Upregulation of microRNA-96 and its oncogenic functions by targeting CDKN1A in bladder cancer
Wu et al. Cancer Cell Int
Upregulation of microRNA-96 and its oncogenic functions by targeting CDKN1A in bladder cancer
Ziyu Wu 2
Kun Liu 0 1
Yunyan Wang 0 1
Zongyuan Xu 0 1
Junsong Meng 0 1
Shuo Gu 0 1
0 Department of Urology, Huai'an First People's Hospital, Nanjing Medical University , 6 Beijing Road West, Huai'an 223300, Jiangsu , People's Republic of China
1 Department of Urology, Huai'an First People's Hospital, Nanjing Medical University , 6 Beijing Road West, Huai'an 223300, Jiangsu , People's Republic of China
2 Department of Urology, Huai'an Hospital Affiliated of Xuzhou Medical College and Huai'an Second People's Hospital , 62 Huaihai Road South, Huai'an 223002 , People's Republic of China
Background: Genome-wide miRNA expression profile has identified microRNA (miR)-96 as one of upregulated miRNAs in clinical bladder cancer (BC) tissues compared to normal bladder tissues. The aim of this study was to confirm the expression pattern of miR-96 in BC tissues and to investigate its involvement in carcinogenesis. Methods: Quantitative real-time PCR was performed to detect the expression levels of miR-96 in 60 BC and 40 normal control tissues. Bioinformatics prediction combined with luciferase reporter assay were used to verify whether the cyclin-dependent kinase inhibitor CDKN1A was a potential target gene of miR-96. Cell counting kit-8 and apoptosis assays were further performed to evaluate the effects of miR-96-CDKN1A axis on cell proliferation and apoptosis of BC cell lines. Results: We validated that miR-96 was significantly increased in both human BC tissues and cell lines. According to the data of miRTarBase, CDKN1A might be a candidate target gene of miR-96. In addition, luciferase reporter and Western blot assays respectively demonstrated that miR-96 could bind to the putative seed region in CDKN1A mRNA 3′UTR, and significantly reduce the expression level of CDKN1A protein. Moreover, we found that the inhibition of miR-96 expression remarkably decreased cell proliferation and promoted cell apoptosis of BC cell lines, which was consistent with the findings observed following the introduction of CDKN1A cDNA without 3′UTR restored miR-96. Conclusions: Our data reveal that miR-96 may function as an onco-miRNA in BC. Upregulation of miR-96 may contribute to aggressive malignancy partly through suppressing CDKN1A protein expression in BC cells.
Bladder cancer; microRNA-96; CDKN1A; Proliferation; Apoptosis
Bladder cancer (BC) ranks the 9th most common
malignancy worldwide and is one of the costliest to clinically
]. It represents a main cause of morbidity and
mortality. Histologically, about 98 % of BC belong to
epithelial malignancies, with the vast majority being
transitional cell carcinomas (TCC), followed by squamous cell
carcinoma (5 %) and adenocarcinoma (2 %) [
]. BC can
be divided into two forms: low grade superficial tumors
and high grade invasive tumors. Surgical resection is the
primary and most effective treatment for patients with
low grade superficial tumors [
]. However, there are still
no efficient therapeutic strategies for high grade
invasive tumors. Although chemotherapy based on BCG may
reduce recurrence in BC patients, more than 70 % will
recur and approximately 30 % will eventually progress to
muscle invasive disease or distant metastasis [
Therefore, it is of great significance to identify novel and
effective molecular markers for the early-stage diagnosis and
for the evaluation of prognosis in patients with BC.
MicroRNAs (miRNAs) are small, endogenous and
non-protein-coding RNAs that control gene expression
post-transcriptionally by interacting with partially
complementary target sites in the 3′-untranslational region
(UTR) of specific mRNA targets, either inducing their
degradation or impairing their translation [
Functionally, miRNAs are implicated in various cellular processes,
including cell proliferation, differentiation, cell cycle and
]. Emerging data have shown the abnormal
expression of miRNAs may be dramatically associated
with several diseases, including cancers [
]. MiRNAs act
as either oncogenes or tumor suppressors, and play
crucial roles in carcinogenesis, progression, and metastases.
The tissue-specific nature of miRNA expression implies
that different tumors would express specific miRNA
]. Especially, previous study of Puerta-Gil et al.
identified miR-143, miR-222, and miR-452 as tumor
stratification and noninvasive diagnostic biomarkers for
]; Majid et al. [
] reported that miR-23b could
function as a tumor suppressor that may confer a
proliferative advantage and promote bladder cancer cell
migration and invasion; Wu et al. [
] also found that miR-99a
could inhibit cell proliferation, migration and invasion by
targeting fibroblast growth factor receptor 3 in BC. These
findings suggest that it is of critical to identify the targets
of miRNAs for understanding the function of miRNAs in
cancer development and progression.
Genome-wide miRNA expression profile reported by
Yoshino et al. has identified miR-96 as one of
upregulated miRNAs in clinical BC tissues compared to normal
bladder tissues [
]. Guo et al. [
] identified that
miR96 was downregulated in transitional cell carcinoma
tissues compared to normal bladder tissues, and regulated
FOXO1-mediated cancer cell apoptosis, while Wang
et al. [
] reported that miR-96 was expressed at higher
levels in human bladder urothelial carcinomas compared
to normal tissues, and found that miR-96 increased
invasion and differentiation of human bladder T24 cells and
promoted cell growth. These converse findings prompt us
to perform this study to confirm the expression pattern
of miR-96 in BC tissues and to investigate its involvement
Upregulation of miR‑96 in BC tissues
Using quantitative real-time PCR, we found that the
expression level of miR-96 was significantly increased in
BC tissues compared with that in NBTC tissues as shown
in Fig. 1a (BC vs. NBTC: 3.69 ± 0.91 vs. 1.86 ± 0.52,
P < 0.001), offering us an initial evidence that miR-96
might be involved in the development of human BC.
miR‑96 negatively regulates the expression of CDKN1A
in BC cells and tissues
We collected the candidate target genes of miR-96 from
the experimentally validated microRNA-target
interactions database (miRTarBase, http://mirtarbase.mbc.
nctu.edu.tw/, Release 4.5), which has accumulated more
than 50,000 miRNA–target interactions (MTIs). The
data in miRTarBase are collected by manually
surveying pertinent literature after data mining of the text
systematically to filter research articles related to
functional studies of miRNAs [
]. All MTIs in this database
are validated experimentally by reporter assay,
Western blot, microarray and next-generation sequencing
experiments. In the current study, only the MTIs which
are validated experimentally by reporter assay, Western
blot and quantitative real-time PCR were collected. As a
result, a total of seven genes, including FOXO1, FOXO3,
HTR1B, Mitf, CDKN1A, PRMT5 and KRAS, have been
regarded as candidate target gene of miR-96 as shown
in Table 1. According to our literature retrieval,
miR-96FOXO1 axis has been indicated to play a crucial role in
bladder carcinogenesis. Li et al. [
Western blotting and dual-luciferase reporter assays to
demonstrate that miR-96 could bind to the putative seed
region in FOXO3 mRNA 3′UTR, and could significantly
decrease the expression of FOXO3 in non-small cell
lung cancer cells. Jensen et al. [
] identified an element
(A-element) within HTR1B mRNA that confers
repression by miR-96. Xu et al. [
] performed target
prediction and in vitro functional studies showed that MITF,
a transcription factor required for the establishment
and maintenance of retinal pigmented epithelium, was
a direct target of miR-96. Pal et al. [
] also revealed
that the decreased miR-96 expression could augment
PRMT5 translation. Recent studies reported that the
involvement of miR-96 in colorectal and pancreatic
carcinogenesis might be related to its suppression on
KRAS expression [
]. In order to validate whether
CDKN1A was a potential target of miR-96, luciferase
reporter assay was performed. As shown in Fig. 1b, c,
co-transfection with miR-96 inhibitor significantly
increased the luciferase activity of the reporter
containing the wild-type 3′-UTR (P < 0.05). Moreover, Western
blot analysis also showed that the expression level of
CDKN1A protein was significantly up-regulated in two
human BC cell lines T24 and EJ transfected with
miR96 inhibitor (both P < 0.001, Fig. 1d). Importantly, the
expression levels of CDKNIA mRNA in BC tissues were
dramatically lower than those in NBTC tissues (BC vs.
NBTC: 2.42 ± 1.05 vs. 3.71 ± 0.90, P < 0.001, Fig. 1e).
The spearman correlation analysis also showed the
negative correlation between miR-96 and CDKNIA mRNA
expression levels in human BC tissues (rs = −0.34,
P = 0.01, Fig. 1f ).
Taken together, these data demonstrated that miR-96
could negatively regulate the expression of CDKN1A in
both BC cells and tissues.
Downregulation of miR‑96 inhibits cell proliferation of BC
cells via regulating CDKN1A mRNA
To determine the effect of miR-96 on cell proliferation of
BC cells, T24 and EJ cells were transfected with miR-96
inhibitor and CCK8 assay was performed. As shown in
Fig. 2a, b, the downregulation of miR-96 could
remarkably inhibit the growth of both T24 and EJ cells (both
P < 0.05). After that, recombinant CDKN1A without the
3′UTR sequence (pcDNA3.1(+)-CDKN1A) was
transfected into two BC cells. Similar with the observations
following the transfection of miR-96 inhibitor, the cell
proliferation of both T24 and EJ cells transfected with
pcDNA3.1(+)-CDKN1A was significantly suppressed
(both P < 0.05, Fig. 2c, d).
Downregulation of miR‑96 promotes apoptosis of BC cells
via regulating CDKN1A mRNA
To determine the effect of miR-96 on cell apoptosis, T24
and EJ cells transfected with miR-96 inhibitor or NC
were double-stained with Annexin V-FITC and PI and
detected using flow cytometry. As shown in Fig. 3a, b, the
downregulation of miR-96 could significantly promote
apoptosis of both T24 and EJ cells (both P < 0.05), which
was consistent with the observation after the
transfection of the recombinant CDKN1A without the 3′UTR
sequence (pcDNA3.1(+)-CDKN1A) (both P < 0.05,
Fig. 3c, d).
Growing evidence show a direct link between miRNAs
and human cancers. miRNAs often play a role in the
control of carcinogenesis, cell proliferation and
apoptosis of malignant cells by regulating the expression of
the corresponding target genes. In the current study, we
confirmed the upregulation of miR-96 in human BC
tissues, which was in line with the previous genome-wide
miRNA expression profile reported by Yoshino et al. [
In addition, bioinformatics prediction, combined with
luciferase reporter and Western blot assays, identified
CDKN1A as a potential target of miR-96, which could
negative regulate the expression level of CDKN1A in
both BC cells and tissues. Moreover, we found that the
inhibition of miR-96 expression remarkably decreased
cell proliferation and promoted cell apoptosis of BC cell
lines, consisting with the findings observed following the
introduction of CDKN1A cDNA without 3′UTR restored
MiR-96, together with miR-182 and miR-183, belongs
to the miR-183 family, which is located proximally on
human chromosome 7, a region containing several
oncogenes and commonly amplified in cancers [
miR-182 and miR-183 share the same transcription start
site (chr7: 129207158), suggesting that these miRNAs
may be coordinately expressed and function together
during carcinogenesis [
]. Recent studies showed that
miR-96 was frequently increased in several human
cancers. MiR-96 has been reported to exert an oncogenic
effect in non-small cell lung cancer, esophageal
cancer, breast cancer, hepatocellular carcinoma, and
prostate cancer, respectively by inhibiting the expression of
FOXO3, RECK, FOXO3a and FOXO1 [
the other hand, miR-96 has been reported to function
as a tumor suppressor in several cancers. For instance,
miR-96 directly targeted the GTPase Kras (KRAS)
oncogene in pancreatic cancer cells and ectopic expression of
miR-96 through a synthetic miRNA precursor inhibited
KRAS, dampened Akt signaling, and triggered apoptosis
in cells [
]. Regarding to BC, the conflict results on
the expression patterns of miR-96 in human BC tissues
have been reported by different research groups [
Liu et al. [
] also indicated that miR-183/96/182
cluster might play oncogenic roles in BC. Similarly, we here
found that the upregulation of miR-96 could enhance
cellular proliferation and inhibit apoptosis of BC cells,
implying that miR-96 might contribute to the
carcinogenesis and aggressive progression of BC.
In addition, we validated that CDKN1A (p21), which is
an important inhibitor of the cell-cycle, regulator of the
DNA damage response and effector of the tumor
suppressor p53 [
], was a potential target gene of miR-96
in BC cells. As a major player in mammalian cell cycle
progression, CDKN1A inhibits cyclin/cdk2 complexes
and plays a crucial role in mediating growth arrest when
cells are exposed to DNA damaging agents, implying its
tumor suppressive role [
]. Several factors, including
Ap2, BRCA1, C/EBPa, E2F-1/E2F-3, Sp1/Sp3, Smads and
STAT, activate the transcription of CDKN1A [
Moreover, CDKN1A is also involved in terminal differentiation,
replicative senescence and protection from
p53-dependent and -independent apoptosis [
]. Cazier et al. [
performed the whole-genome sequencing of BC revealed
somatic CDKN1A mutations and clinicopathological
associations with mutation burden. Liu et al. [
found that TP53/CDKN1A double-mutant BC cells had
a unique dependence on Chk1 activity for the G2-M
cellcycle checkpoint in response to chemotherapy-induced
DNA damage. CDKN1A could be regulated by several
miRNAs in many cancers, including miR-519d,
miR375, miR-31 and miR-663 [
]. In the current study,
we found that miR-96 negatively regulated CDKN1A
expression, and downregulation of miR-96 inhibited cell
proliferation and promoted apoptosis in BC cells by
In conclusion, our data reveal that miR-96 may
function as an onco-miRNA in BC. Upregulation of miR-96
may contribute to aggressive malignancy partly through
suppressing CDKN1A protein expression in BC cells.
Ethics, consent and permissions
This work was approved by the Research Ethics
Committee of Huai’an Hospital Affiliated of Xuzhou Medical
College, Huai’an Second People’s Hospital and Huai’an First
People’s Hospital, Nanjing Medical University. Written
informed consent was obtained from all of the patients.
All specimens were handled and made anonymous
according to the ethical and legal standards.
Consent to publish
We have obtained consent to publish from the
participant (or legal parent or guardian for children) to report
individual patient data.
Patients and tissue samples
Sixty TCC samples included 40 non muscle-invasive TCC
samples which was collected by transurethral resection of
bladder tumor and 20 muscle-invasive TCC tissue which
was collected by radical cystectomy. Histological
identification of TCC was evaluated based on the World Health
Organization criteria. The clinicopathological features
of 60 patients with TCC were summarized in Table 2. In
addition, 40 age-matched patients undergoing
suprapubic transvesical prostatectomy, lithocystotomy and
cystostomy for non-malignant diseases were collected for
Two human bladder cancer cell lines, T24 and EJ, were
purchased from the Type Culture Collection of the
Chinese Academy of Sciences (Shanghai, China) and
cultured in RPMI-1640 medium supplemented with 10 %
fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA)
under a humidified air atmosphere of 5 % CO2 at 37 °C.
A specific miR-96-5p inhibitor (sequence: 5′-AGC AAA
AAU GUG CUA GUG CCA AA-3′, Shanghai
GenePharma Co. Ltd, Shanghai, China) and a negative
control (NC, sequence: 5′-CAG UAC UUU UGU GUA
GUA CAA-3′, Shanghai GenePharma Co. Ltd,
Shanghai, China) were commercially purchased. Two human
BC cell lines T24 and EJ were both plated at a
density of 1.8 × 105 cells/well in six-well plates. Transient
transfection was conducted using Lipofectamine™ 2000
(Invitrogen, Carlsbad, CA, USA) following the
manufacturer’s instructions which the cells reached ~60 %
confluence. Forty-eight hours after transfection, the cells were
harvested for further experiments.
RNA extraction and quantitative real‑time PCR
To detect the expression levels of miR-96 and CDKN1
mRNA in TCC and NBTC tissues, and two human BC
cell lines, total RNAs were extracted by Trizol reagent
(Invitrogen, Carlsbad, CA, USA), and 1 μg of RNA was
reversely transcripted. For miR-96, quantitative
realtime PCR was performed using a high-specificity miR-96
Detection Kit (Stratagene Corp., La Jolla, CA, USA) in
conjunction with an ABI 7500 thermal cycler, according
to the manufacturer’s instruction. For CDKN1 mRNA,
the PCR primer was: 5′-CAG AGG AGG CGC CAA
GAC AG-3′ (forward) and 5′-CCT GAC GGC GGA
AAA CGC-3′ (reverse). The RT-PCR kit (BD Biosciences,
NJ, USA) was used in conjunction with an ABI 7500
thermal cycler following the manufacturer’s protocol. U6 and
GAPDH were used internal controls for the expression of
miR-96 and CDKN1 mRNA, respectively. Relative
miR96 and CDKN1 mRNA expression levels were calculated
using the comparative threshold cycle (Ct) method [
Western blot analysis
To detect the expression levels of CDKN1A protein in
human BC cell lines T24 and EJ transfected with
miR96 inhibitor or NC, the Western blot analysis was
performed according to the previous studies [
brief, total proteins of transfected cells were extracted
using RIPA buffer containing
phenylmethanesulfonyl fluoride (PMSF) and were quantified by a BCA kit
(Thermo, Waltham, MA, USA). Proteins were separated
by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to polyvinylidene
difluoride (PVDF) membranes. The membranes were
incubated overnight at 4 °C with diluted primary
antibodies (mouse anti-human CDKN1A antibody, 1:100,
Cat. #AM2134b, Abgent Inc., San Diego, CA, USA; or
mouse anti-human GAPDH, 1:100, Cat. #AM1020b,
Abgent Inc., San Diego, CA, USA). Following extensive
washing with Tris-buffered saline containing Tween 20
(TBST), the secondary antibodies were incubated at
room temperature for 1 h (HRP labeled goat anti-mouse
IgG, 1:1000, Sangon Biotech, Shanghai, China) for
1 h, and the membranes were detected by ECL system
(Pierce, Rockford, IL, USA).
Luciferase reporter assay
To demonstrate whether CDKN1A mRNA was a target
of miR-96, the luciferase reporter assay was performed
according to the previous studies [
]. In brief, the
human CDKN1A mRNA fragments containing putative
seed regions for miR-96 were amplified by PCR using
the following primers: sense 5′-TTC TTC TAG AGG
AAG CCC TAA TCC-3′ and antisense 5′-TCC CTT CTA
GAA AGA TCT ACT CCC C-3′. The PCR product was
inserted into psiCHECK2 luciferase miRNA expression
reporter vector (Promega, Madison, WI, USA).
Mutation experiment was performed using a fast mutation kit
(NEB, Ipswich, Canada). Two human BC cell lines T24
and EJ were cultured in 24-well plate for 24 h and
cotransfected with 150 nM of miR-96 inhibitor or NC and
WT or Mut 3′-UTR of CDKN1A using Lipofectamine
2000. After that, transfected cells were collected, and
the relative luciferase activity was assayed using
DualLuciferase Reporter Assay System (Promega,
Wisconsin, WI, USA). The results were normalized with Renilla
luciferase. Each reporter plasmid was transfected at least
three times (on different days) and each sample was
assayed in triplicate.
Cell counting kit‑8 assay
Cell counting kit-8 (CCK8) assay was performed to
observe the cell proliferation of human BC cell lines
T24 and EJ transfected with miR-96 inhibitor and NC
using CCK8 solution (Dojindo, Gaithersburg, MD, USA)
according to the manufacturer’s protocol. Optical density
was measured at 450 nm using a microplate reader.
Apoptosis assay was performed to observe the cell
apoptosis of human BC cell lines T24 and EJ transfected with
miR-96 inhibitor and NC using Annexin V-FITC
Apoptosis Detection Kit I (BestBio, Shanghai, China) according
to the manufacturer’s protocol. The cells were analyzed
using flow cytometry (FACSCalibur, Beckman Coulter,
Miami, FL, USA).
Statistical analysis in the current study was performed by
the software of SPSS version 13.0 for Windows (SPSS Inc,
IL, USA). All the experiments were done in triplicate and
continuous variables were expressed as Mean ± SD.
Student’s t test and one-way analysis of variance (ANOVA)
were used to determine the statistical significance of
differences, which were considered statistically significant
when p was less than 0.05.
GS: participated in study design and coordination, analysis and interpretation
of data, and supervised study. WZ: performed most of the experiments and
statistical analysis and drafted the manuscript. Other authors: carry out the
experiment and sample collection. All authors read and approved the final
Compliance with ethical guidelines
The authors declare that they have no competing interests.
1. Siegel R , Naishadham D , Jemal A . Cancer statistics, 2012 . CA Cancer J Clin. 2012 ; 62 : 10 - 29 .
2. Klotz L , Brausi MA . World urologic oncology federation bladder cancer prevention program: a global initiative . Urol Oncol . 2015 ; 33 : 25 - 9 .
3. Carneiro BA , Meeks JJ , Kuzel TM , Scaranti M , Abdulkadir SA , Giles FJ . Emerging therapeutic targets in bladder cancer . Cancer Treat Rev . 2015 ; 41 : 170 - 8 .
4. Ye F , Wang L , Castillo-Martin M , McBride R , Galsky MD , Zhu J , Boffetta P , Zhang DY , Cordon-Cardo C . Biomarkers for bladder cancer management: present and future . Am J Clin Exp Urol . 2014 ; 2 : 1 - 14 .
5. Xue J , Niu J , Wu J , Wu ZH . MicroRNAs in cancer therapeutic response: friend and foe . World J Clin Oncol . 2014 ; 5 : 730 - 43 .
6. Phuah NH , Nagoor NH . Regulation of microRNAs by natural agents: new strategies in cancer therapies . Biomed Res Int . 2014 ; 2014 : 804510 .
7. Cheng G. Circulating miRNAs: roles in cancer diagnosis, prognosis and therapy . Adv Drug Deliv Rev . 2015 ;81C: 75 - 93 .
8. Berindan-Neagoe I , Monroig Pdel C , Pasculli B , Calin GA . MicroRNAome genome: a treasure for cancer diagnosis and therapy . CA Cancer J Clin . 2014 ; 64 : 311 - 36 .
9. Puerta-Gil P , García-Baquero R , Jia AY , Ocana S , Alvarez-Múgica M , Alvarez-Ossorio JL , Cordon-Cardo C , Cava F , Sánchez-Carbayo M. miR- 143 , miR- 222 , and miR-452 are useful as tumor stratification and noninvasive diagnostic biomarkers for bladder cancer . Am J Pathol . 2012 ; 180 : 1808 - 15 .
10. Majid S , Dar AA , Saini S , Deng G , Chang I , Greene K , Tanaka Y , Dahiya R , Yamamura S. MicroRNA-23b functions as a tumor suppressor by regulating Zeb1 in bladder cancer . PLoS One . 2013 ; 8 : e67686 .
11. Wu D , Zhou Y , Pan H , Zhou J , Fan Y , Qu P. microRNA-99a inhibiting cell proliferation, migration and invasion by targeting fibroblast growth factor receptor 3 in bladder cancer . Oncol Lett . 2014 ; 7 : 1219 - 24 .
12. Yoshino H , Seki N , Itesako T , Chiyomaru T , Nakagawa M , Enokida H . Aberrant expression of microRNAs in bladder cancer . Nat Rev Urol . 2013 ; 10 : 396 - 404 .
13. Guo Y , Liu H , Zhang H , Shang C , Song Y. miR -96 regulates FOXO1-mediated cell apoptosis in bladder cancer . Oncol Lett . 2012 ; 4 : 561 - 5 .
14. Wang Y , Luo H , Li Y , Chen T , Wu S , Yang L . hsa-miR-96 up-regulates MAP4K1 and IRS1 and may function as a promising diagnostic marker in human bladder urothelial carcinomas . Mol Med Rep . 2012 ; 5 : 260 - 5 .
15. Hsu SD , Tseng YT , Shrestha S , Lin YL , Khaleel A , Chou CH , Chu CF , Huang HY , Lin CM , Ho SY , Jian TY , Lin FM , Chang TH , Weng SL , Liao KW , Liao IE , Liu CC , Huang HD . miRTarBase update 2014 : an information resource for experimentally validated miRNA-target interactions . Nucleic Acids Res . 2014 ; 42 (Database issue): D78 - 85 .
16. Li J , Li P , Chen T , Gao G , Chen X , Du Y , Zhang R , Yang R , Zhao W , Dun S , Gao F , Zhang G. Expression of microRNA-96 and its potential functions by targeting FOXO3 in non-small cell lung cancer . Tumour Biol . 2015 ; 36 : 685 - 92 .
17. Jensen KP , Covault J , Conner TS , Tennen H , Kranzler HR , Furneaux HM . A common polymorphism in serotonin receptor 1B mRNA moderates regulation by miR-96 and associates with aggressive human behaviors . Mol Psychiatry . 2009 ; 14 : 381 - 9 .
18. Xu S , Witmer PD , Lumayag S , Kovacs B , Valle D. MicroRNA (miRNA) transcriptome of mouse retina and identification of a sensory organ-specific miRNA cluster . J Biol Chem . 2007 ; 282 : 25053 - 66 .
19. Pal S , Baiocchi RA , Byrd JC , Grever MR , Jacob ST , Sif S . Low levels of miR92b/96 induce PRMT5 translation and H3R8/H4R3 methylation in mantle cell lymphoma . EMBO J . 2007 ; 26 : 3558 - 69 .
20. Saud SM , Li W , Morris NL , Matter MS , Colburn NH , Kim YS , Young MR . Resveratrol prevents carcinogenesis in mouse model of Kras activated sporadic colorectal cancer by suppressing oncogenic Kras expression . Carcinogenesis . 2014 ; 35 : 2778 - 86 .
21. Yu S , Lu Z , Liu C , Meng Y , Ma Y , Zhao W , Liu J , Yu J , Chen J . miRNA -96 suppresses KRAS and functions as a tumor suppressor gene in pancreatic cancer . Cancer Res . 2010 ; 70 : 6015 - 25 .
22. Xia H , Chen S , Chen K , Huang H , Ma H. MiR -96 promotes proliferation and chemo- or radioresistance by down-regulating RECK in esophageal cancer . Biomed Pharmacother . 2014 ; 68 : 951 - 8 .
23. Zhang J , Kong X , Li J , Luo Q , Li X , Shen L , Chen L , Fang L . miR -96 promotes tumor proliferation and invasion by targeting RECK in breast cancer . Oncol Rep . 2014 ; 31 : 1357 - 63 .
24. Feng J , Yu J , Pan X , Li Z , Chen Z , Zhang W , Wang B , Yang L , Xu H , Zhang G , Xu Z. HERG1 functions as an oncogene in pancreatic cancer and is downregulated by miR-96 . Oncotarget. 2014 ; 5 : 5832 - 44 .
25. Guo H , Li Q , Li W , Zheng T , Zhao S , Liu Z. MiR -96 downregulates RECK to promote growth and motility of non-small cell lung cancer cells . Mol Cell Biochem . 2014 ; 390 : 155 - 60 .
26. Liu Y , Han Y , Zhang H , Nie L , Jiang Z , Fa P , Gui Y , Cai Z. Synthetic miRNAMowers targeting miR- 183 -96 -182 cluster or miR-210 inhibit growth and migration and induce apoptosis in bladder cancer cells . PLoS One . 2012 ; 7 : e52280 .
27. Kreis NN , Louwen F , Zimmer B , Yuan J . Loss of p21Cip1/CDKN1A renders cancer cells susceptible to Polo-like kinase 1 inhibition . Oncotarget. 2015 ; 6 : 6611 - 26 .
28. Bianco S , Jangal M , Garneau D , Gévry N. LRH-1 controls proliferation in breast tumor cells by regulating CDKN1A gene expression . Oncogene . 2015 ; 34 : 4509 - 18 .
29. Wang X , Lin Y , Lan F , Yu Y , Ouyang X , Liu W , Xie F , Wang X , Huang Q. BAX and CDKN1A polymorphisms correlated with clinical outcomes of gastric cancer patients treated with postoperative chemotherapy . Med Oncol . 2014 ; 31 : 249 .
30. Newbold A , Salmon JM , Martin BP , Stanley K , Johnstone RW . The role of p21(waf1/cip1) and p27(Kip1) in HDACi-mediated tumor cell death and cell cycle arrest in the Eμ-myc model of B-cell lymphoma . Oncogene . 2014 ; 33 : 5415 - 23 .
31. Cazier JB , Rao SR , McLean CM , Walker AL , Wright BJ , Jaeger EE , Kartsonaki C , Marsden L , Yau C , Camps C , Kaisaki P , Oxford-Illumina WGS500 Consortium, Taylor J , Catto JW , Tomlinson IP , Kiltie AE , Hamdy FC . Whole-genome sequencing of bladder cancers reveals somatic CDKN1A mutations and clinicopathological associations with mutation burden . Nat Commun . 2014 ; 5 : 3756 .
32. Liu Y , Kwiatkowski DJ . Combined CDKN1A/TP53 mutation in bladder cancer is a therapeutic target . Mol Cancer Ther . 2015 ; 14 : 174 - 82 .
33. Jung HM , Phillips BL , Chan EK . miR -375 activates p21 and suppresses telomerase activity by coordinately regulating HPV E6/E7, E6AP, CIP2A, and 14-3-3ζ . Mol Cancer . 2014 ; 13 : 80 .
34. Sun D , Yu F , Ma Y , Zhao R , Chen X , Zhu J , Zhang CY , Chen J , Zhang J . MicroRNA -31 activates the RAS pathway and functions as an oncogenic MicroRNA in human colorectal cancer by repressing RAS p21 GTPase activating protein 1 (RASA1) . J Biol Chem . 2013 ; 288 : 9508 - 18 .
35. Yi C , Wang Q , Wang L , Huang Y , Li L , Liu L , Zhou X , Xie G , Kang T , Wang H , Zeng M , Ma J , Zeng Y , Yun JP . MiR- 663 , a microRNA targeting p21(WAF1/ CIP1), promotes the proliferation and carcinogenesis of nasopharyngeal carcinoma . Oncogene . 2012 ; 31 : 4421 - 33 .
36. Al-Shanti N , Saini A , Stewart CE . Two-Step versus one-step RNA-to-CT 2-step and one-step RNA-to-CT 1-step: validity, sensitivity, and efficiency . J Biomol Tech . 2009 ; 20 : 172 - 9 .
37. Segura MF , Hanniford D , Menendez S , Reavie L , Zou X , Alvarez-Diaz S , Zakrzewski J , Blochin E , Rose A , Bogunovic D , Polsky D , Wei J , Lee P , Belitskaya-Levy I , Bhardwaj N , Osman I . Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmiaassociated transcription factor . Proc Natl Acad Sci USA . 2009 ; 106 : 1814 - 9 .
38. Wang Y , Huang JW , Calses P , Kemp CJ , Taniguchi T . MiR -96 downregulates REV1 and RAD51 to promote cellular sensitivity to cisplatin and PARP inhibition . Cancer Res . 2012 ; 72 : 4037 - 46 .