miR-150-5p represses TP53 tumor suppressor gene to promote proliferation of colon adenocarcinoma
miR-150-5p represses TP53 tumor suppressor gene to promote proliferation of colon adenocarcinoma
Xiao Di Wang
OPEN MicroRNAs (miRNAs) play a critical role in regulation of numerous biological processes and pathogenesis of a variety of diseases. In addition, miRNAs contribute to carcinogenesis by acting as oncogenic or tumor suppressive. Circulating miRNAs including miR-150-5p are associated with colorectal cancer progression, and the putative targets of miR-150-5p include tumor suppressor gene, TP53. Here we sought to investigate the role of miR-150-5p-TP53 signaling pathway in proliferation of colon cancer and to determine expression levels of miR-miR-150-5p and TP53 in colon adenocarcinoma and adjacent non-cancerous tissue samples, or in human colon adenocarcinoma cell lines. MTT assay was used to determine proliferation and apoptosis in cell lines. Furthermore, we used Western blot to determine levels of cell cycle regulators with anti-miR-150-5p or apoptosis with overexpression of TP53. Our results show that expression levels of miR-150-5p were significantly elevated in clinical specimens from cancer patients. We further showed that inhibition of miR-150-5p increased TP53, and in turn, suppression of proliferation of colon adenocarcinoma. Moreover, inhibition of miR-150-5p or overexpression of TP53 caused cell arrest or apoptosis in colon adenocarcinoma. Our results support that miR-150-5p-TP53 pathway plays an important role in regulation of proliferation, cell arrest, and apoptosis in colon cancer, and could be an attractive target for therapy.
Colorectal cancer (CRC) is a common malignant tumor with an incidence rate of a million people worldwide
every year, and a major cause of mortality and morbidity in developed countries1?3. In addition, CRC ranks as the
third most common cancer by incidence and the fourth most common cause of cancer-related deaths world-wide.
Presently, surgical resection is the only approach for curative treatment of CRC4. Despite numerous efforts in
the past few decades, CRC survival rates have not significantly improved, and half of the CRCs relapse and the
patients die within five years. As such, identifying novel and more effective drug targets in treatment of CRC are
MicroRNA (miRNAs) usually silence target genes by binding to their target messenger RNAs at 3?-UTR5. It
has been estimated that miRNAs can regulate one-third of all human genes and influence hundreds of
potential gene targets6. Dysregulation of miRNA expression is closely linked to cancer initiation and progression.
Mechanistically, miRNAs can inhibit oncogenes or tumor suppressor genes to play a tumor suppressor or
oncogenic role, respectively7. In addition, miRNAs can serve as biomarkers for early cancer diagnosis, because
miRNAs are stable in body fluids, either in secreted microvesicles or bound to carrier proteins, which provides a
unique opportunity to exploit the role of miRNAs as biomarkers8. Indeed, expression levels of circulating
miRNAs have shown some potential at differentiating cancer patients and healthy controls for prostate9, ovarian10,
lung11 and breast12 cancers. Moreover, multiples studies support the prognostic and diagnostic value of some
circulating exosomal microRNAs in colon cancer, particularly, miR-150-5p13,14.
The tumor suppressor gene, TP53, is one of the most frequently mutated genes in many cancers including
CRC15. TP53 is generally expressed at low levels, in part through negative feedback loops that involve MDM216.
Low abundance of T53 expression preserves homoeostasis of the cell cycle and cell death. In response to stress
factors, such as hypoxia, UV irradiation, oxidative free radicals, and activation of oncogenes, TP53 protein is
activated, and such an activation of p53 can either transactivate or repress downstream target genes that in turn
regulate cell cycle arrest, apoptosis, DNA repair, and angiogenesis and even metastasis17.
Although multiple studies have revealed the potential tumor-suppressive effect of TP53 in human tumor, the
molecular function of TP53 in CRC has remained further defined. Expression of miR-150-5p has been linked
with TP53-downstream target genes15. Interestingly, a preliminary bioinformatics analysis indicates that TP53
is a potential target of miR-150-5p18. In the present study, we sought to investigate the role of miR-150-5p-TP53
signaling in CRC, and we demonstrate that such a signaling plays a critical role in proliferation and progression
Materialas and Methods
Subjects. We collected specimens from a total of 10 CRC patients after surgical resection. The study and all
methods were carried out in accordance with relevant guidelines and regulations. all experimental protocols were
approved by the Ethic Committee of China-Japan Friendship Hospital. The general information for patients is
provided in Table?1. Informed consent was obtained from all subjects.
Cell culture. Normal human colon epithelial cell line CCD 841 CoN (ATCC, USA), and human colon
carcinoma cell lines HT29, T84, and LS174 (ATCC, USA) were maintained in RPMI-1640 medium supplemented
with 10% fetal bovine serum, as recently described19. Cells were cultured in a humidified incubator at 37 ?C and
Reagents. Antibodies to TP53, COX-2, CDK1, Cyclin B1, Caspase-3 and -9 were obtained from Santz Cruz
Biotech (TX, USA). TP53 siRNA, miR-150-5p mimics or anti-miR-150-5p inhibitors were purchased from
Quantitative RT-PCR. Total RNAs were extracted by Trizol method (Invitrogen, CA, USA) and
re-suspended in nuclease-treated H2O. cDNA synthesis was prepared by the miR-150-5p-, U6 snRNA-specific
or oligo-dT primers method using the Superscript II Reverse Transcription kit (Invitrogen, USA). Quantitative
PCR was performed using an ABI7300 machine (Applied Biosystems, USA). miR-150-5p or TP53 mRNA
levels were determined, relative to U6 or GAPDH expression using the SYBR Green PCR kit (Qiagen, USA),
respectively. Fold change in expression was determined by the method of cycle threshold (CT) using the
formula of 2???CT. U6 forward, 5?-TGCGGGTGCTCGCTTCGGCAGC-3?. U6 reverse, 5?-GGGTCCGAGG
TGCACTGGATACGACAAAATATGG-3?; GAPDH forward, 5?-CTCCCGCTTCGCTCTCTG-3?, GAPDH
reverse, 5?- CTGGCGACGCAAAAGAAG-3?, TP53 forward: 5?-GGCCCACTTCACCGTACTAA-3?; TP53
Western blot. Whole cell lysates were suspended in 1 ? SDS loading buffer, boiled at 95 ?C for 5 min, and
centrifuged. Supernatants were separated on SDS?10% PAGE and transferred onto PVDF membranes (Bio-Rad,
USA). Membranes were blocked in 5% milk in PBST (10 mM phosphate buffer, pH 7.2; 150 mM NaCl; and
0.1% Tween-20) for 60 min, washed three times, and incubated with appropriate antibody at 4 ?C overnight.
Membranes were washed three times with PBST, incubated with horseradish peroxidase-conjugated secondary
antibody at 1:5,000, and developed by Immun-Star HRP Substrate (Bio-Rad, CA, USA). Blots were probed with
?-actin antibody (Sigma-Aldrich, USA) as the loading control.
Dual Luciferase Assay. 3?-UTR segment of TP53 gene corresponding to predicted target site was amplified
by PCR from human genomic DNA using primers that included a XbaI and EcoRI tails on the 5? and 3? strands,
respectively, as previously described20. PCR products were restriction digested with both XbaI and EcoRI DNA
restriction endonucleases, gel purified, and ligated into pGL3 vector (Promega, USA). HT29 cells were transfected
with the firefly luciferase UTR-report vector, control Renilla luciferase pRL-TK vector (Promega, USA) with
Lipofectamine 2000 reagent, according to the manufacturer?s protocol (Invitrogen, USA). Twenty-four hours after
transfection, cells were lysed with a 1x passive lysis buffer and the activity of both Renilla and firefly luciferases
were assayed using the dual-luciferase reporter assay system (Promega, USA), according to the manufacturer?s
Cell proliferation Assay. MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbr-omide] - based
assay was performed to estimate the effect of miR-150-5p mimics, anti-miR-150-5p, or TP53 siRNA on human
colonic adenocarcinoma cells? proliferation, as previously described21. Cells were seeded into 96-well plates (5,000
cells/well in 200 ? L medium) and incubated for 24 hrs. HT29 cells were transfected with miR-150-5p mimics,
anti-miR-150-5p, or TP53 siRNA using Lipofectamine 2000 Reagent (ThermoFisher Scientific, USA) as indicated.
Cells cultured in complete medium were used as control. At the end of incubation, 20 ? L of 5 mg/ml MTT (Sigma,
USA) solution was added per well, and the cells were incubated for another 4 hr at 37 ?C. Supernatants were
removed and formazan crystals were dissolved in 150 ? L of DMSO (Sigma-Aldrich, USA). OD was determined at
490 nm using multi-microplate test system (InfiniteM200Pro,USA).
Statistical analysis. All results were expressed as mean ? standard deviation. We used Student?s t-test
or ANONA (One-way) to compare two and three groups, respectively. P < 0.05 was considered significantly
miR-150-5p directly targets TP53 in CRC. TP53 is a tumor suppressor gene as well as putative target of
miR-150-5p18. To investigate whether miR-150-5p targets TP53 in CRC, we first performed bioinformatic
analysis, using TargetScan and found that there are multiple miR-150-5p target sites in TP53 mRNA (Fig.?1A).
To validate a direct interaction between miR-150-5p and TP53, we cloned a miR-150-5p target site at 3?-UTR
of TP53 mRNA upstream of firefly luciferase (FL) reporter gene, so that it could be regulated by miR-150-5p. In
addition, a mutated version of this target site was used as the negative control. Colon adenocarcinoma HT29 cells
were transfected with either the control FL plasmid or FL plasmid with miR-150-5p target site or the FL plasmid
with mutation of miR-150-5p target site. Also, Renilla luciferase reporter plasmid was co-transfected as an
internal reference. As shown in Fig.?1B, a significant decrease in FL activity was observed in cells transfected with FL
reporter with wild type of miR-150-5p target site. In contrast, no repression of FL activity was obtained in cells
transfected with miR-150-5p-mutation FL reporter plasmid (Fig.?2B). Further, we confirmed a direct targeting of
TP53 mRNA by miR-150-5p in HT29 cells. We found that TP53 increased at both protein and mRNA levels when
cells were treated with miR-150-5p specific inhibitors (Fig.?1C). Taken together, these results indicate that TP53
is a direct target of miR-150-5p in CRC cells.
Coupled increase of miR-150-5p and decrease of TP53 in CRC. Next, we sought to determine miR
150-5p and TP53 levels in in tissue specimens derived from CRC patients. Total RNAs were extracted from 10
colon cancer and adjacent non-cancerous tissue samples for the assessment of miR-150-5p and TP53 mRNA
levels by quantitative RT-PCR. TP53 protein levels were assessed by Western blots as well. In the meantime, the
same tissues were subjected to purification of colon cancer cells followed by immunofluorescence staining (IF).
We found that miR-150-5p was significantly up-regulated in CRC cancer tissues (Fig.?2A). In contrast, TP53
mRNA and protein levels decreased by an average of >45% (Fig.?2B,C), compared to the non-cancerous adjacent
colon mucosa. In addition, weak cytoplasmic staining for TP53 was observed in the cancerous tissues, in contrast
to strongly positive staining of COX-2, another biomarker for CRC22 (Fig.?2D).
Next, we used RT-PCR to determine levels of miR-150-5p and TP53 in different colon adenocarcinoma cell
lines and similar results were obtained in all three colon adenocarcinoma cell lines as found in the primary tissues
(Fig.?2E). Collectively, these results suggest that miR-150-5p and TP53 are negatively correlated in CRC,
suggesting a possible regulation of TP53 by miR-150-5p.
miR-150-5p stimulates cell proliferation in vitro. HT29 cells were transfected with non-specific
miRNA mimics, anti-miR-150-5p, or anti-miR-150-5p combined with TP53 siRNAs, as indicated, and MTT
assays were performed to determine cell proliferation. As shown here, miR-150-5p mimics promoted, but
miR150-5p inhibitors inhibited cancer cell growth (Fig.?3A). Consistent with this, depletion of TP53 also promoted
cancer growth, and abolished anti-miR-150-5p mediated inhibition of cell proliferation (Fig.?3B,C). These results
further confirm that miR-150-5p directly inhibits TP53 to exert oncogene functions and promote CRC cancer
Inhibition of miR-150-5p affects the expression of key cell cycle factors. TP53 has been found
to induce cell cycle arrest in G2/M phase in clear cell renal cell carcinoma (ccCRC)23. Here we tested the role
of miR-150-5p-TP53 signaling in regulation of cell cycle in CRC. The expression of cell cycle regulators
associated with G2/M phase transformation was investigated in HT29 cells treated with either anti-miR-150-5p or the
control non-specific miRNA mimics. As shown in Fig.?4, protein levels of cyclinB1 and CDK1 were significantly
decreased in cells transfected with anti-miR-150-5p, in contrast to upregulation of TP53. These results support
that miR-150-5p-TP53 signaling pathway modulates cell cycle progression via expression of cell cycle regulators.
TP53 triggers cell apoptosis in CRC cells. Evasion of apoptosis is a critical event during malignant
transformation, and TP53 has been found to induce apoptosis in various cancer cells24. Hence, we examined the effects
of TP53 on induction of CRC cell apoptosis. As shown in Fig.?5, the expression levels of cleaved caspase-3 and
caspase-9, which are molecular hallmarks for cell apoptosis, were significantly increased in HT29 cells transfected
with pc-DNA3-TP53. Collectively, these results suggest that TP53 overexpression exerts significant effects on the
proliferation of CRC cells.
The molecular mechanisms underlying CRC initiation and progression remain largely unknown, and,
therefore, a comprehensive detailing of all such mechanisms is critical. This alone will facilitate discovery of novel
biomarkers for early diagnosis and therapy, thus improving the outcome of CRC patients. Over the past decade,
miRNAs have emerged as a new and promising class of gene regulators involved in cancer progression25,26. Here,
we show, for the first time, that miR-150-5p plays a critical role in colon tumorigenesis by inhibiting the tumor
suppressor gene TP53 to promote cancer proliferation. Our results suggest that miR-150-5p can be used for early
stage diagnosis and a therapeutic target for therapy of CRC. These collective findings show an important and new
role of miR-150-5p-TP53 signaling pathway in carcinogenesis of CRC, and provide a molecular detaining for the
correlation between elevation of miR-150-5p and cell proliferation in CRC. Therefore, our study offers a concrete
base for further understanding of the complex biology involved in CRC cancer progression.
miRNAs are well-known to play a role in cancer progression. As demonstrated previously by several groups, the
same miRNA might play a distinct role in different types of cancer. For example, miR-146a acts as oncogenic as well
as tumor suppressive in distinct cell types27,28. Consistent with this observation, patients with higher miR-150-5p
levels usually have a worse survival in melanoma and inhibition of miR-150-5p enhanced cell apoptosis via
upregulation of PDCD4-mediated activation of caspase-8 and p2129. However, another study has shown that miR-150
might serve as a potential therapeutic sensitizer through inhibition of the AKT pathway in NK/T cell lymphoma
treatment30. Therefore, more studies are needed to elucidate the role of miR-150-5p in other types of cancer.
Our results are consistent with the earlier published literature that TP53 plays a protective role in
blocking a decisive step in oncogenesis. Activation of TP53 can induce both the mitochondrial and the
death-receptor-induced apoptotic pathways31. Activation of TP53 leads to expression of pro-apoptotic Bcl-2
(B-cell lymphoma-2) family proteins, mainly Bax, Noxa and PUMA, but inhibits the pro-survival Bcl-2, and in
turn, permeabilization of outer mitochondrial membrane. Then cytochrome c releases from the mitochondria
binds to APAF-1, and induces the activation of the initiator caspase-9, eventually resulting in the activation of
executioner caspase-3, -6 and -7. On the other hand, activated TP53 increases some death receptors and form
death-inducing signaling complex with caspase-8, to induce apoptosis. The progression of cell cycle is tightly
controlled by cyclins and cyclin-dependent kinases (CDK). p21 is one member of CDK inhibitor family, which
stimulates cell cycle transition from G1 to S phase, as well as one of the major mediator of p53-induced growth arrest.
In response to DNA damage, p53 induces not only cell cycle G1 phase arrest, but also G2/M checkpoint arrest32.
In conclusion, our results support a role of miR-150-5p-TP53 signaling pathway in the proliferation, cell cycle
progress, cell apoptosis and invasion/migration of CRC cells. Furthermore, miR-150-5p acts an oncogene and its
target TP53 acts as a tumor suppressor in CRC pathogenesis. Due to current lack of appropriate biomarkers and
therapeutic targets in CRC, these results suggest that miR-150-5p-TP53 signaling may provide novel insights into
the improvement of clinical therapeutic strategies for CRC patients.
All the data is available within this manuscript.
F.L. and X.D.W. performed experiments; X.D.W. analyzed results; F.L. put together first draft; X.D.W. developed
idea, provided resources and approved manuscript.
Competing Interests: The authors declare no competing interests.
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