Xianhui Wen1, Hebin Xie2, Rong Gui1, Xinmin Nie3, Dongyong Shan4, Rong Huang1, Hongyu Deng4, Junhua Zhang5. 1. Department of Blood Transfusion, The Third Xiangya Hospital of Central South University, Changsha, China. 2. Research and Teaching Department, Changsha Central Hospital, Changsha, Hunan, China. 3. The Third Xiangya Hospital of Central South University, Changsha, China. 4. Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, China. 5. Department of Blood Transfusion, The Third Xiangya Hospital of Central South University, Changsha, China. Email: jhzhangjunhua@163.com.
Bone marrow transplantation (BMT) remains the
fundamental treatment for various hematological
malignancies, aplastic anemia, severe thalassemia,
and some congenital immune deficiency or metabolic
diseases (1). Total body irradiation (TBI) is one of the
most important pretreatment methods for BMT in the
patients with leukemia (2). While TBI pretreatment
induces bone mesenchymal stem cells (BMSCs) injury,
BMT may seriously interfere with the implantation and
transplantation of hematopoietic stem cells (HSCs). Also,
TBI pretreatment will inevitably cause other normal cells
damages as well as body tissue cells in the bone marrow,
and consequently, destroy the hematopoietic system
(3). When the body is exposed to irradiation, a series
of adaptive stress responses are triggered to repair the
damage and reduce the effects of injury (4). At present,
the damage mechanism in these patients before BMT radiotherapy is not fully understood. Further study
on the regulatory mechanism of irradiation stress and
elucidation of the molecular mechanism of irradiation
biological effect will lay a theoretical foundation for the
protection and treatment of irradiation injury in the TBI
treated patients.CircRNAs are a particular type of non-coding RNAs characterized by a closed ring lacking a
5ˊ cap and a 3ˊ end poly (A) features. They regulate gene expression in the eukaryotes via
RNA-RNA interaction at post-transcriptional levels (5). Some circRNAs are molecular sponges
of their target miRNAs, which can chelate and inhibit miRNAs activity (6). The interaction
between circRNAs and miRNAs implicated in numerous processes suggests their critical
regulatory roles in these processes (7, 8). In our previous studies, we found that
circ-011235 and circ-016901 are significantly
up-regulated in the mice BMSCs follow of TBI irradiation. Bioinformatics analysis, we
concluded that circRNA-011235 (circ-011235) can be acted
as a miR-741-3p sponge (9). Previous studies showed that
miR-741-3p can be used as a biomarker for non-alcoholic fatty liver
disease (10) and attention-deficit/ hyperactivity disorder (11). However, there are limited
studies concerning circ-011235 and miR-741-3p and
irradiation-induced BMSCs injury.The cell cycle is a process tightly regulated by cyclins and cyclin-dependent kinases
(CDKs) (12). CDK6, a catalytic subunit of the CDK complex, drives the G1 phase process and
G1/S transition of the cell cycle. CDK6 activity first appeared in the mid-G1 phase, which
is precisely controlled by many regulatory subunits such as family members of inhibitor of
cyclin-dependent kinase 4 (INK4) and D-type cyclins. Cyclin D1 plays an
essential regulatory role in the cell cycle and is a key regulatory protein of the G1 phase
(13). Cyclin D1 overexpression may lead to an uncontrolled cell
proliferation (14). Studies have shown that CDK4 and CDK6 form a complex with cyclin D1 to
promote G1-S phase transformation via promoting the phosphorylation of tumor suppressor
retinoblastoma (Rb) and formation of CDK2/Cyclin E complex (15).Here, we attempted to explore the potential role of circ-011235 in the
proliferation of BMSC followingirradiation injury. And also, we investigated the
irradiation-induced self-protection mechanism of circ-011235 in the cell
cycle regulation, which may provide novel clinical targets for protecting BMSCs from
radiation damage.
Materials and Methods
Ethical consideration
In this experimental study, all animal procedures
complied with the guidelines permitted by the Animal
Care and Use Committee of XiangYa School of Medicine
(No. 2019-S534).
Isolation, culture and identification of bone mesenchymal
stem cells
Twenty healthy 2-month-old male BALB/c mice (21 ±
3 g) were provided from the XiangYa School of Medicine
of Central South University, China.Isolation, culture and identification of BMSCs from femurs and tibias were performed as
described in our previous study (9). Briefly, after exposure to CO2 , the
femurs and tibias of euthanized mice were separated and freshly isolated bone marrow cells
were washed and then, cultured in the Dulbecco’s Modified Eagle’s medium (DMEM, Cat No.
12634010, ThermoFisher Scientific, Shanghai, China) containing 10% fetal bovine serum
(FBS, Cat No. 10099133C, ThermoFisher Scientific, Shanghai, China) and incubated at 37˚C
in 5% CO2 for 24 hours. The DMEM was replaced every three days. Using CytoFLEX
V2-B2-R0 Flow Cytometer (model No. C09752, Beckman Coulter, Miami, FL, USA), the cells
were sorted with FITC-labeled CD34, CD45, CD90 and Sca-1 after obtaining 80% confluency.
Isolated cells that were identified to be CD34(−), CD45(−), CD90(+) and Sca-1(+) were
considered as BMSCs and used in subsequent experiments.
Radiation treatment
A cell suspension of BMSCs was prepared by trypsinization with 0.25% trypsin (Cat No.
25300120, ThermoFisher Scientific, Shanghai, China), and the cell density was maintained
at 2×106 cells/mL. Subsequently, the BMSCs were exposed to different doses of
irradiation (0, 2, 4, 6 GY) for 6 hours by using 6 MV X-rays of a 137 Cs-γ source (Sangyo
Kagaku, Japan) at an irradiation rate of 0.4 Gy/minutes and a distance of 100 cm from the
source.
Circ-011235 siRNA interference assay
Small interfering RNA (siRNA) targeting circ-011235
(siRNA-011235) and scrambled siRNA negative control
(siNC) were commercially available from KangChen Bio-tech (Shanghai,
China). Using the Lipofectamine 2000 kit (Cat No. 11-668-500, Invitrogen, Carlsbad, CA,
USA), BMSCs in the logarithmic phase were transfected with siRNA-011235
and siNC, according to the protocols provided by the manufacturer.
Briefly, the siRNAs were mixed for 20 min with Lipofectamine 2000 to prepare the
Lipofectamine 2000/siRNA complexes. Meanwhile, the BMSCs were incubated with DMEM medium
at 37˚C for 24 hours in 96-well plates. After that, the medium was refreshed and
Lipofectamine 2000/siRNA complexes were added dropwise to the BMSCs while gently shaking
the plate. Next, after incubation in 5% CO2 incubator for 5 hours, the
transfection efficiency was examined by Reverse-transcription quantitative polymerase
chain reaction (RT-qPCR).
Dual luciferase reporter assay
The potential negative regulatory relationship between circ-011235 and
miR-741-3p was found by Bioinformatics analysis. Also, predicted
databases, Targetscan and miRBase, were used to identify a binding site between the
CDK6 gene and miR-741-3p. The respective fragments of
circ-011235 and CDK6 harboring
miR-741-3p binding sites, miR-741-3p mimic, and miRNA
negative control (mimic control) were synthesized by GenePharma (Shanghai, China).
Subsequently, the fragments were subcloned into the psiCHECK-2 Renilla
(Cat No. C8021, Promega, Madison, WI, USA) luciferase reporter vector following the
manufacturer’s instructions. BMSCs were cotransfected with
circ-011235/CDK6 wild type (WT) vectors or
circ-011235/CDK6 mutated (MUT) vectors with miR-741-3p
mimic via the Lipofectamine 2000 (Cat No. 11-668-500, Invitrogen, Carlsbad, CA, USA)
transfection approach. The Dual-Luciferase Reporter Assay System (Promega, Madison, WI,
USA) was employed for assessing renilla luciferase activity.
Circ-011235 and CDK6 overexpression assay
Sequences of circ-011235 and CDK6 gene were synthesized
by KangChen Bio-tech (Shanghai, China) and cloned into the expression vector
pcDNA3.1 (Cat No. V79020, Invitrogen, Carlsbad, CA, USA) to generate
the overexpression recombinant plasmids pcDNA-circ-011235 and
pcDNA-CDK6, and the empty vector pcDNA3.1 was used as
a negative control. The recombinant plasmids were identified by RT-qPCR and DNA
sequencing. Then, recombinant plasmids were transfected into the logarithmic phase BMSCs
by Lipofectamine 2000 kit (Cat No. 11-668-500, Invitrogen, Carlsbad, CA, USA) based on the
manufacturer’s protocol. RT-qPCR was performed to detect the transfection efficiency 48
hours after transfection.
Cell proliferation assay
Following of 6 GY of irradiation treatment, transfected cells were cultured in 96-well
plates and the cell concentration was maintained at 5×103 cells/well. The cell
proliferation at 12, 24, 36 and 48 hours after seeding was evaluated using the CCK8 assay
(Cat No. GK10001, Glpbio, Montclair, CA, USA). In brief, we added 10 μL CCK8 solution to
each sample and incubated for 2 hours. Subsequently, the absorbance was detected by the
spectrophotometric approach at 450 nm wavelength.
Cell cycle analysis
Twenty four hours after transfection, BMSCs were harvested by centrifugation at 1200 rpm
for 5 minutes. The number of BMSCs was calculated using a haemocytometer, and the final
density of cells was maintained at 1×106 cells/mL with phosphate buffer saline
(PBS, Cat No. C791P76, Thomas Scientific, Swedesboro, NJ, USA). Subsequently, the cells
were fixed with 70% ethanol (3 mL) at 4˚C overnight, and collected by centrifugation at
1200 rpm for 5 minutes. Next, 200 μL of RNase (Cat No. AM2288, ThermoFisher Scientific,
Shanghai, China) was added, followed by incubation at 37 ˚C for 30 minutes. Afterwards,
800 μL of propidium iodide (PI, 20 mg/mL, Cat No. P1304MP, ThermoFisher Scientific,
Shanghai, China) was added, followed by incubation for 30 minutes at 25˚C in the dark. The
distribution of cells was detected by flow cytometry (Beckman, Los Angeles, CA, USA).
Real time-quantitative polymerase chain reaction
After transfection, total RNA of BMSCs was extracted by TRIzol Reagent (Cat No. 15596018,
ThermoFisher Scientific, Shanghai, China), based on the manufacturer’s directives. The
isolated total RNA (20 ng/μL) was employed to synthesize the cDNA by reverse transcription
using the GoScript reverse transcriptase (Promega, Charbonnièresles-Bains, France). For
circ-011235, the RNase R digestion reaction was performed at a ratio of
3U enzyme/1μg RNA before reverse transcription. The cDNA library was amplified using the
Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). The reaction was
started at 95˚C for 2 minutes and then subjected to 40 cycles of 95˚C for 10 seconds and
60 ˚C for 60 seconds. The primer sequences were synthetically produced by KangChen
Bio-tech (Shanghai, China). The primer sequences were displayed in Table 1. The
2-ΔΔCt formula was applied to measure the relative expression of
circ-011235, miR-741-3p, CDK6 and CDK4 (16).The primer sequences used in this study
Protein extraction, purification and western blotting
Radio-immunoprecipitation assay (RIPA) lysis buffer (Cat
No. 20-188, Merck, Shanghai, China) was used to extract the
total cellular RNA while this contains an inhibitor cocktail
mixture of phosphatase and protease (Cat No. ab201119,
Abcam, Waltham, MA, USA). Next, protein concentration
of the collected supernatant was measured by Pierce
BCA assay kit (Cat No. 23225, ThermoFisher Scientific,
Rockford, IL, USA). The extracted proteins were transferred
to polyvinylidene fluoride (PVDF) membranes (Cat No.
IPVH00010, Merck, Shanghai, China) after purification on
10% SDS-PAGE (Cat No. MT-46040CI, Fisher Scientific,
Loughborough, USA), and then, 5% skim milk (Cat No.
100518-0201, Medallion Milk Co., Winnipeg, MB, USA)
was used to block the membranes. Next, the membranes
were reacted with our primary antibodies at 4˚C overnight.
Our primary antibodies were included : CDK4 (1:1000;
Cat No. 12790, Cell Signaling Technology, Beverly, MA,
USA), cyclin D1 (1:1000; Cat No. 55506, Cell Signaling
Technology, Beverly, MA, USA) and GAPDH (1:2000;
Cat No. 5174, Cell Signaling Technology, Beverly, MA,
USA). Then, the membranes were incubated at 37˚C for 60
min with the HRP-labeled secondary antibody (1:2000; Cat
No. 7074, Cell Signaling Technology, Beverly, MA, USA).
Subsequently, a chemiluminescence imaging system (model
No. GeneGnome XRQ, Syngene, Cambridge, UK) was used
to examine the immunoblot signals of the target proteins,
and GAPDH was chosen as a housekeeping endogenous control for these proteins. The analysis of protein bands was
performed using the software Image J (National Institutes of
Health, Bethesda, MD, USA).
Statistical analysis
The results were analysed by SPSS 28.0.1 software (IBM,
Armonk, NY, USA). All data were presented as mean ±
standard deviation (SD). All data were analyzed using one-way or two-way analysis of variance (ANOVA) followed
by Turkey’s Post Hoc multiple comparison test to detect the
differences among the groups. Pearson correlation analysis
was achieved using the R package Hmisc. Also, P<0.05 was
considered for statistical significance.
Results
Irradiation increased the expression of circ-011235 and
CDK6
To examine the relationship between the expression of circ-011235,
miR-741-3p and CDK6, the correlation of their expression
levels in the BMSCs treated with irradiation was analyzed. It was found that the
irradiation treatment increased the expression of circ-011235 and
CDK6 in the BMSCs in comparison with the control group (0 GY)
(P<0.01, Fig .1A, B) but decreased the expression of miR-741-3p
(P<0.01, Fig .1C); there was a significant dose-dependent effect (2 GY<4
GY<6 GY) in the irradiation treated BMSCs. The Pearson correlation analysis found a
strong correlation between circ-011235 and CDK6 (r=0.99,
P<0.05, Fig .1D). Additionally, miR-741-3p expression was
negatively interrelated with the expression levels of circ-011235
(r=-0.98, P<0.05) and CDK6 (r=-0.97, P<0.05, Fig .1D).
Fig.1
RT-qPCR detection of gene expression in the BMSCs after irradiation treatment with different
doses (0, 2, 4, 6 GY). A. Relative expression of
circ-011235. B. Relative expression of
miR-741-3p. C. Relative mRNA expression of
CDK6 in the BMSCs. D. Pearson correlation analysis of
the correlation among circ-011235, miR-741-3p, and
CDK6 in the BMSCs after irradiation treatment. Data were
represented by mean ± SD. Independent experiments were replicated three times, **;
P<0.01, vs. the control group, RT-PCR; Real time-quantitative polymerase chain
reaction, and BMSCs; Bone mesenchymal stem cells.
RT-qPCR detection of gene expression in the BMSCs after irradiation treatment with different
doses (0, 2, 4, 6 GY). A. Relative expression of
circ-011235. B. Relative expression of
miR-741-3p. C. Relative mRNA expression of
CDK6 in the BMSCs. D. Pearson correlation analysis of
the correlation among circ-011235, miR-741-3p, and
CDK6 in the BMSCs after irradiation treatment. Data were
represented by mean ± SD. Independent experiments were replicated three times, **;
P<0.01, vs. the control group, RT-PCR; Real time-quantitative polymerase chain
reaction, and BMSCs; Bone mesenchymal stem cells.
Circ-011235 regulated miR-741-3p/CDK6 pathway in
irradiation-treated bone mesenchymal stem cells
Using online databases, IntaRNA, TargetScan, and miRBase, we observed that
miR-741-3p have a potential binding site for
circ-011235 (Fig .2A), and a unique binding site with 7 base pairs
between 3ˊ-UTR of CDK6 gene and miR-741-3p (Fig .2B). The
results showed that circ-011235 siRNA significantly up-regulated the
expression of miR-741-3p, but down-regulated the expression of
CDK6 (P<0.01, Fig .2C, D). The transfection efficiency of
miR-741-3p mimic was examined using RT-qPCR, and the results indicated
that miR-741-3p overexpression significantly upregulated the expression
of miR-741-3p (P<0.01, Fig .2E). In addition,
miR-741-3p overexpression significantly decreased luciferase activity
in the WT-circ-011235 and WT-CDK6 transfected cells
(P<0.01) but there was no significant effect on luciferase activity in the
MUT-circ-011235 and MUT-CDK6 transfected cells
(P>0.05, Fig .2F, G).
Fig.2
Circ-011235/miR-741-3p/CDK6 signal pathway. A. IntaRNA prediction of
miR-741-3p as a target for circ-011235.
B. TargetScan prediction of 3ˊ-UTR of CDK6 gene as a
target for miR-741-3p. C.
Circ-011235 silencing upregulates the miR-741-3p
expression in the BMSCs. D.
Circ-011235 silencing decreases the CDK6 expression
in the BMSCs. E. Evaluation by RT-qPCR of the transfection efficiency of
miR-741-3p mimic. F. Luciferase reporter assay
illustrating the interactions between circ-011235 and
miR-741-3p. G. Luciferase reporter assay illustrating
the interactions between CDK6 and miR-741-3p in the
BMSCs. Data were represented by mean ± SD. Independent experiments were replicated
three times, **; P<0.01, vs. the control group, RT-PCR; Real time-quantitative
polymerase chain reaction, and BMSCs; Bone mesenchymal stem cells.
Circ-011235/miR-741-3p/CDK6 signal pathway. A. IntaRNA prediction of
miR-741-3p as a target for circ-011235.
B. TargetScan prediction of 3ˊ-UTR of CDK6 gene as a
target for miR-741-3p. C.
Circ-011235 silencing upregulates the miR-741-3p
expression in the BMSCs. D.
Circ-011235 silencing decreases the CDK6 expression
in the BMSCs. E. Evaluation by RT-qPCR of the transfection efficiency of
miR-741-3p mimic. F. Luciferase reporter assay
illustrating the interactions between circ-011235 and
miR-741-3p. G. Luciferase reporter assay illustrating
the interactions between CDK6 and miR-741-3p in the
BMSCs. Data were represented by mean ± SD. Independent experiments were replicated
three times, **; P<0.01, vs. the control group, RT-PCR; Real time-quantitative
polymerase chain reaction, and BMSCs; Bone mesenchymal stem cells.
Overexpression of circ-011235 and CDK6 increased
the irradiation-treated bone mesenchymal stem cells proliferation
To investigate the transfection efficiency of pcDNA-circ-011235 and
pcDNA-CDK6, we detected the expression levels of
circ-011235 and CDK6 in BMSCs. The results showed that
pcDNA-circ-011235 significantly up-regulated
circ-011235 in comparison with the control group (P<0.01,
Fig .3A), and pcDNA-CDK6 treatment led to the similar results for the
CDK6 cell (Fig .3B). Furthermore, we also examined the overexpression
effect of circ-011235 and CDK6 on the proliferation
after exposure to 6 Gy irradiation. It was found that irradiation treatment hindered the
proliferation of BMSCs compared with the control group (P<0.01); however,
overexpression of circ-011235 or CDK6 significantly
reversed this inhibitory effect after 24 hours seeding (Fig .3C).
Fig.3
Effect of circ-011235 and CDK6 on the proliferation of
irradiation-treated BMSCs. A. RT-qPCR detection of transfection
efficiency of pcDNA-circ-011235. B. RT-qPCR detection of
transfection efficiency of pcDNA-CDK6. C. CCK-8 assay
detection of the effects of pcDNA-circ-011235 and
pcDNA-CDK6 on the proliferation of irradiation-treated BMSCs.
Values were represented by the mean ± SD. Separated experiments were repeated three
times. **; P<0.01, vs. the control group, #; P<0.05, ##; P<0.01,
vs. the irradiation treatment group and control vector group, RT-PCR; Real
time-quantitative polymerase chain reaction, BMSCs; Bone mesenchymal stem cells, and
h; Hour
Effect of circ-011235 and CDK6 on the proliferation of
irradiation-treated BMSCs. A. RT-qPCR detection of transfection
efficiency of pcDNA-circ-011235. B. RT-qPCR detection of
transfection efficiency of pcDNA-CDK6. C. CCK-8 assay
detection of the effects of pcDNA-circ-011235 and
pcDNA-CDK6 on the proliferation of irradiation-treated BMSCs.
Values were represented by the mean ± SD. Separated experiments were repeated three
times. **; P<0.01, vs. the control group, #; P<0.05, ##; P<0.01,
vs. the irradiation treatment group and control vector group, RT-PCR; Real
time-quantitative polymerase chain reaction, BMSCs; Bone mesenchymal stem cells, and
h; Hour
Overexpression of circ-011235 and CDK6 affected
the cell cycle of irradiation-treated bone mesenchymal stem cells
To investigate the overexpression effect of circ-011235 and
CDK6 on cell proliferation, cell cycle analysis was performed. When
compared with the control group, the results showed that irradiation treatment
significantly increased the percentage of cells in the G1 phase (P<0.01), while the
proportion of cells in the S phase was significantly declined (P<0.01, Fig .4A).
However, compared with the irradiation-treated group, circ-011235
overexpression significantly reduced the percentage of cells in the G1 phase and elevated
the percentage of cells in the S phase (P<0.01, Fig .4A). Similar results were found
with the CDK6 overexpression.
Fig.4
Effects of circ-011235 and CDK6 on the cell cycle in the
irradiation-treated BMSCs. A. Flow cytometry analysis of the effects of
pcDNA-circ-011235 and pcDNA-CDK6 on the cell cycle
in the irradiation-treated BMSCs. B. RT-qPCR detection of the expression
of cyclin D1 and CDK4 at the gene level.
C. Western blot detection of the expression of cyclin
D1 and CDK4 at the protein levels. D.
Densitometry analysis of western blot bands of the expression of cyclin D1 and
CDK4. Data were represented by mean ± SD. Separate experiments were replicated three
times, **; P<0.01, vs. the control group, ##; P<0.01, vs. the
irradiation treatment group and control vector group, RT-PCR; Real time-quantitative
polymerase chain reaction, and BMSCs; Bone mesenchymal stem cells.
Also, the expression analysis of cyclin D1 showed that cyclin D1 was significantly
up-regulated in the circ-011235 overexpression group and
CDK6 overexpression group compared to the irradiation-treated group
(P<0.01), while there was no significant change in CDK4 expression
(Fig .4B-D).Effects of circ-011235 and CDK6 on the cell cycle in the
irradiation-treated BMSCs. A. Flow cytometry analysis of the effects of
pcDNA-circ-011235 and pcDNA-CDK6 on the cell cycle
in the irradiation-treated BMSCs. B. RT-qPCR detection of the expression
of cyclin D1 and CDK4 at the gene level.
C. Western blot detection of the expression of cyclin
D1 and CDK4 at the protein levels. D.
Densitometry analysis of western blot bands of the expression of cyclin D1 and
CDK4. Data were represented by mean ± SD. Separate experiments were replicated three
times, **; P<0.01, vs. the control group, ##; P<0.01, vs. the
irradiation treatment group and control vector group, RT-PCR; Real time-quantitative
polymerase chain reaction, and BMSCs; Bone mesenchymal stem cells.
Discussion
HSC transplantation has been achieved a huge success
in the treatment of blood system diseases and is currently
known as a most effective cell replacement therapy.
TBI is one of the necessary pretreatments for HSC
transplantation (17). Whether the injury of TBI to BMSCs
affects the hematopoietic function of HSCs. Although,
its regulatory mechanism is not fully known, rational
treatment plans will direct to high efficiency and low
toxicity in the clinical phase.The hematopoietic support of BMSCs mainly regulates the survival, self-renewal, migration
and differentiation of hematopoietic stem or progenitor cells through intercellular
interactions and secretion of growth factors, chemical factors, and extracellular matrix
(18). Co-infusion of HSCs and MSCs heterogeneity have been observed to promote hematopoietic
reconstruction (19). BMSCs with low immunogenicity and immunomodulation can avoid and
alleviate host immune responses, induce the formation of specific immune tolerance, and
promote the transplantation of HSCs. It repairs tissue injury caused by pre-transplant
pretreatment, which can reduce the incidence of severe GVHD and transplant-related mortality
(20). Compared with HSCs, BMSCs are highly resistant to irradiation and can survive acute
exposure (21), which is an important hematopoietic support cell for hematopoietic recovery
after irradiation. The study of irradiation-induced injury in the BMSCs has significant
implications to improve the HSCs transplantation survival rate . In the present study, the
expression of circ-011235, miR-741-3p, and CDK6 was
dose-dependent after irradiation for 6 h, indicating that irradiation can change the
molecular profile of BMSCs.Studies have shown that non-coding RNAs such as circRNAs and miRNAs act as gene expression
regulators and have been confirmed to be involved in the regulation of the cell
proliferation such as cancer cells (22). CircRNA contains miRNA response elements, which act
as a competitive endogenous RNA to compete binding site between miRNA and its target gene,
thereby may be acted as a eliminating agent against the inhibitory effect of miRNA on their
target gene (23). In this study, our results showed that circ-011235
increases the cell proliferation and progresses cell cycle of irradiation-treated BMSCs by
down-regulating miR-741-3p expression and up-regulating
CDK6 expression. This suggests that circ-011235
derepresses CDK6 gene by acting as a sponge of miR-741-3p
to counteract the irradiation-induced damage of BMSCs. This is the first study to explore
the role and function of circ-011235 and miR-741-3p in the
irradiation-induced BMSCs injury.As an oncogene, CDK6 can promote the cell proliferation and play a
regulatory role in the occurrence and development of various cancers, such as bladder
cancer, glioma, and medulloblastoma (24). Cyclin D-associated kinases inhibitors such as
CDK4 and CDK6 can be used as a potential cancer therapeutic targets. The function and
regulation mechanism of CDK6 in the BMSCs are still not clearly explained.
CDK6, together with CDK4, acts as a switching signal in the G1 phase that, directing cells
towards the S phase (25). CDK6 is an important driving factor in the shift of the cell cycle
from G1 to S stage. However, a previous study showed that the cell cycle is regulated by
complex regulatory pathways, and CDK6 is not necessary for the proliferation of every cell
type (26). In addition, CDK4 or CDK2 are protein kinases that compensate the effects of
CDK6. Moreover, CDK6 is primarily associated with cyclins proteins such as cyclins D1, D2,
and D3 (27). The positive activation of CDK6 can be achieved by phosphorylation of the
177th conserved threonine residue by CDK activated kinase (CAK) (28). In
addition, Kaposi’s sarcoma-associated herpesvirus can phosphorylate and overactive CDK6, and
cause uncontrolled cell proliferation (29). In the present study, our results demonstrated
that circ-011235 and CDK6 were activated by irradiation
and their expression associated with a dose-dependent increase effect. Similarly, a previous
study in mice found that the cell viability was reduced by ultraviolet light C (UVC)
treatment, but loss of Runx2 could counteract UVC induced cell death by
increasing the expression of cyclins and related CDK activities (30). Moreover, Zou et al.
(31) showed that the Cell Division Cycle 25A (CDC25A), an activator of G1
CDKs in the nucleus, could inhibit the oxidant-triggered gamma irradiation induced apoptosis
via diminishing the activation of the oxidative stress kinase cascades. Furthermore, the
complex of cyclin D1 with CDK4 and CDK6 is involved in the regulation of the cell cycle,
which phosphorylates the Rb protein, thereby promoting the cell cycle from G1 to S stage
(32). Previous studies showed that CDK6 activity elevates in the cultured mouse astrocytes
without alteration of CDK4 activity (33). Also, we found that overexpressions of circ-011235
and CDK6 both promoted the proliferation and cell cycle through increasing the expression of
cyclin D1 in the irradiation treated BMSCs, which suggested its vital role in the
self-protection mechanism of BMSCs in response to irradiation.We did not same results in the
overexpression of CDK4.Finally, our study presents some limitations. In cell cycle analysis, only cyclin D1 and
CDK4 protein and mRNA expression were detected, and further cyclins such as cyclin E, CDK2,
and p27 kipl are required to increase the confidence of our findings. Moreover, animal
experiments are needed to explore the potential role of circ-011235,
miR-741-3p, and CDK6. However, further measurement methods are
still needed to confirm our findings.
Conclusion
In this study, we uncovered the regulatory function of circ-011235 on the
cell cycle in the irradiation-treated BMSCs. Our results showed that
circ-011235 increases the irradiation-treated BMSCs proliferation and
promotes cell cycle progression through down-regulating miR-741-3p and
up-regulating CDK6. Also, Circ-011235 and CDK6
overexpression could effectively reverse the inhibitory effect of irradiation on
the proliferation and cell cycle arrest of the BMSCs through promoting the expression of
cyclin D1. This is the first study to demonstrate the protective role of
circ-011235/miR-741- 3p/CDK6 axis, especially
circ-011235 and miR-741-3p, against irradiation-induced
damage of BMSCs. The circ-011235/miR-741-3p/CDK6 axis may be a probable
therapeutic target in the clinical application of TBI-induced BMSCs injury.
Authors: E K von der Heide; M Neumann; S Vosberg; A R James; M P Schroeder; J Ortiz-Tanchez; K Isaakidis; C Schlee; M Luther; K Jöhrens; I Anagnostopoulos; L H Mochmann; D Nowak; W K Hofmann; P A Greif; C D Baldus Journal: Leukemia Date: 2016-11-11 Impact factor: 11.528
Authors: N L Davis; C E Stewart; A D Moss; W W W Woltersdorf; L P Hunt; R A Elson; J M Cornish; M C G Stevens; E C Crowne Journal: Clin Endocrinol (Oxf) Date: 2015-05-05 Impact factor: 3.478
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