- Email: [email protected]
P53 signaling pathway
Journal Pre-proof MiR-1258 promotes the apoptosis of cervical cancer cells by regulating the E2F1/P53 signaling pathway
Xing Peng, Yumei Zhang, Jinyu Gao, Chunyan Cai PII:
S0014-4800(19)30719-1
DOI:
https://doi.org/10.1016/j.yexmp.2020.104368
Reference:
YEXMP 104368
To appear in:
Experimental and Molecular Pathology
Received date:
18 September 2019
Revised date:
11 December 2019
Accepted date:
3 January 2020
Please cite this article as: X. Peng, Y. Zhang, J. Gao, et al., MiR-1258 promotes the apoptosis of cervical cancer cells by regulating the E2F1/P53 signaling pathway, Experimental and Molecular Pathology(2019), https://doi.org/10.1016/ j.yexmp.2020.104368
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof MiR-1258 promotes the apoptosis of cervical cancer cells by regulating the E2F1/P53 signaling pathway
Xing Peng, Yumei Zhang, Jinyu Gao, Chunyan Cai
Department of Gynaecology, The Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University, Huaian, Jiangsu, China
of
Correspondence to: Chunyan Cai, Department of Gynaecology, The Affiliated Huaian
ro
No.1 People’s Hospital of Nanjing Medical University, 6 Beijing Road West, Huaian,
-p
Jiangsu Province, 223300, China. E-mail: [email protected]
lP
re
Key words: miR-1258, apoptosis, cervical cancer, E2F1/P53 signaling pathway
Abstract.
na
Running title: Role of miR-1258 in cervical cancer
Jo ur
Objective: Cervical cancer is the most common malignant tumor in gynecology with high mortality. MiRNA has been reported to regulate cell biological processes in cervical cancer. This study aimed to explore the expression of miR-1258 and role of miR-1258 by targeting E2F1 in cervical cancer cells. Methods: The expression of miR-1258 and E2F1 in cervical cancer cells and transfection effects was determined by RT-qPCR analysis. The expression of E2F1, MMP2, MMP7, MMP9, Bcl2, Bax, cleaved caspase3, caspase3, KI67, p-AKT, cyclinD1, CDK2, P53 and AKT in cervical cancer cells was detected by western blot analysis. The proliferation, invasion, migration and apoptosis were respectively analyzed by CCK-8 assay, transwell assay, wound healing assay and flow cytometry analysis. E2F1 was a potential target of miR-1258, which demonstrated by a dual-luciferase reporter assay. Results: miR-1258 expression was decreased while E2F1 expression was increased in
Journal Pre-proof cervical cancer cells. MiR-1258 overexpression could down-regulate the E2F1 expression. Overexpression of miR-1258 inhibited the proliferation, invasion and migration and promoted the apoptosis of cervical cancer cells by AKT and P53 signal pathway. And, Overexpression of miR-1258 also suppressed the tumor growth by AKT and P53 signal pathway. Overexpression of E2F1 reduced the inhibition effects of miR-1258 in cervical cancer. Conclusion: Taken together, miR-1258 overexpression exerts its inhibition effects on the proliferation, invasion and migration and promotion effects on the apoptosis of
of
cervical cancer cells by targeting the E2F1, which might provide new ideas for
ro
clinical treatment of cervical cancer.
-p
Introduction
re
Cervical cancer is the second most common female malignancy, with more than 1.5 million new cases diagnosed each year and more than 300,000 deaths (1). The
lP
main cause of cervical cancer such as human papillomavirus (HPV) infection has been identified, so early implementation of surgery combined with chemotherapy can
na
significantly improve the survival rate of patients. However, clinical studies have found that for patients with advanced cervical cancer, intra-pelvic lymph node
Jo ur
metastasis is the main factor affecting the prognosis of patients (2). In addition, the 5-year survival rate of cervical cancer is below 40%, and poor prognosis of cervical cancer is a major public health problem, leading to high hospital costs. Therefore, in-depth study of the molecular mechanism of the occurrence and development of cervical cancer and search for effective molecular markers are conducive to explore methods that can block the proliferation and metastasis of tumor cells and promote the apoptosis of tumor cells. In recent years, more attention has been paid to the role of miRNA in the occurrence, development and metastasis of cervical cancer. MicroRNAs (miRNAs), non-coding small RNA molecules (20-24 nucleotides), are bound to the 3′UTR of the target genes (3). A single miRNA can interact with amounts of mRNA targets to affect the important biological processes (4, 5). MiR-874 overexpression suppressed the
Journal Pre-proof proliferation, migration and invasion of cervical cancer cells (6). MiR-203 also inhibited the cervical cancer cell proliferation, tumor growth and angiogenesis in nude mice (7). However, there are no research about miR-1258 in the field of cervical cancer. MiR-1258 inhibited the proliferation of osteosarcoma cells by targeting AKT3 (8). MiR-1258 suppressed the proliferation of oral squamous cell carcinoma cells and the occurrence of epithelial-mesenchymal transition by targeting SP1 (9). MiR-1258 inhibited the proliferation and cell cycle progression by targeting E2F8 in colorectal cancer (10). Filippova, EA et al (11) identified that miR-1258 was associated with
of
ovarian cancer metastasis and expression of hypermethylated miR-1258 was valuable
ro
for the prediction and personalized treatment of ovarian cancer. MiR-1258 commonly hypermethylated (2852% ) in tumor tissues vs 47% in paired histologically normal
-p
tissues, were identified in a representative set of 54 ovarian cancer samples using
re
methylation-specific PCR. It was shown that miR-1258 was significantly related to
lP
the metastasis of ovarian cancer (12). MiR-1258 was methylated in the majority of tumor specimens compared with adjacent tissues in ovarian cancer, which predicted
na
the prognostic potential (13). MiR-1258 could inhibit the expression and activity of heparanase in brain metastatic breast cancer (BMBC) cells, thereby suppress the cell
Jo ur
invasion and experimental brain metastasis (14, 15).Therefore, we speculated that miR-1258 might also affected the cell biological processes of cervical cancer. E2F1 gene might be related to the susceptibility of cervical cancer (16). Yueli Wu et al (17) found that silencing of MSK2 inhibited the E2F1 expression to decrease the cell proliferation and tumor growth in cervical cancer. E2F1 overexpression potentially leaded to cell transformation, and tumor was more likely happened in E2F1 transgenic mice (18). E2F1 upregulation promoted apoptosis via both p53-dependent and p53-independent pathways (19). E2F-1 is overexpressed in Hodgkin lymphoma (HL) and functional p53 regulated the E2F-1 expression and tumor kinetics in HL (20). X. Wu et al (21) demonstrated that E2F-1 and p53 cooperated to mediate apoptosis. Timmers et al (22) reported that E2F-1 disruption induced the expression of p53-target genes. Nip et al (23) found that E2F-1 suppressed the transcriptional activity of p53. E2F1 regulated the expression of Cyclin
Journal Pre-proof D1 because Cyclin D1 promoter contained an E2F1 consensus site and E2F1 could suppressed Cyclin D1 gene expression (24). Annalisa P et al (25) indicated that uL3 regulated E2F1 expression which in turn controlled the expression of Cyclin D1. The effects of E2F1 on cervical cancer is still unknown. Therefore, the expression and related role of miRNA-1258 and E2F1 in cervical cancer cells was investigated in this study. Materials and methods
of
Cell culture
ro
Normal cervical cell lines Ect1/E6E7 and cervical cell lines (Hela, SiHa, C-33A and Caski) were purchased from American Type Culture Collection (ATCC, Manassas,
-p
VA, USA). All cells were then incubated in DMEM medium containing 10% fetal
re
bovine serum (FBS) in incubators with 5% CO2 at 37 °C.
lP
Xenograft model in vivo
The female nude mice (6-8 weeks) were randomly assigned to five groups, including
na
control group, miR-mimic NC group, miR-1258 mimic group, miR-1258 mimic+NC group and miR-1258 mimic+E2F1 group. The mice were injected subcutaneously
Jo ur
with 2×106 cells per 100 microliters on the right flanks. The tumor size was observed and mice weight was weighed at 1th, 5th, 10th, 15th and 20th Day. RT-qPCR analysis
According to the instructions of TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), total RNA in each cell line was extracted by chloroform-isopropanol method. 200 μL chloroform was added to TRIzol and then the mixture was centrifuged at high speed to obtain the supernatant. Next, the supernatant was added to isopropanol, which centrifuged at high speed to obtain the RNA. 2 μg RNA was reversed into cDNA by using the M-MLV reverse transcriptase system (Promega) and detected by a 2×SYBR Mix and ABI 7500 fast quantitative PCR instrument with the synthesized miR-1258 and E2F1 primers. U6 and GAPDH were
Journal Pre-proof used as internal controls for determining the expression of miR-1258 and E2F1 with the 2−△△Ct method. Western blot analysis Total intracellular protein was extracted with RIPA lysate, which was measured by Bradford assay to determine the protein concentration. 40 μg protein per lane was performed with 10% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Billerica, MA, USA). 5% skim milk was
of
added to the PVDF (polyvinylidene fluoride) membrane for blocking for 1 h. Then 5
ro
ml of different primary antibodies was added to the membranes, which were incubated overnight at 4˚C. The next day, the membranes were incubated with
-p
horseradish peroxidase-linked IgG secondary antibody (Sigma-Aldrich) at room
re
temperature for 1h. The exposal and develop with ECL liquid was carried out. The gray analysis was conducted with ImageLab4.0 software. The primary antibodies
lP
were including anti-E2F1 (ab112580; Abcam; dilution, 1:1000), anti-MMP2 (ab97779; Abcam; dilution, 1:1000), anti-MMP7 (ab5706; Abcam; dilution, 1:1000),
na
anti-MMP9 (ab38898; Abcam; dilution, 1:1000), anti-Bcl2 (ab32124; Abcam; dilution, 1:1000), anti-Bax (ab32503; Abcam; dilution, 1:1000), anti-cleaved caspase3 (ab2302;
Jo ur
Abcam; dilution, 1:1000), anti-caspase3 (ab13847; Abcam; dilution, 1:1000), anti-KI67 (ab16667; Abcam; dilution, 1:1000), anti-p-AKT (#4060; Cell Signaling Technology, Inc.; dilution, 1:2000), anti-cyclinD1 (ab16663; Abcam; dilution, 1:1000), anti-CDK2 (ab32147; Abcam; dilution, 1:1000), anti-P53 (ab32389; Abcam; dilution, 1:1000), anti-AKT (#9272; Cell Signaling Technology, Inc.; dilution, 1:1000). Blots were stripped and reprobed with a GAPDH antibody (ab9485; Abcam; dilution, 1:1000) as a loading control. Dual-luciferase reporter assay miRDB software (http://www.mirdb.org/) predicted that E2F1 was a potential target of miR-1258. The c-33A cells were inoculated into a 96-well plate at the density of 1× 104 cells/well, which was cultured for 24h. The c-33A cells were co-transfected with
Journal Pre-proof pGL3-E2F1 3′UTR plasmid (containing mutant E2F1 3′UTR or wild-type E2F1 3′UTR) and a miR-1258 mimic or miR-control vector using Lipofectamine® 2000 reagent. After 48 h, the cells per hole were washed with PBS for one time and lysed with 25 μL lysates. After 30 min of cell lysis at room temperature, 100 μL firefly luciferase activity detection reagent was added to each hole and multifunctional microplate reader detected the value of each hole. The renilla luciferase activity detection reagent was added to each hole, and the value of each hole was measured by a multifunctional microplate reader. Luciferase activity = firefly luciferase activity/
of
renilla luciferase activity.
ro
Cell transfection
-p
After c-33A cells confluence reached to 80%, miR-1258 mimic, miR-mimic NC,
re
overexpression-E2F1 and overexpression-NC (RiboBio Co., LTD, Guangzhou, China) were transfected into the c-33A cells using Lipofectamine® 2000 reagent (Invitrogen,
na
CCK-8 assay
lP
USA). The c-33A cells in the control group received no treatment.
After transfection, c-33A cells were seeded into a 24-well plate with 1×103 cells per
Jo ur
well. Subsequently, each well was added with 10μL CCK8 solution and then the 24-well plate was further incubated for 4h. The OD value of each well at 450 nm was detected by a multifunctional microplate reader at 24, 48 and 72h. Wound healing assay
The c-33A cells were inoculated into a 6-well plate with 2×104 cells per well, and each well was marked vertically with 200μL sterilizing gun head. The initial distance of scratch was measured under the microscope (0 h), and c-33A cells were incubated in a constant temperature incubator for 72 h. The scratch distance at 72 h was measured again to calculate the cell migration rate. Cell migration rate= (0 h migration distance-72 h migration distance)/0 h migration distance. Transwell assay
Journal Pre-proof Transwell chambers were placed in a 24-well plate and matrigel liquid was applied to the inner surface of the transwell upper chambers. Then, c-33A cells were diluted into suspension with serum-free medium at a density of 2.5×104/ml and 20μL cell suspension was added to each well. At the same time, 500μL DMEM containing 10% FBS was added to lower chambers of transwell. The 24-well plate was cultured in 37 ˚C incubators for 24 h and cells on the inner membrane of upper chamber were removed with cotton swabs. The c-33A cells on the exterior membrane of upper
of
chamber were fixed with formaldehyde and stained with crystal violet. The cell
ro
invasion ability was observed under electron microscope. Flow cytometry analysis
-p
After cell transfection for 48h, c-33A cells were inoculated on 6-well plates with
re
2×104 cells per well. The cells were digested and collected with trypsin and centrifuged at 1000 r/min for 5 min. After washing with PBS twice, cells were
lP
incubated in the darkness with 5μL Annexin V-FITC and 5μL propidium iodide (PI)
h.
Jo ur
Statistical analysis
na
for 30min. Finally, the c-33A cells apoptosis was analyzed by flow cytometry within 1
Each experiment was repeated three times to ensure the reliability of the results. All data were represented by mean ± standard deviation (SD), and statistical analysis was conducted by SPSS 19.0. The independent sample T test compared the differences between the two groups and one-way analysis of variance (ANOVA) compared the differences in the multiple groups. The value of P<0.05 represented the differences between data was statistically significant. Results Expression of miR-1258 and E2F1 in cervical cancer cell lines. RT-qPCR analysis was conducted to determine the expression of miR-1258 in cervical cancer cell lines in which E2F1 expression was measured by RT-qPCR analysis and
Journal Pre-proof Western blot analysis. Results in Fig. 1A showed that miR-1258 expression was obviously decreased in cervical cancer cell lines compared with normal Ect1/E6E7 cells. Results in Fig. 1B and 1C showed that E2F1 expression was significantly higher than that in normal Ect1/E6E7 cells. By comprehensive consideration, c-33A was chosen for the forthcoming experiment. miR-1258 directly targets E2F1. The binding sites of miR-1258 and E2F1 were predicted by a bioinformatics approach
of
(Fig. 2A). Luciferase reporter assays were performed to validate that E2F1 bound to
ro
miR-1258 directly. The luciferase activity was obviously decreased in cells co-transfected with miR-1258 mimic and E2F1-WT compared with cells
-p
co-transfected with miR-control and E2F1-WT. Instead, there was no effect on the
re
luciferase activity in cells transfected with E2F1-MUT (Fig. 2B). The results of RT-qPCR analysis showed that miR-1258 overexpression obviously inhibited the
lP
expression of E2F1 (Fig. 2C). Collectively, the above data demonstrated that
na
miR-1258 directly targeted E2F1 and suppressed the expression of E2F1. miR-1258 overexpression inhibits the proliferation of cervical cancer cells.
Jo ur
RT-qPCR analysis was applied to determine the transfection effects of miR-1258. The results showed that miR-1258 expression was obviously up-regulated in the cells transfected with miR-1258 mimic (Fig. 3A). The transfection effects of E2F1 were determined by RT-qPCR analysis and Western blot analysis and the results showed that E2F1 expression was increased when cells transfected with overexpression-E2F1 plasmids (Fig. 3B and 3C). CCK-8 assays were performed to observe the cell viability, and we observed that cell viability was notably decreased in miR-1258 mimic transfected cells and E2F1 overexpression weakened the inhibition effects of miR-1258 mimic on cell viability (Fig. 3D). miR-1258 overexpression inhibits the invasion and migration of cervical cancer cells. Wound healing assays and transwell assays were conducted (Fig. 4A and 4B). The
Journal Pre-proof ability of cell migration and invasion were the strongest in the control group and cells transfected with miR-mimic-NC. MiR-1258 overexpression leaded to decreased ability of cell migration and invasion. However, E2F1 was discovered partially in antagonizing the inhibition effects of miR-1258 overexpression. The expression of MMP2, MMP7 and MMP9 was found obviously decreased in cells transfected with miR-1258 mimic while slightly increased in cells transfected with miR-1258 mimic and E2F1 (Fig. 4C). Taken together, the above data strongly indicated that miR-1258
of
overexpression inhibited the invasion and migration of cervical cancer cells.
ro
miR-1258 overexpression promotes the apoptosis of cervical cancer cells. The flow cytometry analysis was performed to determine the cell apoptosis and the
-p
results showed that cell apoptosis was obviously inhibited in cells transfected with
re
miR-1258 mimic. However, the inhibition effects of miR-1258 overexpression on the cell apoptosis were weakened in cells transfected with miR-1258 mimic and E2F1
lP
(Fig.5A). Subsequently, we examined the expression of apoptosis-related proteins by Western blot assay. Overexpression of miR-1258 in cells significantly up-regulated
na
the expression of Bax and cleaved caspase3 and down-regulated the Bcl2 expression compared with the control group. And, E2F1 overexpression could slightly antagonize
Jo ur
the effects of miR-1258 overexpression (Fig.5B). miR-1258 overexpression inhibits AKT and activates P53 signaling pathways. The changes of proliferation and apoptosis signaling pathways were determined by Western blot analysis. The expression of p-AKT, cyclinD1 and CDK2 was decreased and p53 expression was increased in cells transfected with miR-1258 mimic. However, overexpression of E2F1 was found to weaken the effects of miR-1258 overexpression (Fig.6). Taken together, miR-1258 overexpression inhibited AKT and activated P53 signaling pathways. miR-1258 overexpression inhibits the development of tumor in vivo. The growth of the tumor and the weight change of the mice affected with miR-1258 mimic and E2F1 were observed in vivo. With the extension of time, the tumor volume
Journal Pre-proof was all gradually increased in all groups while the tumor growth rate in miR-1258 mimic group and miR-1258 mimic+NC group were the lowest. Overexpression of E2F1 increased the tumor growth rate when cells transfected with miR-1258 mimic and E2F1 (Fig.7A). The changes of mice weight showed the same trend with the changes of tumor volume (Fig.7B). The expression of Bax and cleaved caspase3 was increased and the expression of KI67 and Bcl2 was decreased in tumor of miR-1258 mimic group and miR-1258 mimic+NC group compared with the control group and miR-mimic NC group. And, E2F1 overexpression could slightly reverse the effects of
of
miR-1258 overexpression (Fig.7C). Therefore, miR-1258 overexpression changed the
ro
expression of apoptosis-related proteins to suppress the growth of tumor.
-p
miR-1258 overexpression inhibits AKT and activates P53 signaling pathways in
re
vivo.
That miR-1258 overexpression inhibits AKT and activates P53 signaling pathways in
lP
cells was validated in vivo. The expression of p-AKT, cyclinD1 and CDK2 was decreased and P53 expression was increased in tumor of miR-1258 mimic group and
na
miR-1258 mimic+NC group compared with the control group and miR-mimic NC group. However, overexpression of E2F1 was found to weaken the effects of
Jo ur
miR-1258 overexpression (Fig.8). Taken together, miR-1258 overexpression suppressed AKT and promoted P53 signaling pathways in vivo. Discussion
Although researchers gradually understood the potential mechanisms of cervical cancer, the high morbidity and fatality rates were not decreased accordingly (26). In recent years, molecular biology research has been further deepen. MiRNAs become the main targets of new biological targeted therapies for malignant tumors including cervical cancer and rectal cancer (27, 28). It has been reported that miRNAs can regulate the development of tumors by targeting the mRNAs. MiRNAs can be used as tumor suppressor to downregulate the downstream oncogenic target genes, thus further inhibiting the growth of tumors.
Journal Pre-proof Similarly, miRNAs can also be used as tumor promoter to increase the role of oncogenes (29). In the cervical cancer, considerable achievements have been made in the research on miRNAs. Many miRNAs could suppress the cell proliferation, invasion and migration by targeting their downstream oncogenic target genes in cervical cancer (30-32). However, there are no suitable miRNA targets for diagnosis, treatment or prognosis of cervical cancer. MiR-1258 has been demonstrated to generate tumor suppressor activity in a variety of cancers. Liu et al (33) found that miR-1258 could be the key to the treatment of lung cancer metastasis. Shi et al (34)
of
found that miR-1258 could inhibit the invasion and metastasis of gastric cancer by
ro
targeting heparanase. However, research of miR-1258 in cervical cancer still not be existed. Therefore, it is necessary to study the expression, role and mechanism of
-p
miR-1258 in cervical cancer. In this study, we found that miR-1258 expression was
re
down-regulated in cervical cancer cell lines. The up-regulated miR-1258 could inhibit
lP
the proliferation, invasion and migration and promote apoptosis of cervical cancer cells in vitro and also inhibit the proliferation and promote apoptosis of cervical
na
cancer cells in vivo by AKT and P53 signaling pathway. To further investigate the mechanism of miR-1258 in cervical cancer, we used
Jo ur
bioinformatics tools to predict the potential targets of miR-1258. E2F1 was demonstrated to be a potential target of miR-1258. Transcription factor E2F1 is the first protein cloned from the E2F family and plays a key role in cell proliferation, differentiation and apoptosis. E2F1 can induce the rapid transformation of cell cycle from G1 phase to S phase (35-37). In recent years, more and more studies have found that E2F1 is involved in the regulation of multiple malignant tumors. Inhibition of the expression of E2F1 in lymphoma cells caused tumor cell growth retardation and shortened the survival time of tumor (38). Loss of E2F1 could induce Rb deficient epidermal cells to develop into tumor spontaneously (39). E2F1 transcription activated the expression of beta-catenin binding protein 1 (ICAT) in colon cancer cells to negatively regulate the Wnt/beta-catenin signaling pathway (40). We found that E2F1 was up-regulated in cervical cancer cells and E2F1 overexpression could weaken the effects of miR-1258 overexpression for cell proliferation, invasion,
Journal Pre-proof migration and apoptosis by AKT and P53 signaling pathway. In summary, miR-1258 showed low expression in cervical cancer cells, and up-regulated expression of miR-1258 could inhibit proliferation, invasion and migration and promote apoptosis of cervical cancer cells. The function mechanisms of miR-1258 regulating the biological progresses of cervical cancer cells are related to E2F1 and E2F1 overexpression could reverse the role of miR-1258 overexpression in the biological progresses of cervical cancer cells.
of
Funding
ro
Not applicable.
-p
Conflicts of Interest
re
The authors declare they have no competing interests. Reference
3. 4.
5. 6.
7.
8.
9.
lP
na
2.
Ferlay J, I I, Dikshit R, Eser S, Rebelo M, Parkin D, Forman D,Bray F: Cancer Incidence and Mortality Worldwide: sources, methods and major patterns in GLOBOCAN 2012. International Journal of Cancer 136:2014. Berman TA,Schiller JT: Human papillomavirus in cervical cancer and oropharyngeal cancer: One cause, two diseases. Cancer 123:2219, 2017. Carthew RW,Sontheimer EJ: Origins and Mechanisms of miRNAs and siRNAs. Cell 136:642-655, 2009. Marco Antonio VS, Jidong L, Hannon GJ,Roy P: Control of translation and mRNA degradation by miRNAs and siRNAs. Genes & Development 20:515-524, 2006. Bartel DP: MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell 116:281-297, 2004. Liao HH, Pan YF, Pan YF, Shen JJ, Qi Q, Zhong LP, Han WS, Wang Q,Jiang YZ: MicroRNA-874 is downregulated in cervical cancer and inhibits cancer progression by directly targeting ETS1. Oncology Reports 40:2389-2398, 2018. Zhu XY, Er KJ, Mao CY, Yan Q, Xu HJ, Zhang YB, Zhu JH, Cui F, Zhao WX,Shi H: miR-203 Suppresses Tumor Growth and Angiogenesis by Targeting VEGFA in Cervical Cancer. Cell. Physiol. Biochem. 32:64-73, 2013. Liu W, Zhou ZM, Zhang Q, Rong YL, Li LW, Luo YJ, Wang JX, Yin GY, Lv CT,Cai WH: Overexpression of miR-1258 inhibits cell proliferation by targeting AKT3 in osteosarcoma. Biochem. Biophys. Res. Commun. 510:479-486, 2019. Zhang H, Jiang S, Guo L,Li X: MicroRNA‐1258, regulated by c‐Myb, inhibits growth and epithelial ‐ to ‐ mesenchymal transition phenotype via
Jo ur
1.
Journal Pre-proof
10.
11.
16. 17.
18.
19. 20.
21.
lP
na
15.
Jo ur
14.
re
-p
13.
ro
of
12.
targeting SP1 in oral squamous cell carcinoma. Journal of Cellular and Molecular Medicine 23:2019. Zhang ZY, Li J, Huang YJ, Peng W, Qian WW, Gu JO, Wang QY, Hu T, Ji DJ, Ji B, et al: Upregulated miR-1258 regulates cell cycle and inhibits cell proliferation by directly targeting E2F8 in CRC. Cell Proliferation 51:14, 2018. Filippova EA, Loginov VI, Burdennyi AM, Braga EA, Pronina IV, Kazubskaya TP, Kushlinskii DN, Utkin DO, Fridman MV, Khodyrev DS, et al: Hypermethylated Genes of MicroRNA in Ovarian Carcinoma: Metastasis Prediction Marker Systems. Bulletin of Experimental Biology and Medicine 167:79-83, 2019. Loginov VI, Burdennyy AM, Filippova EA, Pronina IV, Kazubskaya TP, Kushlinsky DN, Ermilova VD, Rykov SV, Khodyrev DS,Braga EA: Hypermethylation of miR-107, miR-130b, miR-203a, miR-1258 Genes Associated with Ovarian Cancer Development and Metastasis. Mol. Biol. 52:693-700, 2018. Braga EA, Loginov VI, Burdennyi AM, Filippova EA, Pronina IV, Kurevlev SV, Kazubskaya TP, Kushlinskii DN, Utkin DO, Ermilova VD, et al: Five Hypermethylated MicroRNA Genes as Potential Markers of Ovarian Cancer. Bulletin of Experimental Biology and Medicine 164:351-355, 2018. Tang DB, Zhang QY, Zhao S, Wang JC, Lu KP, Song Y, Zhao L, Kang XM, Wang JX, Xu SQ, et al: The expression and clinical significance of microRNA-1258 and heparanase in human breast cancer. Clinical Biochemistry 46:926-932, 2013. Zhang LX, Sullivan PS, Goodman JC, Gunaratne PH,Marchetti D: MicroRNA-1258 Suppresses Breast Cancer Brain Metastasis by Targeting Heparanase. Cancer Research 71:645-654, 2011. Singh S, Gupta M, Seam RK,Changotra H: E2F1 genetic variants and risk of cervical cancer in Indian women. Int. J. Biol. Markers 33:389-394, 2018. Wu YL, Li HM, Wang H, Zhang FL, Cao H,Xu SY: MSK2 promotes proliferation and tumor formation in squamous cervical cancer via PAX8/RB-E2F1/cyclin A2 axis. Journal of Cellular Biochemistry 120:11432-11440, 2019. Pierce AM, Schneider-Broussard R, ., Gimenez-Conti IB, Russell JL, Conti CJ,Johnson DG: E2F1 has both oncogenic and tumor-suppressive properties in a transgenic model. Molecular & Cellular Biology 19:6408, 1999. Eli B,Doron G: ATM is a target for positive regulation by E2F-1. Oncogene 22:161, 2003. Georgiadi EC, Dimtsas GS, Vassilakopoulos TP, Pangalis GA, Kittas C,Doussis-Anagnostopoulou IA: Functional p53 can modulate the relationship between E2F-1 expression and tumor kinetics in Hodgkin lymphoma. Leukemia & Lymphoma 56:748-754, 2015. X W,AJ L: p53 and E2F-1 cooperate to mediate apoptosis. Proceedings of the National Academy of Sciences of the United States of America 91:3602-3606, 1994.
Journal Pre-proof
Jo ur
na
lP
re
-p
ro
of
22. Timmers C, Sharma N, Opavsky R, Maiti B, Wu L, Wu JJ, Orringer D, Trikha P, Saavedra H,Leone G: E2f1, E2f2, and E2f3 Control E2F Target Expression and Cellular Proliferation via a p53-Dependent Negative Feedback Loop. Molecular and cellular biology 27:65-78, 2007. 23. J N, DK S, CM E, JL C, GP Z,SW H: E2F-1 induces the stabilization of p53 but blocks p53-mediated transactivation. Oncogene 20:910-920, 2001. 24. Klein E,Assoian R: Transcriptional regulation of the cyclin D1 gene at a glance. Journal of cell science 121:3853-3857, 2009. 25. A P, P C, G R,A R: Ribosomal protein uL3 targets E2F1 and Cyclin D1 in cancer cell response to nucleolar stress. Scientific reports 9:15431, 2019. 26. Small W, A Bacon M, Bajaj A, T Chuang L, J Fisher B, M Harkenrider M, Jhingran A, C Kitchener H, R Mileshkin L, Viswanathan A, et al: Cervical cancer: A global health crisis. Cancer 123:2017. 27. Garima S, Pradeep D,Subhash Mohan A: A Comprehensive Review of Dysregulated miRNAs Involved in Cervical Cancer. Current Genomics 15:-, 2014. 28. Eriksen AHM, Andersen RF, Pallisgaard N, Sørensen FB, Jakobsen A,Hansen TF: MicroRNA Expression Profiling to Identify and Validate Reference Genes for the Relative Quantification of microRNA in Rectal Cancer. Plos One 11:e0150593, 2016. 29. Shenouda SK,Alahari SK: MicroRNA function in cancer: oncogene or a tumor suppressor? Cancer & Metastasis Reviews 28:369, 2009. 30. Wang SZ, Gao BH, Yang HL, Liu XJ, Wu X,Wang WJ: MicroRNA-432 is downregulated in cervical cancer and directly targets FN1 to inhibit cell proliferation and invasion. Oncol. Lett. 18:1475-1482, 2019. 31. Sun Y, Cheng Y, Zhang Y,Han K: MicroRNA-889-3p targets FGFR2 to inhibit cervical cancer cell viability and invasion. Exp. Ther. Med. 18:1440-1448, 2019. 32. Feng SJ, Liu W, Sakhautdinova I, Gao B,Tan WH: MicroRNA-505-5p functions as a tumor suppressor by targeting cyclin-dependent kinase 5 in cervical cancer. Bioscience Reports 39:2019. 33. Hongcheng L, Xiaofeng C, Wen G,Gening J: The expression of heparanase and microRNA-1258 in human non-small cell lung cancer. Tumour Biology the Journal of the International Society for Oncodevelopmental Biology & Medicine 33:1327-1334, 2012. 34. Shi J, Chen P, Sun J, Song Y, Ma B, Gao P, Chen X,Wang Z: MicroRNA-1258: An invasion and metastasis regulator that targets heparanase in gastric cancer. Oncol. Lett. 13:3739, 2017. 35. Müller H, ., Bracken AP, Vernell R, ., Moroni MC, Christians F, ., Grassilli E, ., Prosperini E, ., Vigo E, ., Oliner JD,Helin K, . E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis. Genes & Development 15:267, 2001. 36. Yuan H, Jiang F, Wang R, Shen M,Chen N: Lentivirus-mediated RNA interference of E2F-1 suppresses Tca8113 cell proliferation. Molecular medicine reports 5:420-426, 2011.
Journal Pre-proof
of
37. Annette L, Weini S, Helen C, Rachna P, Reena M, J Lewis S, Glen R, Woolley AG, Miller LD,Black MA: YB-1, the E2F pathway, and regulation of tumor cell growth. J Natl Cancer Inst 104:133-146, 2012. 38. Fang H, Gartenhaus RB, Zhao XF, Fang HB, Minkove S, Poss DE,Rapoport AP: c-Myc and E2F1 drive PBK/TOPK expression in high-grade malignant lymphomas. Leuk. Res. 37:447-454, 2013. 39. Costa C, ., Santos M, ., Martínez-Fernández M, ., Due?As M, ., Lorz C, ., García-Escudero R, .,Paramio JM: E2F1 loss induces spontaneous tumour development in Rb-deficient epidermis. Oncogene 32:2937-2951, 2013. 40. Wu Z, Zheng S, Li Z, Tan J,Yu Q: E2F1 suppresses Wnt/beta-catenin activity through transactivation of beta-catenin interacting protein ICAT. Oncogene 30:3979-3984, 2011.
ro
Figure legends
-p
Figure 1. Expression of miR-1258 and E2F1 in cervical cancer cell lines. (A) The
re
miR-1258 expression in normal Ect1/E6E7 cells and cervical cancer cells was detected by RT-qPCR analysis. **P<0.01 and ***P<0.001 vs. Normal group. (B) The
lP
protein level of E2F1 in normal Ect1/E6E7 cells and cervical cancer cells was detected by Western blot analysis. **P<0.01 and ***P<0.001 vs. Normal group. (C)
na
The mRNA level of E2F1 in normal Ect1/E6E7 cells and cervical cancer cells was detected by RT-qPCR analysis. ***P<0.001 vs. Normal group. Assays were
Jo ur
performed in triplicate. ANOVA test was used.
Figure 2. miR-1258 directly targets E2F1. (A) The binding sites of miR-1258 and E2F1 were predicted by miRDB. (B) Luciferase activity was analyzed in cells co-transfected with miR-1258 mimic or miR-control with pmirGLO-E2F1-Wt or pmirGLO-E2F1-Mut. ***P<0.001 vs. E2F1+ miR-control group. (C) E2F1 mRNA expression levels in transfected c-33A cells were analyzed by RT-qPCR analysis. ***P<0.001 vs. control group.
###
P<0.001 vs. miR-mimic NC group. Assays were
performed in triplicate. Independent sample T test and ANOVA test was used.
Figure 3. miR-1258 overexpression inhibits the proliferation of cervical cancer cells. (A) miR-1258 expression levels in transfected c-33A cells were analyzed by RT-qPCR
Journal Pre-proof analysis. ***P<0.001 vs. Control group. ###P<0.001 vs. miR-mimic NC group. (B) The protein level of E2F1 in transfected c-33A cells were analyzed by Western blot analysis. *P<0.05 vs. Control group. #P<0.05 vs. Overexpression-NC group. (C) The mRNA level of E2F1 in transfected c-33A cells were analyzed by RT-qPCR analysis. *P<0.05 vs. Control group. #P<0.05 vs. Overexpression-NC group. (D) The cell viability of transfected c-33A cells was analyzed by CCK-8 assay. ***P<0.001 vs. Control group.
P<0.001 vs. miR-mimic NC group. ∆P<0.05 and
###
∆∆
P<0.01 vs.
of
miR-1258 mimic group. Assays were performed in triplicate. ANOVA test was used.
ro
Figure 4. miR-1258 overexpression inhibits the invasion and migration of cervical cancer cells. (A) The migration of transfected c-33A cells was showed by wound ###
P<0.001 vs. miR-mimic NC group.
-p
healing assay. ***P<0.001 vs. Control group. ∆∆
re
P<0.01 vs. miR-1258 mimic group. (B) The invasion of transfected c-33A cells was
showed by transwell assay. *P<0.05 and ***P<0.001 vs. Control group. ###P<0.001 vs.
lP
miR-mimic NC group. ∆P<0.05 vs. miR-1258 mimic group. (C) The protein level of
na
MMP2, MMP7 and MMP9 in transfected c-33A cells were analyzed by Western blot analysis. *P<0.05, **P<0.01 and ***P<0.001 vs. Control group.
P<0.001 vs.
∆∆∆
P<0.001 vs. miR-1258 mimic group. Assays
Jo ur
miR-mimic NC group. ∆P<0.05 and
###
were performed in triplicate. ANOVA test was used.
Figure 5. miR-1258 overexpression promotes the apoptosis of cervical cancer cells. (A) The cell apoptosis of transfected c-33A cells was detected by flow cytometry analysis. ***P<0.001 vs. Control group.
###
P<0.001 vs. miR-mimic NC group.
∆∆∆
P<0.001 vs. miR-1258 mimic group. (B) The protein level of apoptosis related
proteins was analyzed by Western blot analysis. **P<0.01 and ***P<0.001 vs. Control group. ###P<0.001 vs. miR-mimic NC group. ∆∆∆P<0.001 vs. miR-1258 mimic group. Assays were performed in triplicate. ANOVA test was used.
Figure 6. miR-1258 overexpression inhibits AKT and activates P53 signaling pathways. The protein level of p-AKT, AKT, CDK2, p53 and CyclinD1 was detected
Journal Pre-proof by Western blot analysis. **P<0.01 and ***P<0.001 vs. Control group. ###P<0.001 vs. miR-mimic NC group. ∆P<0.05 and
∆∆∆
P<0.001 vs. miR-1258 mimic group. Assays
were performed in triplicate. ANOVA test was used.
Figure 7. miR-1258 overexpression inhibits the development of tumor in vivo. (A) Photographs of tumors were obtained from different groups of transfected mice and growth curve of tumor volumes was calculated. *P<0.05 and ***P<0.001 vs. Control group. ###P<0.001 vs. miR-mimic NC group.
∆∆∆
P<0.001 vs. miR-1258 mimic group.
of
(B) The weight of tumors was measured. *P<0.05, **P<0.01 and ***P<0.001 vs.
ro
Control group. ##P<0.01 vs. miR-mimic NC group. (C) The protein level of apoptosis related proteins in tumor tissues was analyzed by Western blot analysis. **P<0.01 and ###
P<0.001 vs. miR-mimic NC group. ∆P<0.05 vs.
-p
***P<0.001 vs. Control group.
lP
re
miR-1258 mimic group. Assays were performed in triplicate. ANOVA test was used.
Figure 8. miR-1258 overexpression inhibits AKT and activates P53 signaling
na
pathways in vivo. The protein level of p-AKT, AKT, CDK2, p53 and CyclinD1 in tumor tissues was detected by Western blot analysis. **P<0.01 and ***P<0.001 vs.
∆∆∆
###
P<0.001 vs. miR-mimic NC group.
Jo ur
Control group.
∆
P<0.05,
∆∆
P<0.01 and
P<0.001 vs. miR-1258 mimic group. Assays were performed in triplicate. ANOVA
test was used.
Journal Pre-proof
Dear editor， On behalf of the co-authors, I am now submitting the a revised version of the manuscript entitled “miR-1258 promotes the apoptosis of cervical cancer cells by regulating the E2F1/P53 signaling pathway” to Experimental and Molecular Pathology. We have carefully revised the manuscript according to the reviewers comments
of
and hope this revision can meet the requirements of reviewers.
Yours sincerely,
ro
Chunyan Cai, Department of Gynaecology, The Affiliated Huaian No.1 People’s
-p
Hospital of Nanjing Medical University, 6 Beijing Road West, Huaian, Jiangsu
Jo ur
na
lP
re
Province, 223300, China. E-mail: [email protected]
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Xing Peng, Yumei Zhang, Jinyu Gao, Chunyan Cai PII:
S0014-4800(19)30719-1
DOI:
https://doi.org/10.1016/j.yexmp.2020.104368
Reference:
YEXMP 104368
To appear in:
Experimental and Molecular Pathology
Received date:
18 September 2019
Revised date:
11 December 2019
Accepted date:
3 January 2020
Please cite this article as: X. Peng, Y. Zhang, J. Gao, et al., MiR-1258 promotes the apoptosis of cervical cancer cells by regulating the E2F1/P53 signaling pathway, Experimental and Molecular Pathology(2019), https://doi.org/10.1016/ j.yexmp.2020.104368
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof MiR-1258 promotes the apoptosis of cervical cancer cells by regulating the E2F1/P53 signaling pathway
Xing Peng, Yumei Zhang, Jinyu Gao, Chunyan Cai
Department of Gynaecology, The Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University, Huaian, Jiangsu, China
of
Correspondence to: Chunyan Cai, Department of Gynaecology, The Affiliated Huaian
ro
No.1 People’s Hospital of Nanjing Medical University, 6 Beijing Road West, Huaian,
-p
Jiangsu Province, 223300, China. E-mail: [email protected]
lP
re
Key words: miR-1258, apoptosis, cervical cancer, E2F1/P53 signaling pathway
Abstract.
na
Running title: Role of miR-1258 in cervical cancer
Jo ur
Objective: Cervical cancer is the most common malignant tumor in gynecology with high mortality. MiRNA has been reported to regulate cell biological processes in cervical cancer. This study aimed to explore the expression of miR-1258 and role of miR-1258 by targeting E2F1 in cervical cancer cells. Methods: The expression of miR-1258 and E2F1 in cervical cancer cells and transfection effects was determined by RT-qPCR analysis. The expression of E2F1, MMP2, MMP7, MMP9, Bcl2, Bax, cleaved caspase3, caspase3, KI67, p-AKT, cyclinD1, CDK2, P53 and AKT in cervical cancer cells was detected by western blot analysis. The proliferation, invasion, migration and apoptosis were respectively analyzed by CCK-8 assay, transwell assay, wound healing assay and flow cytometry analysis. E2F1 was a potential target of miR-1258, which demonstrated by a dual-luciferase reporter assay. Results: miR-1258 expression was decreased while E2F1 expression was increased in
Journal Pre-proof cervical cancer cells. MiR-1258 overexpression could down-regulate the E2F1 expression. Overexpression of miR-1258 inhibited the proliferation, invasion and migration and promoted the apoptosis of cervical cancer cells by AKT and P53 signal pathway. And, Overexpression of miR-1258 also suppressed the tumor growth by AKT and P53 signal pathway. Overexpression of E2F1 reduced the inhibition effects of miR-1258 in cervical cancer. Conclusion: Taken together, miR-1258 overexpression exerts its inhibition effects on the proliferation, invasion and migration and promotion effects on the apoptosis of
of
cervical cancer cells by targeting the E2F1, which might provide new ideas for
ro
clinical treatment of cervical cancer.
-p
Introduction
re
Cervical cancer is the second most common female malignancy, with more than 1.5 million new cases diagnosed each year and more than 300,000 deaths (1). The
lP
main cause of cervical cancer such as human papillomavirus (HPV) infection has been identified, so early implementation of surgery combined with chemotherapy can
na
significantly improve the survival rate of patients. However, clinical studies have found that for patients with advanced cervical cancer, intra-pelvic lymph node
Jo ur
metastasis is the main factor affecting the prognosis of patients (2). In addition, the 5-year survival rate of cervical cancer is below 40%, and poor prognosis of cervical cancer is a major public health problem, leading to high hospital costs. Therefore, in-depth study of the molecular mechanism of the occurrence and development of cervical cancer and search for effective molecular markers are conducive to explore methods that can block the proliferation and metastasis of tumor cells and promote the apoptosis of tumor cells. In recent years, more attention has been paid to the role of miRNA in the occurrence, development and metastasis of cervical cancer. MicroRNAs (miRNAs), non-coding small RNA molecules (20-24 nucleotides), are bound to the 3′UTR of the target genes (3). A single miRNA can interact with amounts of mRNA targets to affect the important biological processes (4, 5). MiR-874 overexpression suppressed the
Journal Pre-proof proliferation, migration and invasion of cervical cancer cells (6). MiR-203 also inhibited the cervical cancer cell proliferation, tumor growth and angiogenesis in nude mice (7). However, there are no research about miR-1258 in the field of cervical cancer. MiR-1258 inhibited the proliferation of osteosarcoma cells by targeting AKT3 (8). MiR-1258 suppressed the proliferation of oral squamous cell carcinoma cells and the occurrence of epithelial-mesenchymal transition by targeting SP1 (9). MiR-1258 inhibited the proliferation and cell cycle progression by targeting E2F8 in colorectal cancer (10). Filippova, EA et al (11) identified that miR-1258 was associated with
of
ovarian cancer metastasis and expression of hypermethylated miR-1258 was valuable
ro
for the prediction and personalized treatment of ovarian cancer. MiR-1258 commonly hypermethylated (2852% ) in tumor tissues vs 47% in paired histologically normal
-p
tissues, were identified in a representative set of 54 ovarian cancer samples using
re
methylation-specific PCR. It was shown that miR-1258 was significantly related to
lP
the metastasis of ovarian cancer (12). MiR-1258 was methylated in the majority of tumor specimens compared with adjacent tissues in ovarian cancer, which predicted
na
the prognostic potential (13). MiR-1258 could inhibit the expression and activity of heparanase in brain metastatic breast cancer (BMBC) cells, thereby suppress the cell
Jo ur
invasion and experimental brain metastasis (14, 15).Therefore, we speculated that miR-1258 might also affected the cell biological processes of cervical cancer. E2F1 gene might be related to the susceptibility of cervical cancer (16). Yueli Wu et al (17) found that silencing of MSK2 inhibited the E2F1 expression to decrease the cell proliferation and tumor growth in cervical cancer. E2F1 overexpression potentially leaded to cell transformation, and tumor was more likely happened in E2F1 transgenic mice (18). E2F1 upregulation promoted apoptosis via both p53-dependent and p53-independent pathways (19). E2F-1 is overexpressed in Hodgkin lymphoma (HL) and functional p53 regulated the E2F-1 expression and tumor kinetics in HL (20). X. Wu et al (21) demonstrated that E2F-1 and p53 cooperated to mediate apoptosis. Timmers et al (22) reported that E2F-1 disruption induced the expression of p53-target genes. Nip et al (23) found that E2F-1 suppressed the transcriptional activity of p53. E2F1 regulated the expression of Cyclin
Journal Pre-proof D1 because Cyclin D1 promoter contained an E2F1 consensus site and E2F1 could suppressed Cyclin D1 gene expression (24). Annalisa P et al (25) indicated that uL3 regulated E2F1 expression which in turn controlled the expression of Cyclin D1. The effects of E2F1 on cervical cancer is still unknown. Therefore, the expression and related role of miRNA-1258 and E2F1 in cervical cancer cells was investigated in this study. Materials and methods
of
Cell culture
ro
Normal cervical cell lines Ect1/E6E7 and cervical cell lines (Hela, SiHa, C-33A and Caski) were purchased from American Type Culture Collection (ATCC, Manassas,
-p
VA, USA). All cells were then incubated in DMEM medium containing 10% fetal
re
bovine serum (FBS) in incubators with 5% CO2 at 37 °C.
lP
Xenograft model in vivo
The female nude mice (6-8 weeks) were randomly assigned to five groups, including
na
control group, miR-mimic NC group, miR-1258 mimic group, miR-1258 mimic+NC group and miR-1258 mimic+E2F1 group. The mice were injected subcutaneously
Jo ur
with 2×106 cells per 100 microliters on the right flanks. The tumor size was observed and mice weight was weighed at 1th, 5th, 10th, 15th and 20th Day. RT-qPCR analysis
According to the instructions of TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), total RNA in each cell line was extracted by chloroform-isopropanol method. 200 μL chloroform was added to TRIzol and then the mixture was centrifuged at high speed to obtain the supernatant. Next, the supernatant was added to isopropanol, which centrifuged at high speed to obtain the RNA. 2 μg RNA was reversed into cDNA by using the M-MLV reverse transcriptase system (Promega) and detected by a 2×SYBR Mix and ABI 7500 fast quantitative PCR instrument with the synthesized miR-1258 and E2F1 primers. U6 and GAPDH were
Journal Pre-proof used as internal controls for determining the expression of miR-1258 and E2F1 with the 2−△△Ct method. Western blot analysis Total intracellular protein was extracted with RIPA lysate, which was measured by Bradford assay to determine the protein concentration. 40 μg protein per lane was performed with 10% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Billerica, MA, USA). 5% skim milk was
of
added to the PVDF (polyvinylidene fluoride) membrane for blocking for 1 h. Then 5
ro
ml of different primary antibodies was added to the membranes, which were incubated overnight at 4˚C. The next day, the membranes were incubated with
-p
horseradish peroxidase-linked IgG secondary antibody (Sigma-Aldrich) at room
re
temperature for 1h. The exposal and develop with ECL liquid was carried out. The gray analysis was conducted with ImageLab4.0 software. The primary antibodies
lP
were including anti-E2F1 (ab112580; Abcam; dilution, 1:1000), anti-MMP2 (ab97779; Abcam; dilution, 1:1000), anti-MMP7 (ab5706; Abcam; dilution, 1:1000),
na
anti-MMP9 (ab38898; Abcam; dilution, 1:1000), anti-Bcl2 (ab32124; Abcam; dilution, 1:1000), anti-Bax (ab32503; Abcam; dilution, 1:1000), anti-cleaved caspase3 (ab2302;
Jo ur
Abcam; dilution, 1:1000), anti-caspase3 (ab13847; Abcam; dilution, 1:1000), anti-KI67 (ab16667; Abcam; dilution, 1:1000), anti-p-AKT (#4060; Cell Signaling Technology, Inc.; dilution, 1:2000), anti-cyclinD1 (ab16663; Abcam; dilution, 1:1000), anti-CDK2 (ab32147; Abcam; dilution, 1:1000), anti-P53 (ab32389; Abcam; dilution, 1:1000), anti-AKT (#9272; Cell Signaling Technology, Inc.; dilution, 1:1000). Blots were stripped and reprobed with a GAPDH antibody (ab9485; Abcam; dilution, 1:1000) as a loading control. Dual-luciferase reporter assay miRDB software (http://www.mirdb.org/) predicted that E2F1 was a potential target of miR-1258. The c-33A cells were inoculated into a 96-well plate at the density of 1× 104 cells/well, which was cultured for 24h. The c-33A cells were co-transfected with
Journal Pre-proof pGL3-E2F1 3′UTR plasmid (containing mutant E2F1 3′UTR or wild-type E2F1 3′UTR) and a miR-1258 mimic or miR-control vector using Lipofectamine® 2000 reagent. After 48 h, the cells per hole were washed with PBS for one time and lysed with 25 μL lysates. After 30 min of cell lysis at room temperature, 100 μL firefly luciferase activity detection reagent was added to each hole and multifunctional microplate reader detected the value of each hole. The renilla luciferase activity detection reagent was added to each hole, and the value of each hole was measured by a multifunctional microplate reader. Luciferase activity = firefly luciferase activity/
of
renilla luciferase activity.
ro
Cell transfection
-p
After c-33A cells confluence reached to 80%, miR-1258 mimic, miR-mimic NC,
re
overexpression-E2F1 and overexpression-NC (RiboBio Co., LTD, Guangzhou, China) were transfected into the c-33A cells using Lipofectamine® 2000 reagent (Invitrogen,
na
CCK-8 assay
lP
USA). The c-33A cells in the control group received no treatment.
After transfection, c-33A cells were seeded into a 24-well plate with 1×103 cells per
Jo ur
well. Subsequently, each well was added with 10μL CCK8 solution and then the 24-well plate was further incubated for 4h. The OD value of each well at 450 nm was detected by a multifunctional microplate reader at 24, 48 and 72h. Wound healing assay
The c-33A cells were inoculated into a 6-well plate with 2×104 cells per well, and each well was marked vertically with 200μL sterilizing gun head. The initial distance of scratch was measured under the microscope (0 h), and c-33A cells were incubated in a constant temperature incubator for 72 h. The scratch distance at 72 h was measured again to calculate the cell migration rate. Cell migration rate= (0 h migration distance-72 h migration distance)/0 h migration distance. Transwell assay
Journal Pre-proof Transwell chambers were placed in a 24-well plate and matrigel liquid was applied to the inner surface of the transwell upper chambers. Then, c-33A cells were diluted into suspension with serum-free medium at a density of 2.5×104/ml and 20μL cell suspension was added to each well. At the same time, 500μL DMEM containing 10% FBS was added to lower chambers of transwell. The 24-well plate was cultured in 37 ˚C incubators for 24 h and cells on the inner membrane of upper chamber were removed with cotton swabs. The c-33A cells on the exterior membrane of upper
of
chamber were fixed with formaldehyde and stained with crystal violet. The cell
ro
invasion ability was observed under electron microscope. Flow cytometry analysis
-p
After cell transfection for 48h, c-33A cells were inoculated on 6-well plates with
re
2×104 cells per well. The cells were digested and collected with trypsin and centrifuged at 1000 r/min for 5 min. After washing with PBS twice, cells were
lP
incubated in the darkness with 5μL Annexin V-FITC and 5μL propidium iodide (PI)
h.
Jo ur
Statistical analysis
na
for 30min. Finally, the c-33A cells apoptosis was analyzed by flow cytometry within 1
Each experiment was repeated three times to ensure the reliability of the results. All data were represented by mean ± standard deviation (SD), and statistical analysis was conducted by SPSS 19.0. The independent sample T test compared the differences between the two groups and one-way analysis of variance (ANOVA) compared the differences in the multiple groups. The value of P<0.05 represented the differences between data was statistically significant. Results Expression of miR-1258 and E2F1 in cervical cancer cell lines. RT-qPCR analysis was conducted to determine the expression of miR-1258 in cervical cancer cell lines in which E2F1 expression was measured by RT-qPCR analysis and
Journal Pre-proof Western blot analysis. Results in Fig. 1A showed that miR-1258 expression was obviously decreased in cervical cancer cell lines compared with normal Ect1/E6E7 cells. Results in Fig. 1B and 1C showed that E2F1 expression was significantly higher than that in normal Ect1/E6E7 cells. By comprehensive consideration, c-33A was chosen for the forthcoming experiment. miR-1258 directly targets E2F1. The binding sites of miR-1258 and E2F1 were predicted by a bioinformatics approach
of
(Fig. 2A). Luciferase reporter assays were performed to validate that E2F1 bound to
ro
miR-1258 directly. The luciferase activity was obviously decreased in cells co-transfected with miR-1258 mimic and E2F1-WT compared with cells
-p
co-transfected with miR-control and E2F1-WT. Instead, there was no effect on the
re
luciferase activity in cells transfected with E2F1-MUT (Fig. 2B). The results of RT-qPCR analysis showed that miR-1258 overexpression obviously inhibited the
lP
expression of E2F1 (Fig. 2C). Collectively, the above data demonstrated that
na
miR-1258 directly targeted E2F1 and suppressed the expression of E2F1. miR-1258 overexpression inhibits the proliferation of cervical cancer cells.
Jo ur
RT-qPCR analysis was applied to determine the transfection effects of miR-1258. The results showed that miR-1258 expression was obviously up-regulated in the cells transfected with miR-1258 mimic (Fig. 3A). The transfection effects of E2F1 were determined by RT-qPCR analysis and Western blot analysis and the results showed that E2F1 expression was increased when cells transfected with overexpression-E2F1 plasmids (Fig. 3B and 3C). CCK-8 assays were performed to observe the cell viability, and we observed that cell viability was notably decreased in miR-1258 mimic transfected cells and E2F1 overexpression weakened the inhibition effects of miR-1258 mimic on cell viability (Fig. 3D). miR-1258 overexpression inhibits the invasion and migration of cervical cancer cells. Wound healing assays and transwell assays were conducted (Fig. 4A and 4B). The
Journal Pre-proof ability of cell migration and invasion were the strongest in the control group and cells transfected with miR-mimic-NC. MiR-1258 overexpression leaded to decreased ability of cell migration and invasion. However, E2F1 was discovered partially in antagonizing the inhibition effects of miR-1258 overexpression. The expression of MMP2, MMP7 and MMP9 was found obviously decreased in cells transfected with miR-1258 mimic while slightly increased in cells transfected with miR-1258 mimic and E2F1 (Fig. 4C). Taken together, the above data strongly indicated that miR-1258
of
overexpression inhibited the invasion and migration of cervical cancer cells.
ro
miR-1258 overexpression promotes the apoptosis of cervical cancer cells. The flow cytometry analysis was performed to determine the cell apoptosis and the
-p
results showed that cell apoptosis was obviously inhibited in cells transfected with
re
miR-1258 mimic. However, the inhibition effects of miR-1258 overexpression on the cell apoptosis were weakened in cells transfected with miR-1258 mimic and E2F1
lP
(Fig.5A). Subsequently, we examined the expression of apoptosis-related proteins by Western blot assay. Overexpression of miR-1258 in cells significantly up-regulated
na
the expression of Bax and cleaved caspase3 and down-regulated the Bcl2 expression compared with the control group. And, E2F1 overexpression could slightly antagonize
Jo ur
the effects of miR-1258 overexpression (Fig.5B). miR-1258 overexpression inhibits AKT and activates P53 signaling pathways. The changes of proliferation and apoptosis signaling pathways were determined by Western blot analysis. The expression of p-AKT, cyclinD1 and CDK2 was decreased and p53 expression was increased in cells transfected with miR-1258 mimic. However, overexpression of E2F1 was found to weaken the effects of miR-1258 overexpression (Fig.6). Taken together, miR-1258 overexpression inhibited AKT and activated P53 signaling pathways. miR-1258 overexpression inhibits the development of tumor in vivo. The growth of the tumor and the weight change of the mice affected with miR-1258 mimic and E2F1 were observed in vivo. With the extension of time, the tumor volume
Journal Pre-proof was all gradually increased in all groups while the tumor growth rate in miR-1258 mimic group and miR-1258 mimic+NC group were the lowest. Overexpression of E2F1 increased the tumor growth rate when cells transfected with miR-1258 mimic and E2F1 (Fig.7A). The changes of mice weight showed the same trend with the changes of tumor volume (Fig.7B). The expression of Bax and cleaved caspase3 was increased and the expression of KI67 and Bcl2 was decreased in tumor of miR-1258 mimic group and miR-1258 mimic+NC group compared with the control group and miR-mimic NC group. And, E2F1 overexpression could slightly reverse the effects of
of
miR-1258 overexpression (Fig.7C). Therefore, miR-1258 overexpression changed the
ro
expression of apoptosis-related proteins to suppress the growth of tumor.
-p
miR-1258 overexpression inhibits AKT and activates P53 signaling pathways in
re
vivo.
That miR-1258 overexpression inhibits AKT and activates P53 signaling pathways in
lP
cells was validated in vivo. The expression of p-AKT, cyclinD1 and CDK2 was decreased and P53 expression was increased in tumor of miR-1258 mimic group and
na
miR-1258 mimic+NC group compared with the control group and miR-mimic NC group. However, overexpression of E2F1 was found to weaken the effects of
Jo ur
miR-1258 overexpression (Fig.8). Taken together, miR-1258 overexpression suppressed AKT and promoted P53 signaling pathways in vivo. Discussion
Although researchers gradually understood the potential mechanisms of cervical cancer, the high morbidity and fatality rates were not decreased accordingly (26). In recent years, molecular biology research has been further deepen. MiRNAs become the main targets of new biological targeted therapies for malignant tumors including cervical cancer and rectal cancer (27, 28). It has been reported that miRNAs can regulate the development of tumors by targeting the mRNAs. MiRNAs can be used as tumor suppressor to downregulate the downstream oncogenic target genes, thus further inhibiting the growth of tumors.
Journal Pre-proof Similarly, miRNAs can also be used as tumor promoter to increase the role of oncogenes (29). In the cervical cancer, considerable achievements have been made in the research on miRNAs. Many miRNAs could suppress the cell proliferation, invasion and migration by targeting their downstream oncogenic target genes in cervical cancer (30-32). However, there are no suitable miRNA targets for diagnosis, treatment or prognosis of cervical cancer. MiR-1258 has been demonstrated to generate tumor suppressor activity in a variety of cancers. Liu et al (33) found that miR-1258 could be the key to the treatment of lung cancer metastasis. Shi et al (34)
of
found that miR-1258 could inhibit the invasion and metastasis of gastric cancer by
ro
targeting heparanase. However, research of miR-1258 in cervical cancer still not be existed. Therefore, it is necessary to study the expression, role and mechanism of
-p
miR-1258 in cervical cancer. In this study, we found that miR-1258 expression was
re
down-regulated in cervical cancer cell lines. The up-regulated miR-1258 could inhibit
lP
the proliferation, invasion and migration and promote apoptosis of cervical cancer cells in vitro and also inhibit the proliferation and promote apoptosis of cervical
na
cancer cells in vivo by AKT and P53 signaling pathway. To further investigate the mechanism of miR-1258 in cervical cancer, we used
Jo ur
bioinformatics tools to predict the potential targets of miR-1258. E2F1 was demonstrated to be a potential target of miR-1258. Transcription factor E2F1 is the first protein cloned from the E2F family and plays a key role in cell proliferation, differentiation and apoptosis. E2F1 can induce the rapid transformation of cell cycle from G1 phase to S phase (35-37). In recent years, more and more studies have found that E2F1 is involved in the regulation of multiple malignant tumors. Inhibition of the expression of E2F1 in lymphoma cells caused tumor cell growth retardation and shortened the survival time of tumor (38). Loss of E2F1 could induce Rb deficient epidermal cells to develop into tumor spontaneously (39). E2F1 transcription activated the expression of beta-catenin binding protein 1 (ICAT) in colon cancer cells to negatively regulate the Wnt/beta-catenin signaling pathway (40). We found that E2F1 was up-regulated in cervical cancer cells and E2F1 overexpression could weaken the effects of miR-1258 overexpression for cell proliferation, invasion,
Journal Pre-proof migration and apoptosis by AKT and P53 signaling pathway. In summary, miR-1258 showed low expression in cervical cancer cells, and up-regulated expression of miR-1258 could inhibit proliferation, invasion and migration and promote apoptosis of cervical cancer cells. The function mechanisms of miR-1258 regulating the biological progresses of cervical cancer cells are related to E2F1 and E2F1 overexpression could reverse the role of miR-1258 overexpression in the biological progresses of cervical cancer cells.
of
Funding
ro
Not applicable.
-p
Conflicts of Interest
re
The authors declare they have no competing interests. Reference
3. 4.
5. 6.
7.
8.
9.
lP
na
2.
Ferlay J, I I, Dikshit R, Eser S, Rebelo M, Parkin D, Forman D,Bray F: Cancer Incidence and Mortality Worldwide: sources, methods and major patterns in GLOBOCAN 2012. International Journal of Cancer 136:2014. Berman TA,Schiller JT: Human papillomavirus in cervical cancer and oropharyngeal cancer: One cause, two diseases. Cancer 123:2219, 2017. Carthew RW,Sontheimer EJ: Origins and Mechanisms of miRNAs and siRNAs. Cell 136:642-655, 2009. Marco Antonio VS, Jidong L, Hannon GJ,Roy P: Control of translation and mRNA degradation by miRNAs and siRNAs. Genes & Development 20:515-524, 2006. Bartel DP: MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell 116:281-297, 2004. Liao HH, Pan YF, Pan YF, Shen JJ, Qi Q, Zhong LP, Han WS, Wang Q,Jiang YZ: MicroRNA-874 is downregulated in cervical cancer and inhibits cancer progression by directly targeting ETS1. Oncology Reports 40:2389-2398, 2018. Zhu XY, Er KJ, Mao CY, Yan Q, Xu HJ, Zhang YB, Zhu JH, Cui F, Zhao WX,Shi H: miR-203 Suppresses Tumor Growth and Angiogenesis by Targeting VEGFA in Cervical Cancer. Cell. Physiol. Biochem. 32:64-73, 2013. Liu W, Zhou ZM, Zhang Q, Rong YL, Li LW, Luo YJ, Wang JX, Yin GY, Lv CT,Cai WH: Overexpression of miR-1258 inhibits cell proliferation by targeting AKT3 in osteosarcoma. Biochem. Biophys. Res. Commun. 510:479-486, 2019. Zhang H, Jiang S, Guo L,Li X: MicroRNA‐1258, regulated by c‐Myb, inhibits growth and epithelial ‐ to ‐ mesenchymal transition phenotype via
Jo ur
1.
Journal Pre-proof
10.
11.
16. 17.
18.
19. 20.
21.
lP
na
15.
Jo ur
14.
re
-p
13.
ro
of
12.
targeting SP1 in oral squamous cell carcinoma. Journal of Cellular and Molecular Medicine 23:2019. Zhang ZY, Li J, Huang YJ, Peng W, Qian WW, Gu JO, Wang QY, Hu T, Ji DJ, Ji B, et al: Upregulated miR-1258 regulates cell cycle and inhibits cell proliferation by directly targeting E2F8 in CRC. Cell Proliferation 51:14, 2018. Filippova EA, Loginov VI, Burdennyi AM, Braga EA, Pronina IV, Kazubskaya TP, Kushlinskii DN, Utkin DO, Fridman MV, Khodyrev DS, et al: Hypermethylated Genes of MicroRNA in Ovarian Carcinoma: Metastasis Prediction Marker Systems. Bulletin of Experimental Biology and Medicine 167:79-83, 2019. Loginov VI, Burdennyy AM, Filippova EA, Pronina IV, Kazubskaya TP, Kushlinsky DN, Ermilova VD, Rykov SV, Khodyrev DS,Braga EA: Hypermethylation of miR-107, miR-130b, miR-203a, miR-1258 Genes Associated with Ovarian Cancer Development and Metastasis. Mol. Biol. 52:693-700, 2018. Braga EA, Loginov VI, Burdennyi AM, Filippova EA, Pronina IV, Kurevlev SV, Kazubskaya TP, Kushlinskii DN, Utkin DO, Ermilova VD, et al: Five Hypermethylated MicroRNA Genes as Potential Markers of Ovarian Cancer. Bulletin of Experimental Biology and Medicine 164:351-355, 2018. Tang DB, Zhang QY, Zhao S, Wang JC, Lu KP, Song Y, Zhao L, Kang XM, Wang JX, Xu SQ, et al: The expression and clinical significance of microRNA-1258 and heparanase in human breast cancer. Clinical Biochemistry 46:926-932, 2013. Zhang LX, Sullivan PS, Goodman JC, Gunaratne PH,Marchetti D: MicroRNA-1258 Suppresses Breast Cancer Brain Metastasis by Targeting Heparanase. Cancer Research 71:645-654, 2011. Singh S, Gupta M, Seam RK,Changotra H: E2F1 genetic variants and risk of cervical cancer in Indian women. Int. J. Biol. Markers 33:389-394, 2018. Wu YL, Li HM, Wang H, Zhang FL, Cao H,Xu SY: MSK2 promotes proliferation and tumor formation in squamous cervical cancer via PAX8/RB-E2F1/cyclin A2 axis. Journal of Cellular Biochemistry 120:11432-11440, 2019. Pierce AM, Schneider-Broussard R, ., Gimenez-Conti IB, Russell JL, Conti CJ,Johnson DG: E2F1 has both oncogenic and tumor-suppressive properties in a transgenic model. Molecular & Cellular Biology 19:6408, 1999. Eli B,Doron G: ATM is a target for positive regulation by E2F-1. Oncogene 22:161, 2003. Georgiadi EC, Dimtsas GS, Vassilakopoulos TP, Pangalis GA, Kittas C,Doussis-Anagnostopoulou IA: Functional p53 can modulate the relationship between E2F-1 expression and tumor kinetics in Hodgkin lymphoma. Leukemia & Lymphoma 56:748-754, 2015. X W,AJ L: p53 and E2F-1 cooperate to mediate apoptosis. Proceedings of the National Academy of Sciences of the United States of America 91:3602-3606, 1994.
Journal Pre-proof
Jo ur
na
lP
re
-p
ro
of
22. Timmers C, Sharma N, Opavsky R, Maiti B, Wu L, Wu JJ, Orringer D, Trikha P, Saavedra H,Leone G: E2f1, E2f2, and E2f3 Control E2F Target Expression and Cellular Proliferation via a p53-Dependent Negative Feedback Loop. Molecular and cellular biology 27:65-78, 2007. 23. J N, DK S, CM E, JL C, GP Z,SW H: E2F-1 induces the stabilization of p53 but blocks p53-mediated transactivation. Oncogene 20:910-920, 2001. 24. Klein E,Assoian R: Transcriptional regulation of the cyclin D1 gene at a glance. Journal of cell science 121:3853-3857, 2009. 25. A P, P C, G R,A R: Ribosomal protein uL3 targets E2F1 and Cyclin D1 in cancer cell response to nucleolar stress. Scientific reports 9:15431, 2019. 26. Small W, A Bacon M, Bajaj A, T Chuang L, J Fisher B, M Harkenrider M, Jhingran A, C Kitchener H, R Mileshkin L, Viswanathan A, et al: Cervical cancer: A global health crisis. Cancer 123:2017. 27. Garima S, Pradeep D,Subhash Mohan A: A Comprehensive Review of Dysregulated miRNAs Involved in Cervical Cancer. Current Genomics 15:-, 2014. 28. Eriksen AHM, Andersen RF, Pallisgaard N, Sørensen FB, Jakobsen A,Hansen TF: MicroRNA Expression Profiling to Identify and Validate Reference Genes for the Relative Quantification of microRNA in Rectal Cancer. Plos One 11:e0150593, 2016. 29. Shenouda SK,Alahari SK: MicroRNA function in cancer: oncogene or a tumor suppressor? Cancer & Metastasis Reviews 28:369, 2009. 30. Wang SZ, Gao BH, Yang HL, Liu XJ, Wu X,Wang WJ: MicroRNA-432 is downregulated in cervical cancer and directly targets FN1 to inhibit cell proliferation and invasion. Oncol. Lett. 18:1475-1482, 2019. 31. Sun Y, Cheng Y, Zhang Y,Han K: MicroRNA-889-3p targets FGFR2 to inhibit cervical cancer cell viability and invasion. Exp. Ther. Med. 18:1440-1448, 2019. 32. Feng SJ, Liu W, Sakhautdinova I, Gao B,Tan WH: MicroRNA-505-5p functions as a tumor suppressor by targeting cyclin-dependent kinase 5 in cervical cancer. Bioscience Reports 39:2019. 33. Hongcheng L, Xiaofeng C, Wen G,Gening J: The expression of heparanase and microRNA-1258 in human non-small cell lung cancer. Tumour Biology the Journal of the International Society for Oncodevelopmental Biology & Medicine 33:1327-1334, 2012. 34. Shi J, Chen P, Sun J, Song Y, Ma B, Gao P, Chen X,Wang Z: MicroRNA-1258: An invasion and metastasis regulator that targets heparanase in gastric cancer. Oncol. Lett. 13:3739, 2017. 35. Müller H, ., Bracken AP, Vernell R, ., Moroni MC, Christians F, ., Grassilli E, ., Prosperini E, ., Vigo E, ., Oliner JD,Helin K, . E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis. Genes & Development 15:267, 2001. 36. Yuan H, Jiang F, Wang R, Shen M,Chen N: Lentivirus-mediated RNA interference of E2F-1 suppresses Tca8113 cell proliferation. Molecular medicine reports 5:420-426, 2011.
Journal Pre-proof
of
37. Annette L, Weini S, Helen C, Rachna P, Reena M, J Lewis S, Glen R, Woolley AG, Miller LD,Black MA: YB-1, the E2F pathway, and regulation of tumor cell growth. J Natl Cancer Inst 104:133-146, 2012. 38. Fang H, Gartenhaus RB, Zhao XF, Fang HB, Minkove S, Poss DE,Rapoport AP: c-Myc and E2F1 drive PBK/TOPK expression in high-grade malignant lymphomas. Leuk. Res. 37:447-454, 2013. 39. Costa C, ., Santos M, ., Martínez-Fernández M, ., Due?As M, ., Lorz C, ., García-Escudero R, .,Paramio JM: E2F1 loss induces spontaneous tumour development in Rb-deficient epidermis. Oncogene 32:2937-2951, 2013. 40. Wu Z, Zheng S, Li Z, Tan J,Yu Q: E2F1 suppresses Wnt/beta-catenin activity through transactivation of beta-catenin interacting protein ICAT. Oncogene 30:3979-3984, 2011.
ro
Figure legends
-p
Figure 1. Expression of miR-1258 and E2F1 in cervical cancer cell lines. (A) The
re
miR-1258 expression in normal Ect1/E6E7 cells and cervical cancer cells was detected by RT-qPCR analysis. **P<0.01 and ***P<0.001 vs. Normal group. (B) The
lP
protein level of E2F1 in normal Ect1/E6E7 cells and cervical cancer cells was detected by Western blot analysis. **P<0.01 and ***P<0.001 vs. Normal group. (C)
na
The mRNA level of E2F1 in normal Ect1/E6E7 cells and cervical cancer cells was detected by RT-qPCR analysis. ***P<0.001 vs. Normal group. Assays were
Jo ur
performed in triplicate. ANOVA test was used.
Figure 2. miR-1258 directly targets E2F1. (A) The binding sites of miR-1258 and E2F1 were predicted by miRDB. (B) Luciferase activity was analyzed in cells co-transfected with miR-1258 mimic or miR-control with pmirGLO-E2F1-Wt or pmirGLO-E2F1-Mut. ***P<0.001 vs. E2F1+ miR-control group. (C) E2F1 mRNA expression levels in transfected c-33A cells were analyzed by RT-qPCR analysis. ***P<0.001 vs. control group.
###
P<0.001 vs. miR-mimic NC group. Assays were
performed in triplicate. Independent sample T test and ANOVA test was used.
Figure 3. miR-1258 overexpression inhibits the proliferation of cervical cancer cells. (A) miR-1258 expression levels in transfected c-33A cells were analyzed by RT-qPCR
Journal Pre-proof analysis. ***P<0.001 vs. Control group. ###P<0.001 vs. miR-mimic NC group. (B) The protein level of E2F1 in transfected c-33A cells were analyzed by Western blot analysis. *P<0.05 vs. Control group. #P<0.05 vs. Overexpression-NC group. (C) The mRNA level of E2F1 in transfected c-33A cells were analyzed by RT-qPCR analysis. *P<0.05 vs. Control group. #P<0.05 vs. Overexpression-NC group. (D) The cell viability of transfected c-33A cells was analyzed by CCK-8 assay. ***P<0.001 vs. Control group.
P<0.001 vs. miR-mimic NC group. ∆P<0.05 and
###
∆∆
P<0.01 vs.
of
miR-1258 mimic group. Assays were performed in triplicate. ANOVA test was used.
ro
Figure 4. miR-1258 overexpression inhibits the invasion and migration of cervical cancer cells. (A) The migration of transfected c-33A cells was showed by wound ###
P<0.001 vs. miR-mimic NC group.
-p
healing assay. ***P<0.001 vs. Control group. ∆∆
re
P<0.01 vs. miR-1258 mimic group. (B) The invasion of transfected c-33A cells was
showed by transwell assay. *P<0.05 and ***P<0.001 vs. Control group. ###P<0.001 vs.
lP
miR-mimic NC group. ∆P<0.05 vs. miR-1258 mimic group. (C) The protein level of
na
MMP2, MMP7 and MMP9 in transfected c-33A cells were analyzed by Western blot analysis. *P<0.05, **P<0.01 and ***P<0.001 vs. Control group.
P<0.001 vs.
∆∆∆
P<0.001 vs. miR-1258 mimic group. Assays
Jo ur
miR-mimic NC group. ∆P<0.05 and
###
were performed in triplicate. ANOVA test was used.
Figure 5. miR-1258 overexpression promotes the apoptosis of cervical cancer cells. (A) The cell apoptosis of transfected c-33A cells was detected by flow cytometry analysis. ***P<0.001 vs. Control group.
###
P<0.001 vs. miR-mimic NC group.
∆∆∆
P<0.001 vs. miR-1258 mimic group. (B) The protein level of apoptosis related
proteins was analyzed by Western blot analysis. **P<0.01 and ***P<0.001 vs. Control group. ###P<0.001 vs. miR-mimic NC group. ∆∆∆P<0.001 vs. miR-1258 mimic group. Assays were performed in triplicate. ANOVA test was used.
Figure 6. miR-1258 overexpression inhibits AKT and activates P53 signaling pathways. The protein level of p-AKT, AKT, CDK2, p53 and CyclinD1 was detected
Journal Pre-proof by Western blot analysis. **P<0.01 and ***P<0.001 vs. Control group. ###P<0.001 vs. miR-mimic NC group. ∆P<0.05 and
∆∆∆
P<0.001 vs. miR-1258 mimic group. Assays
were performed in triplicate. ANOVA test was used.
Figure 7. miR-1258 overexpression inhibits the development of tumor in vivo. (A) Photographs of tumors were obtained from different groups of transfected mice and growth curve of tumor volumes was calculated. *P<0.05 and ***P<0.001 vs. Control group. ###P<0.001 vs. miR-mimic NC group.
∆∆∆
P<0.001 vs. miR-1258 mimic group.
of
(B) The weight of tumors was measured. *P<0.05, **P<0.01 and ***P<0.001 vs.
ro
Control group. ##P<0.01 vs. miR-mimic NC group. (C) The protein level of apoptosis related proteins in tumor tissues was analyzed by Western blot analysis. **P<0.01 and ###
P<0.001 vs. miR-mimic NC group. ∆P<0.05 vs.
-p
***P<0.001 vs. Control group.
lP
re
miR-1258 mimic group. Assays were performed in triplicate. ANOVA test was used.
Figure 8. miR-1258 overexpression inhibits AKT and activates P53 signaling
na
pathways in vivo. The protein level of p-AKT, AKT, CDK2, p53 and CyclinD1 in tumor tissues was detected by Western blot analysis. **P<0.01 and ***P<0.001 vs.
∆∆∆
###
P<0.001 vs. miR-mimic NC group.
Jo ur
Control group.
∆
P<0.05,
∆∆
P<0.01 and
P<0.001 vs. miR-1258 mimic group. Assays were performed in triplicate. ANOVA
test was used.
Journal Pre-proof
Dear editor， On behalf of the co-authors, I am now submitting the a revised version of the manuscript entitled “miR-1258 promotes the apoptosis of cervical cancer cells by regulating the E2F1/P53 signaling pathway” to Experimental and Molecular Pathology. We have carefully revised the manuscript according to the reviewers comments
of
and hope this revision can meet the requirements of reviewers.
Yours sincerely,
ro
Chunyan Cai, Department of Gynaecology, The Affiliated Huaian No.1 People’s
-p
Hospital of Nanjing Medical University, 6 Beijing Road West, Huaian, Jiangsu
Jo ur
na
lP
re
Province, 223300, China. E-mail: [email protected]
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8