reporter vector. Luciferase assay was performed by co-transfecting either control miRNA or miR-298 mimics with the wild-type or mutant UCA1 into SW- 13 and NCI-H295R cells. The result showed that overexpression of miR- 298 significantly decreased the luciferase activity of wild-type but not the mutant UCA1 in ACC cells (Fig. 3B and C). To evaluate the reg- ulation of UCA1 on the expression of miR-298, SW-13 and NCI-H295R cells were transfected with UCA1 and the level of miR-298 was detected by the RT-qPCR analysis. The data showed that overexpression of UCA1 significantly decreased the abundance of miR-298 in ACC cells (Fig. 3D). These results indicated that UCA1 bound miR-298 and ne- gatively regulated the expression of miR-298 in ACC cells.
3.4. UCA1 up-regulated the expression of CDK6
To further investigate the consequence for the interaction between UCA1 and miR-298, the downstream targets of miR-298 were predicted with the TargetScan database. The result uncovered that there was potential binding sites of miR-298 at the 3’-UTR of CDK6 (Fig. 4A). CDK6 is a key regulator that controls the cell passage from G1 to S phase (Andisheh-Tadbir et al., 2018). To confirm this observation, lu- ciferase assay was performed by introducing the reporter vector con- taining wild type or mutant 3’-UTR of CDK6 into SW-13 and NCI- H295R cells. The data showed that highly expressed miR-298 sig- nificantly decreased the luciferase activity of wild-type but not the mutant 3’-UTR of CDK6 (Fig. 4B and C). To investigate whether the binding of miR-298 with the 3’-UTR of CDK6 affected the mRNA sta- bility of CDK6, ACC cells were transfected with control miRNA or miR- 298 mimics and the mRNA level of CDK6 was examined by the RT- qPCR assay. The result showed that transfection of miR-298 sig- nificantly reduced the mRNA level of CDK6 (Fig. 4D). Furthermore, the
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Sequence of miR-298: 3’-ACCCUCUUGGAGGGACGAAGACGA
UCA1 (location of 1185-1210):5’- TATCTCTTCTGCATAG
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Relative luciferase activity (Normalized to the control group)
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protein abundance of CDK6 was also evaluated by western blot with SW-13 and NCI-H295R cells expressing control miRNA or miR-298. As shown in Fig. 4E, in comparison with the control group, overexpression of miR-298 remarkably decreased the protein level of CDK6 in SW-13 and NCI-H295R cells. These results indicated that highly expressed miR-298 negatively modulated the expression of CDK6 in ACC cells.
As ectopic expressed UCA1 reduced the expression of miR-298 in ACC cells, to detect whether UCA1 affected the level of CDK6, both SW- 13 and NCI-H295R cells were transfected with control lncRNA or UCA1 and the mRNA level of CDK6 was detected. The data indicated that overexpression of UCA1 significantly up-regulated the mRNA abun- dance of CDK6 in SW-13 and NCI-H295R cells (Fig. 4F). Consistently, the protein level of CDK6 was also increased with the transfection of UCA1 (Fig. 4G). These results suggested that UCA1 acted as sponge of miR-298 and enhanced the level of CDK6 in SW-13 cells.
3.5. UCA1 promoted the cell cycle progression of ACC cells
It has been documented that CDK6 regulated the progression of cell cycle. As UCA1 increased the expression of CDK6 in ACC cells, to in- vestigate whether UCA1 modulated the cell cycle progression, SW-13 cells were transfected with control lncRNA or UCA1 and the cell cycle distribution was detected by the FACS analysis. The result showed that transfection of UCA1 significantly enhanced the cell cycle transition
from G1 to S phase of ACC cells (Fig. 5A). The conclusion was further supported with the incorporation of BrdU. The data showed that overexpression of UCA1 significantly promoted the cell cycle progres- sion from G1 to S phase (Supplementary Fig. 1A and B). To further confirm this observation, the endogenous UCA1 was down-regulated by transfecting UCA1-shRNA into the cells. The knockdown efficiency of UCA1 was confirmed by the RT-qPCR assay (Fig. 5B). Firstly, the ex- pression of CDK6 was detected in SW-13 and NCI-H295R cells with depleted UCA1. The data showed that depletion of UCA1 decreased both the mRNA and protein levels of CDK6 in ACC cells (Fig. 5C and D). Consistent with these data, the influence of down-regulation of UCA1 on the cell cycle distribution of SW-13 and NCI-H295R cells was ana- lyzed and the result showed that depletion of UCA1 led to cell cycle arrest at G1 phase (Fig. 5E). These results demonstrated that UCA1 modulated the expression of CDK6 and regulated the cell cycle pro- gression of ACC cells.
4. Discussion
As a large part of the genome, lncRNAs regulate the expression of protein-coding genes at epigenetic, transcriptional and post-transcrip- tional levels (Marques and Ponting, 2014; Luo et al., 2016; Chen et al., 2018). Increasing evidence has suggested that a variety of lncRNAs were aberrantly expressed in human diseases, especially cancers, which
A
Predicted consequential pairing of target region (top) and miRNA (bottom)
| Position 1265-1271 of CDK6 3'-UTR | 5'-GGUUUAUGGCCUCGA ---- UUCUGCAA |
| Has-miR-298 | 3'-ACCCUCUUGGAGGGACGAAGACGA |
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Negative control miRNA miR-298 mimics
Relative luciferase activity
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modulated the cancer invasion, metastasis and drug resistance (Chen et al., 2017; Yang et al., 2018). The oncogenic or tumor suppressive function of lncRNAs encouraged us to investigate the underlying mo- lecular mechanisms by which lncRNAs regulated the progression of cancers.
In this study, we uncovered that lncRNA UCA1 was overexpressed in ACC tissues and correlated with the metastasis of ACC patients. It is worth mentioning that the up-regulation of UCA1 was observed in several cancers, including breast cancer, colon cancer and bladder cancer (Fan et al., 2014; Tuo et al., 2015; Xiao et al., 2016; Xie et al.,
2016). These data suggested the potential oncogenic function of UCA1 in cancers. Here, overexpression of UCA1 significantly promoted the proliferation, colony formation and inhibited the apoptosis of ACC cells. Further studies might be necessary to detect the promotion effects of UCA1 on the growth of ACC cells by in vivo studies. Consistent with these data, previous findings demonstrated that down-regulation of UCA1 significantly decreased the cell viability of bladder cancer (Zhen et al., 2017). These results indicated that UCA1 is a promising novel diagnostic and therapeutic target of ACC.
Different from small miRNAs that specially bind the targeted
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mRNAs, the functional mechanisms of lncRNAs are more complicated and diverse. Beyond the classic roles of lncRNAs in regulating the chromatin modification, structure rearrangement and RNA processing, recent studies proposed the ceRNA hypothesis to explain the molecular
mechanisms by which lncRNA regulated both the physiological and pathological conditions (Yao et al., 2017). With the ceRNA model, lncRNA act as miRNA sponges to interact with miRNAs and regulate the expression of miRNA-targeted genes (Li et al., 2015). It has been
reported that UCA1 directly interacted with the tumor suppressing miR- 143, which attenuated the function of miR-143 and enhanced the growth of breast cancer cells (Tuo et al., 2015). In this study, we also found that UCA1 decreased the expression of miR-143 in ACC cells (Supplementary Fig. 1C). The involvement of UCA1-miR-143 axis in the development of ACC requires further study. Additionally, the up-regu- lated UCA1 inhibited miR-216 and activated the FGFR1/ERK pathway in hepatocellular carcinoma (Wang et al., 2015). In this study, we found that UCA1 acted as the sponge of miR-298 and decreased the expression of miR-298 in ACC cells. Recent publication illustrated that miR-298 suppressed the malignancy of epithelial ovarian cancer by modulating the expression of EZH2, which indicated the potential tumor suppres- sive function of miR-298 in cancers (Zhou et al., 2016). To further understand the consequent effects of the decreased miR-298 by UCA1, the downstream targets of miR-298 were predicted. Our results showed that miR-298 bound the 3’-UTR of CDK6 and negatively regulated the expression level of CDK6 in ACC cells. Notably, CDK6 interacted with cyclin D to regulate the cell cycle progression and represent attractive therapeutic targets in cancers (Tadesse et al., 2015). Increased activity of CDK6 has been observed in human cancers. In the present data, overexpression of UCA1 promoted the abundance of CDK6, which consequently promoted the cell cycle progression of ACC cells. Our results provided the possible mechanism by which UCA1 modulated the growth of ACC cells via regulating the miR-298-CDK6 axis. Notably, other potential targets of miR-298 that might be involved in the on- cogenic function of UCA1 needs further studies. In the current study, we confirmed that STAT3 was a target of miR-298 in ACC cells. Over- expression of miR-298 decreased both the mRNA and protein expres- sion of STAT3 (Supplementary Fig. 1D and E). And transfection of UCA1 promoted the level of STAT3 in ACC cells (Supplementary Fig. 1F and G). These results indicated the potential important role of STAT3 in the UCA1/miR-298 modulated growth of ACC cells.
5. Conclusions
Our study demonstrated the highly expressed UCA1 in ACC patients. Overexpression of UCA1 facilitated the growth of ACC cells, which suggested the potential oncogenic function of UCA1 in ACC. Furthermore, the molecular study uncovered that UCA1 sponged miR- 298 and up-regulated CDK6, which indicated that critical involvement of miR-298-CDK6 pathway in mediating the promoting effect of UCA1 on the growth of ACC cells.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2019.03.066.
Conflicts of interest
The authors declare that they have no conflicts of interests.
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