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miR-214 down-regulates MKK3 and suppresses malignant phenotypes of cervical cancer cells
Journal Pre-proofs Research paper miR-214 down-regulates MKK3 and suppresses malignant phenotypes of cervical cancer cells Ruiqing Peng, Xiangshan Cheng, Yue Zhang, Xin Lu, Zhidong Hu PII: DOI: Reference:
S0378-1119(19)30805-4 https://doi.org/10.1016/j.gene.2019.144146 GENE 144146
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Gene Gene
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22 August 2019 24 September 2019 25 September 2019
Please cite this article as: R. Peng, X. Cheng, Y. Zhang, X. Lu, Z. Hu, miR-214 down-regulates MKK3 and suppresses malignant phenotypes of cervical cancer cells, Gene Gene (2019), doi: https://doi.org/10.1016/j.gene. 2019.144146
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© 2019 Published by Elsevier B.V.
miR-214 down-regulates MKK3 and suppresses malignant phenotypes of cervical cancer cells Ruiqing Penga, Xiangshan Chengb, Yue Zhanga, Xin Lua, Zhidong Hua* aLaboratory Center, Tianjin Medical University General Hospital, Tianjin, 300052, PR China bDepartment of Hematology, Heze Municipal Hospital, Heze, Shandong, 274031, PR China
*Corresponding author Address: Laboratory Center, Tianjin Medical University General Hospital, Tianjin, 300052, PR China E-mail address: [email protected]
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Abstract: miRNA mediated genetic regulation is widely involved in carcinogenesis of cervical cancer. In this study, miR-214 was confirmed to directly regulate MKK3 via imperfect base-pairing to its 3’UTR, resulting down-regulation of its expression level. Compared to normal tissues, a down-regulated level of miR-214 was observed in cervical cancer, while MKK3 was up-regulated. Next, we demonstrated that over-expression of miR-214 or knockdown of MKK3 can inhibit the growth, proliferation, invasion and migration of cervical cancer in vitro and in vivo. Moreover, the effects of miR-214 in HeLa cells were rescued by the restoration of MKK3. In conclusion, our results laid new foundations for investigating the pathogenesis and diagnosis of cervical cancer. Keywords: miR-214, MKK3, cervical cancer, proliferation, migration, invasion
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1. Introduction Cervical cancer, a female reproductive cancer, is the second popular malignant tumors in females, only next to breast cancer(Schiffman and Solomon, 2013). Incidence of cervical cancer is increasing gradually with younger population age, severely threatening female health and life(Duenas-Gonzalez and Campbell, 2016). Many tumor suppressor genes and oncogenes are abnormally expressed in this complex disease. For example, p53 and Bax both can serve as tumor suppressors in the pathogenesis of cervical cancer(Karlidag et al., 2007). Although the mechanisms underneath known genes have provided new insights, detailed investigation of microRNAs (miRNAs) would provide some new information of the pathogenesis of cervical cancer. miRNAs, a length of 22-25 nt, are a class of non-coding RNA, which regulate gene expression via binding the 3’-untranslated region (3’UTR) of target transcripts in a complete or incomplete complementary binding manner. miRNA, which occupies cervical cancer only 1% of human genome, can regulate the expression of more than 30% of human target genes(Gui and Shen, 2012). Recent studies have reported that aberrant regulation of microRNA-214 (miR-214) can result in multiple carcinomas, including NSCLC(Dandan et al., 2019), colorectal cancer(Li et al., 2019; Xu et al., 2019), papillary thyroid carcinoma(Liu et al., 2018), osteosarcoma(Rehei et al., 2018), hepatocellular carcinoma(Tian et al., 2018), esophageal squamous cell carcinoma(Guanen et al., 2018) and so on. In addition, miR-214 regulates specific cell functions including cell growth, proliferation, apoptosis, invasion and migration(Chen et al., 2018; Guanen et al., 2018; Tian et al., 2018; Wang et al., 2018; Zhang et al., 2018). Various studies showed significantly decreased miR-214 expression in cervical cancer tissues, indicating its potential tumor-suppressing role in cervical cancer pathogenesis(Peng et al., 2017; Wang et al., 2017). The mitogen-activated protein kinase kinase 3 (MKK3), localized on chromosome 17q11.2, can specifically phosphorylate p38 MAPK to activate the mitogen-activated protein kinase (MAPK) signaling pathway, affecting the cell differentiation, motility, division, and death(Derijard et al., 1995). Bioinformatics analysis revealed the existence of complementary binding sites between miR-214 and 3’UTR of MKK3 mRNA. This work investigated whether miR-214 played a role in mediating MKK3 expression to regulate the growth, proliferation, migration and invasion of cervical cancer. 2. Material and methods 2.1. Prediction of the targets of miR-214 The potential targets of miR-214 were predicted using three public databases: PicTar, TargetScan, and miRBase Targets. 2.2. Construction of plasmids Construction of the pcDNA3/pri-miR-214 and ASO-miR-214 was as previously described (Peng et al., 2017). The 3’UTR fragment of MKK3 with the miR-214 putative binding site predicted above and binding site mutated fragment were PCR amplified (Table 1), and inserted into the pcDNA3/EGFP plasmid (Hind III/BamH I sites) to generate pcDNA3/EGFP-MKK3 3’UTR 3
and pcDNA3/EGFP-MKK3 3’UTR mut, which were sequenced to confirm the correction of the sequence. A 70-bp double-strand fragment which expresses MKK3 siRNA was generated by annealing two single-strand DNAs (Table 1) and inserted into the pSilencer2.1/neo vector (Ambion), and named as pSilencer/shRNA-MKK3 (siR-MKK3) A PCR amplified full-length human MKK3 cDNA (NM_145109) was cloned into the pcDNA3 vector (XhoI/XbaI sites) and named as pcDNA3/MKK3. 2.3. Cell culture and transfection HeLa and C33A, were maintained in RPMI-1640 medium containing FBS (10%), penicillin (100 IU/ml) and streptomycin (100 μg/ml) in a incubator with 5% CO2 at 37 ℃. For transfection, Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA) was applied. 2.4. RNA isolation from the human tissue samples The preparation of the 30 pairs of human cervical cancer tissues and adjacent normal tissues were approved by the by the Ethics Committee of Tianjin Medical University General Hospital. All of the patients were provided with informed consents. The obtained tissues were used for RNA isolation using the mirVana miRNA Isolation Kit (Ambion, USA). 2.5. Quantitative real-time polymerase chain reaction To quantify the expression of miR-214, a stem-loop qRT-PCR was conducted. Briefly, the cDNA was generated using small RNA (2 μg), stem-loop RT primer, and M-MLV reverse transcriptase (Promega, USA). For MKK3 gene quantification, cDNA was generated with large RNA (5 μg). The resulted products were then applied for qPCR on an Bio-Rad iQ5 apparatus with SYBR Kit (TaKaRa, Japan). To amplify miR-214 and U6 snRNA (as an endogenous control), thermo cycles were as follows: 6 min at 94 ℃, followed by 40 cycles of 30 s at 94 ℃, 30 s at 56 ℃ and 30 s at 72 ℃. To amplify the MKK3 gene and β-actin (as an endogenous control gene, the thermo cycles were 5 min at 94 ℃, followed by 40 cycles of 30 s at 94 ℃, 30 s at 58 ℃ and 30 s at 72 ℃. The 2–ΔΔCt method was used for calculating the relative expression of miR-214 or MKK3. All primers are listed in Table 1, which were provided from AuGCT, Inc. (Beijing, China). 2.6. Western blot Protein samples were separated with 10% SDS-PAGE, followed by transferring to a nitrocellulose membranes, which were incubated with 3% skim-milk for 60 min at room temperature and MKK3 antibody (Saier Biotech, Tianjin, China) or GAPDH antibody (Saier Biotech, Tianjin, China) at 4 ℃ overnight, followed by washing the membranes for 3 times at a 5 min interval and incubating secondary antibody labelled with HRP (Saier Biotech, Tianjin, China). The signals were captured on a chemiluminescent film via the enhanced chemiluminescence and quantified via the Lab Works image acquisition and analysis software (Version 3.0, UVP, Upland, CA). 2.7. Fluorescent reporter assay
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After seeding HeLa cells into a 24-well plate for 24 h, the cells were co-transfecting with plasmids which were divided into 8 groups: pcDNA3/EGFP-MKK3 3’UTR+pcDNA3, pcDNA3/EGFP-MKK3 3’UTR+pri-miR-214, pcDNA3/EGFP-MKK3 3’UTR+ASO-NC, pcDNA3/EGFP-MKK3 3’UTR+ASO-miR-214, pcDNA3/EGFP-MKK3 3’UTR mutant+pcDNA3, pcDNA3/EGFP-MKK3 3’UTR mut+pri-miR-214, pcDNA3/EGFP-MKK3 3’UTR mut+ASO-NC, pcDNA3/EGFP-MKK3 3’UTR mut+ASO-miR-214. pDsRed2-N1 (Clontech, USA) was co-transfected into each group as an internal control. After 48 h, proteins were obtained via cells lysis, followed by detection of the EGFP and RFP fluorescent protein intensity with an F-4500 Fluorescence Spectrophotometer (Hitachi, Tokyo, Japan). 2.8. Cell viability assays HeLa cells (6,000 cells/well) or C33A cells (10,000 cells/well) were seeded in 96-well plates. MTT was used to analyze the cell viability at the indicated time points. 2.9. Colony formation assay HeLa (200 cells/well) or C33A (300 cells/well) cells were seeded in 12-well plates in triplicate, and stained with crystal violet for 12 or 15 days, respectively, the colony formation ability of the cells were assessed via counting the colonies (with more than 50 cells) of each group. 2.10. Transwell assays After coating the chamber membranes (pore size of 8 μm; Corning, USA) with (for invasion assay) or without (for migration assay) 40 μl Matrigel (2 mg/ml, Growth Factor Reduced BD Matrigel TM Matrix) for 1 h, the upper chambers were added with 200 μl FBS-free RPMI-1640 containing HeLa (6×104 cells/well) or C33A (1.2×105 cells/well). The lower part of the chambers were added with 600 μl RPMI 1640 with 20%, which were incubated with 24 h and 48 h for invasion assay and 18 h and 30 h for migration assay, followed by treating the membranes with 2% crystal violet. After 10 min, the membranes were then cut and placed on glass slides, followed by counting the cell numbers with a light microscope in five random visual fields. All assays were conducted with three independent times in triplicate. 2.11. Xenograft tumor formation assay Female nude mice were purchased from the Laboratory Animal Research Institute of the Beijing Chinese Academy of Medical Sciences and raised in specific pathogen-free conditions. Each of the 24 mice was randomly assigned to one of four cages (6 mice per cage). HeLa cells were stably transfected with pcDNA3 or pri-miR-214, siR-Ctrl or siR-MKK3. About 2×106 transfected cells, resuspended in 100 μl serum-free RPMI 1640 culture medium, were subcutaneously injected into the oxter of the 5 week-old nude mice. Mouse tumor sizes were measured every 2 days after 9 days of injection. The tumor volume was calculated as follows: length×width2×1/2. All the mice were sacrificed at day 21 after injection. The tumors were isolated from the mice and stored at -80 °C. All animal experiments were approved by the Animal Ethics Committees of Tianjin Medical University General Hospital. 5
2.12. Statistical analysis All the data were performed Student’s t test analysis, and presented as means ± standard deviation (SD). Differences were considered statistically significant with p < 0.05 (*p < 0.05, **p < 0.01). 3 Results 3.1. miR-214 represses the expression of MKK3 via directly binding its 3’UTR To investigate whether the 3’UTR of MKK3 was regulated by miR-214, algorithm programs was used for prediction, which harbors a potential target site (nucleotides 1521– 1545) of miR-214 (Fig. 1A). Then the transfection efficiency was conducted, and showed that pri-miR-214 increased the expression of miR-214 by 11.7- and 8.8-fold in HeLa and C33A cells, while ASO-miR-214 decreased its expression by nearly 67% and 70% (Fig. 1B). Furthermore, a significant reduction of the intensity of EGFP fluorescence was mediate by the pri-miR-214, while the intensity of EGFP fluorescence was increased by ASO-miR-214. However, the fluorescence activity of pcDNA3/EGFP-MKK3 3’UTR mut was not affected by either pri-miR-214 or ASO-miR-214 (Fig. 1C), indicating that miR-214 repressed the expression of MKK3 via binding directly to its 3’UTR. We then performed qRT-PCR and western blot to confirm whether the endogenous expression of MKK3 can be regulated by miR-214, the results illustrated that ectopic expression of miR-214 decreased 56% mRNA and 66% protein of MKK3, whereas blocking miR-214 led to an increase of 5.1-fold mRNA and 2.1-fold protein of MKK3 in HeLa cells (Fig. 1D, E). In C33A cells, the over-expression of miR-214 led to a decrease of 64% mRNA and 72% protein levels of MKK3, conversely, blocking miR-214 led to an increase of 3.6-fold mRNA and 0.9-fold protein levels of MKK3 (Fig. 1D, E). These results indicated that miR-214 can inhibited the level of MKK3 via binding to its 3’UTR directly in HeLa and C33A cells. Moreover, we performed qRT-PCR to analyze the transcripts level of MKK3 and miR-214 with 30 pairs of human cervical cancer tissues and adjacent normal tissues to analyze, which showed that the expression of miR-214 was lower (Fig. 1F), while MKK3 was higher in human cervical cancer tissues (Fig. 1G). Therefore, these results further provide evidence for the previous results that MKK3 was negatively regulated by miR-214. 3.2.
miR-214 suppresses the malignant phenotypes in cervical cancer in vitro and in vivo
We further detected whether the malignant phenotypes can be regulated by miR-214 in cervical cancer cells. The viability of HeLa cells was repressed in the cells over-expressed with miR-214 by 19%, while increased with ASO-miR-214 by 37% (Fig. 2A). The pri-miR-214 decreased the colony numbers by 52% and 54%, while ASO-miR-214 led to the increase of colony numbers by 1.1-fold and 0.78-fold in HeLa and C33A cells, respectively (Fig. 2B). Furthermore, pri-miR-214 gave rise to a reduction of the migration and invasion abilities by 53% and 59%, while ASO-miR-214 increased the migration and invasion abilities by 1.74- and 1.5-fold in HeLa cells. We also observed similar results in C33A cells (Fig. 2C, D). 6
To further examine the effect of miR-214 on tumor growth in vivo, miR-214 over-expressed-HeLa cells and control cells were injected into the oxter of nude mice. At 21 days after injection, the nude mice were sacrificed. The volume of tumors for the miR-214 group was decreased compared with the control group (Fig 2E). Together, these results demonstrated that the malignant phenotypes of cervical cancer can be inhibited by miR-214 in vitro and in vivo. 3.3. Knockdown of MKK3 inhibits the malignant phenotypes of in cervical cancer in vitro and in vivo To detect the effect of MKK3 in cervical cancer cells, pSilencer/shRNA-MKK3 (siR-MKK3) was constructed, which decreased the HeLa cells’ MKK3 by 68% and C33A cells’ MKK3 by 66% (Fig. 3A), and inhibited viability (Fig. 3B), colony formation (Fig. 3C), the rate of cell migration (Fig. 3D) and invasion (Fig. 3E). The in vivo results were consistent with the in vitro results (Fig. 3F). The results demonstrated that the effects of MKK3 down-regulation on malignant phenotypes were consistent with the effects of over-expression of miR-214 in vitro and in vivo. 3.4. MKK3 mediates the downstream function of miR-214 in vitro We further confirmed whether MKK3 mediates the function of miR-214, the results showed that over-expression of MKK3 by pcDNA3/MKK3 partly restored the decreased endogenous MKK3 caused by over-expression of miR-214 (Fig. 4A). Furthermore, pri-miR-214 induced inhibition of cell growth (Fig. 4B), migration (Fig. 4C) and invasion (Fig. 4D) were abrogated by the over-expression of MKK3. Taken together, these data demonstrated that the up-regulation of MKK3 countered the effect of miR-214 on malignant phenotypes of cervical cancer cells. 4 Discussion Cervical cancer has relatively insidious disease onset, as no obvious symptom can be observed at early stage. It is frequently being misdiagnosed, leading to lower early diagnostic rate. Although combined therapy, including surgical resection and chemo-/radio-therapy, have achieved a big progress, the treatment efficacy is still not satisfactory for patients at the terminal stage. Therefore, investigation and identification of critical regulatory factors are of critical importance for early diagnosis, treatment efficacy and prognosis. Our results indicated that down-regulation of miR-214 exerted the facilitating effects on MKK3 expression and malignant phenotypes of cervical cancer in vitro and in vivo. Recent findings(Peng et al., 2017) found lower level of miR-214 was expressed in cervical cancer tissues and up-regulation of miR-214 inhibited proliferation of HeLa or C33A, and decreased their migration or invasion potency via suppression of ARL2 gene expression. Chandrasekaran et al(Chandrasekaran et al., 2016)found cervical cancer tissues had lower level of miR-214 and higher level of its target gene HMGA1, and miR-214 up-regulation could target HMGA1 to decrease the malignant phenotypes of cervical cancer cells. Qiang et al(Qiang et al., 2011)found that, compared to normal cervical tissues, cervical cancer tissues 7
had abnormally lower expression of miR-214, which was correlated with peripheral tissue infiltration and distal metastasis and showed that the expression of Plexin-B1 was inhibited by up-regulation of miR-214, which inhibited in vivo growth of HeLa cells inside BALB/c nude mice. Wang et al(Wang et al., 2013) also showed that miR-214 over-expression inhibited Bcl2 expression, proliferation or survival of cervical cancer cells, induced cell apoptosis, as well as enhanced their sensitivity against chemotherapy drugs cisplatin. Wen et al(Wen et al., 2014) showed a correlation between miR-214 down-regulation and enhanced malignant biological features of cervical cancer cells. MKK3, a member of MAPK signaling pathway, play a key function in a plethora of cellular programs(Johnson and Lapadat, 2002). Recent study indicated that MKK3 can promote the tumor progression of gliomas and breast tumors(Demuth et al., 2007). Baldariet al(Baldari et al., 2015)found that depletion of MKK3 inhibited the proliferation of cancer cell and increased the response of tumor cell chemotherapeutic drugs in vivo(Baldari et al., 2015). Another report observed increased MKK3 mRNA expression in NSCLC(MacNeil et al., 2014). Gabriely et al(Gabriely et al., 2008)showed that Alpha-4 can bind to MKK3, which directed its site-specific dephosphorylation of Thr193, leading to the activation of p38 MAPK. In this study, we obtained the results that up-regulation of MKK3 promoted cell viability, proliferation, migration, and invasion of cervical cancer cells. In sum, the data here confirmed that as a new negative regulator of MKK3, miR-214 targets MKK3’s 3’UTR in cervical cancer cells. miR-214-inhibited malignant phenotypes of cervical cancer cells can be rescued by up-regulation of MKK3. These findings illustrated novel molecular mechanism of cervical cancer and may provide new diagnosis approach of cervical cancer carcinogenesis.
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Acknowledgements This work was supported by the Youth Incubation Foundation of TMUGH (grant number ZYYFY2015021) and the Science and Technology Foundation of Tianjin Municipal Health Bureau (grant number 2015KZ115).
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References Baldari, S., Ubertini, V., Garufi, A., D'Orazi, G. and Bossi, G., 2015. Targeting MKK3 as a novel anticancer strategy: molecular mechanisms and therapeutical implications. Cell Death Dis 6, e1621. Chandrasekaran, K.S., Sathyanarayanan, A. and Karunagaran, D., 2016. MicroRNA-214 suppresses growth, migration and invasion through a novel target, high mobility group AT-hook 1, in human cervical and colorectal cancer cells. Br J Cancer 115, 741-51. Chen, X., Du, J., Jiang, R. and Li, L., 2018. MicroRNA-214 inhibits the proliferation and invasion of lung carcinoma cells by targeting JAK1. Am J Transl Res 10, 1164-1171. Dandan, W., Jianliang, C., Haiyan, H., Hang, M. and Xuedong, L., 2019. Long noncoding RNA MIR31HG is activated by SP1 and promotes cell migration and invasion by sponging miR-214 in NSCLC. Gene. Demuth, T., Reavie, L.B., Rennert, J.L., Nakada, M., Nakada, S., Hoelzinger, D.B., Beaudry, C.E., Henrichs, A.N., Anderson, E.M. and Berens, M.E., 2007. MAP-ing glioma invasion: mitogen-activated protein kinase kinase 3 and p38 drive glioma invasion and progression and predict patient survival. Mol Cancer Ther 6, 1212-22. Derijard, B., Raingeaud, J., Barrett, T., Wu, I.H., Han, J., Ulevitch, R.J. and Davis, R.J., 1995. Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267, 682-5. Duenas-Gonzalez, A. and Campbell, S., 2016. Global strategies for the treatment of early-stage and advanced cervical cancer. Curr Opin Obstet Gynecol 28, 11-7. Gabriely, G., Wurdinger, T., Kesari, S., Esau, C.C., Burchard, J., Linsley, P.S. and Krichevsky, A.M., 2008. MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol Cell Biol 28, 5369-80. Guanen, Q., Junjie, S., Baolin, W., Chaoyang, W., Yajuan, Y., Jing, L., Junpeng, L., Gaili, N., Zhongping, W. and Jun, W., 2018. MiR-214 promotes cell meastasis and inhibites apoptosis of esophageal squamous cell carcinoma via PI3K/AKT/mTOR signaling pathway. Biomed Pharmacother 105, 350-361. Gui, T. and Shen, K., 2012. miRNA-101: a potential target for tumor therapy. Cancer Epidemiol 36, 537-40. Johnson, G.L. and Lapadat, R., 2002. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298, 1911-2. Karlidag, T., Cobanoglu, B., Keles, E., Alpay, H.C., Ozercan, I., Kaygusuz, I., Yalcin, S. and Sakallioglu, O., 2007. Expression of Bax, p53, and p27/kip in patients with papillary thyroid carcinoma with or without cervical nodal metastasis. Am J Otolaryngol 28, 31-6. Li, S., Li, C. and Fang, Z., 2019. MicroRNA 214 inhibits adipocyte enhancer-binding protein 1 activity and increases the sensitivity of chemotherapy in colorectal cancer. Oncol Lett 17, 55-62. Liu, F., Lou, K., Zhao, X., Zhang, J., Chen, W., Qian, Y., Zhao, Y., Zhu, Y. and Zhang, Y., 2018. miR-214 regulates papillary thyroid carcinoma cell proliferation and metastasis by targeting PSMD10. Int J Mol Med 42, 3027-3036. MacNeil, A.J., Jiao, S.C., McEachern, L.A., Yang, Y.J., Dennis, A., Yu, H., Xu, Z., Marshall, J.S. and Lin, T.J., 2014. MAPK kinase 3 is a tumor suppressor with reduced copy number in breast cancer. Cancer Res 74, 162-72.
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Peng, R., Men, J., Ma, R., Wang, Q., Wang, Y., Sun, Y. and Ren, J., 2017. miR-214 down-regulates ARL2 and suppresses growth and invasion of cervical cancer cells. Biochem Biophys Res Commun 484, 623-630. Qiang, R., Wang, F., Shi, L.Y., Liu, M., Chen, S., Wan, H.Y., Li, Y.X., Li, X., Gao, S.Y., Sun, B.C. and Tang, H., 2011. Plexin-B1 is a target of miR-214 in cervical cancer and promotes the growth and invasion of HeLa cells. Int J Biochem Cell Biol 43, 632-41. Rehei, A.L., Zhang, L., Fu, Y.X., Mu, W.B., Yang, D.S., Liu, Y., Zhou, S.J. and Younusi, A., 2018. MicroRNA-214 functions as an oncogene in human osteosarcoma by targeting TRAF3. Eur Rev Med Pharmacol Sci 22, 5156-5164. Schiffman, M. and Solomon, D., 2013. Clinical practice. Cervical-cancer screening with human papillomavirus and cytologic cotesting. N Engl J Med 369, 2324-31. Tian, C., Wu, H., Li, C., Tian, X., Sun, Y., Liu, E., Liao, X. and Song, W., 2018. Downreguation of FoxM1 by miR-214 inhibits proliferation and migration in hepatocellular carcinoma. Gene Ther 25, 312-319. Wang, F., Liu, M., Li, X. and Tang, H., 2013. MiR-214 reduces cell survival and enhances cisplatin-induced cytotoxicity via down-regulation of Bcl2l2 in cervical cancer cells. FEBS Lett 587, 488-95. Wang, F., Tan, W.H., Liu, W., Jin, Y.X., Dong, D.D., Zhao, X.J. and Liu, Q., 2018. Effects of miR-214 on cervical cancer cell proliferation, apoptosis and invasion via modulating PI3K/AKT/mTOR signal pathway. Eur Rev Med Pharmacol Sci 22, 1891-1898. Wang, J.M., Ju, B.H., Pan, C.J., Gu, Y., Li, M.Q., Sun, L., Xu, Y.Y. and Yin, L.R., 2017. MiR-214 inhibits cell migration, invasion and promotes the drug sensitivity in human cervical cancer by targeting FOXM1. Am J Transl Res 9, 3541-3557. Wen, Z., Lei, Z., Jin-An, M., Xue-Zhen, L., Xing-Nan, Z. and Xiu-Wen, D., 2014. The inhibitory role of miR-214 in cervical cancer cells through directly targeting mitochondrial transcription factor A (TFAM). Eur J Gynaecol Oncol 35, 676-82. Xu, M., Chen, X., Lin, K., Zeng, K., Liu, X., Xu, X., Pan, B., Xu, T., Sun, L., He, B., Pan, Y., Sun, H. and Wang, S., 2019. lncRNA SNHG6 regulates EZH2 expression by sponging miR-26a/b and miR-214 in colorectal cancer. J Hematol Oncol 12, 3. Zhang, K., Zhang, M., Jiang, H., Liu, F., Liu, H. and Li, Y., 2018. Down-regulation of miR-214 inhibits proliferation and glycolysis in non-small-cell lung cancer cells via down-regulating the expression of hexokinase 2 and pyruvate kinase isozyme M2. Biomed Pharmacother 105, 545-552.
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Table 1 Primers and oligonucleotides used in this work. Name
Sequence (5’–3’)
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Figures
Fig.1. MKK3 expression is inhibited by miR-214 directly. (A) miR-214 potentially targets the 3’UTR of MKK3 and its mutated sequence. pri-miR-214 or ASO-miR-214 were transfected into HeLa and C33A cells, and (B) qRT-PCR was performed to detect the expression of miR-214. U6 snRNA was used for normalization. (C) HeLa cells were co-transfected with MKK3 3’UTR or 3’UTR mut, and detected the EGFP intensity. (D, E) The level of MMK3 was analyzed by qRT-PCR and western blot. (F, G) qRT-PCR analysis of the transcripts of miR-214 and MKK3 in the 30 pairs of cervical cancer tissues and matched normal tissues. The results are given as mean ± SD values for three experiments. *p < 0.05, ** p < 0.01.
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Fig.2. miR-214 represses the malignant phenotypes of cervical cancer. pri-miR-214 or ASO-miR-214 were transfected into HeLa and C33A cells, followed by the MTT analysis after 48 h (A), colony formation (B), migration (C) and invasion assays (D). The volume of the tumors in the nude mice xenograft experiment showed the effect of miR-214 on tumor growth in vivo (E). Tumor size were measured every 2 days after 9 days of injection. The tumor volume was calculated as follows: length×width2×1/2. All the mice were sacrificed 21 days after injection. Representative images are shown. The results are given as mean ± SD values. *p < 0.05, ** p < 0.01.
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Fig.3. Repression of MKK3 suppresses the malignant phenotypes of cervical cancer. Cells were transfected with siR-MKK3, followed by the western blot analysis of MKK3 expression after 48 h (A), MTT analysis (B), colony formation (C), migration (D) and invasion assays (E). The volume of the tumors in nude mice xenograft experiment (F) showed the effect of siR-MKK3 on tumor growth in vivo. Representative images are shown. The results are given as mean ± SD values. *p < 0.05, ** p < 0.01.
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Fig.4. miR-214-repressed malignant phenotypes of HeLa cells were rescued by MKK3 over-expression. Cells were co-transfected with the pcDNA3/MKK3 vector with or without pri-miR-214 vector, followed by the western blot detection of MKK3 protein level at 48 h post-transfection (A), MTT (B), migration (C) and invasion assays (D). Representative images of the randomly selected fields are shown. The results are given as mean ± SD values for three experiments. *p < 0.05, ** p < 0.01.
Abstract: miRNA mediated genetic regulation is widely involved in carcinogenesis of cervical cancer. In this study, miR-214 was confirmed to directly regulate MKK3 via imperfect base-pairing to its 3’UTR, resulting down-regulation of its expression level. Compared to normal tissues, a down-regulated level of miR-214 was observed in cervical cancer, while MKK3 was up-regulated. Next, we demonstrated that over-expression of miR-214 or knockdown of MKK3 can inhibit the growth, proliferation, invasion and migration of cervical cancer in vitro and in vivo. Moreover, the effects of miR-214 in HeLa cells were rescued by the restoration of MKK3. In conclusion, our results laid new foundations for investigating the pathogenesis and diagnosis of cervical cancer.
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1miR-214 targets MKK3 2 MKK3 maybe an oncogene in cervical cancer 3 MKK3 rescues miR-214 1
2 3 4 5
miRNAs: microRNAs 3’UTR: 3’-untranslated region miR-214: microRNA-214 MAPK: mitogen-activated protein kinase MKK3: mitogen-activated protein kinase kinase3
Conflict of interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
Ruiqing Peng, Xiangshan Cheng, Yue Zhang, Xin Lu, Zhidong Hu
Declaration of Interest Statement: We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Ruiqing Peng, Xiangshan Cheng, Yue Zhang, Xin Lu, Zhidong Hu
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S0378-1119(19)30805-4 https://doi.org/10.1016/j.gene.2019.144146 GENE 144146
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
22 August 2019 24 September 2019 25 September 2019
Please cite this article as: R. Peng, X. Cheng, Y. Zhang, X. Lu, Z. Hu, miR-214 down-regulates MKK3 and suppresses malignant phenotypes of cervical cancer cells, Gene Gene (2019), doi: https://doi.org/10.1016/j.gene. 2019.144146
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 B.V.
miR-214 down-regulates MKK3 and suppresses malignant phenotypes of cervical cancer cells Ruiqing Penga, Xiangshan Chengb, Yue Zhanga, Xin Lua, Zhidong Hua* aLaboratory Center, Tianjin Medical University General Hospital, Tianjin, 300052, PR China bDepartment of Hematology, Heze Municipal Hospital, Heze, Shandong, 274031, PR China
*Corresponding author Address: Laboratory Center, Tianjin Medical University General Hospital, Tianjin, 300052, PR China E-mail address: [email protected]
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Abstract: miRNA mediated genetic regulation is widely involved in carcinogenesis of cervical cancer. In this study, miR-214 was confirmed to directly regulate MKK3 via imperfect base-pairing to its 3’UTR, resulting down-regulation of its expression level. Compared to normal tissues, a down-regulated level of miR-214 was observed in cervical cancer, while MKK3 was up-regulated. Next, we demonstrated that over-expression of miR-214 or knockdown of MKK3 can inhibit the growth, proliferation, invasion and migration of cervical cancer in vitro and in vivo. Moreover, the effects of miR-214 in HeLa cells were rescued by the restoration of MKK3. In conclusion, our results laid new foundations for investigating the pathogenesis and diagnosis of cervical cancer. Keywords: miR-214, MKK3, cervical cancer, proliferation, migration, invasion
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1. Introduction Cervical cancer, a female reproductive cancer, is the second popular malignant tumors in females, only next to breast cancer(Schiffman and Solomon, 2013). Incidence of cervical cancer is increasing gradually with younger population age, severely threatening female health and life(Duenas-Gonzalez and Campbell, 2016). Many tumor suppressor genes and oncogenes are abnormally expressed in this complex disease. For example, p53 and Bax both can serve as tumor suppressors in the pathogenesis of cervical cancer(Karlidag et al., 2007). Although the mechanisms underneath known genes have provided new insights, detailed investigation of microRNAs (miRNAs) would provide some new information of the pathogenesis of cervical cancer. miRNAs, a length of 22-25 nt, are a class of non-coding RNA, which regulate gene expression via binding the 3’-untranslated region (3’UTR) of target transcripts in a complete or incomplete complementary binding manner. miRNA, which occupies cervical cancer only 1% of human genome, can regulate the expression of more than 30% of human target genes(Gui and Shen, 2012). Recent studies have reported that aberrant regulation of microRNA-214 (miR-214) can result in multiple carcinomas, including NSCLC(Dandan et al., 2019), colorectal cancer(Li et al., 2019; Xu et al., 2019), papillary thyroid carcinoma(Liu et al., 2018), osteosarcoma(Rehei et al., 2018), hepatocellular carcinoma(Tian et al., 2018), esophageal squamous cell carcinoma(Guanen et al., 2018) and so on. In addition, miR-214 regulates specific cell functions including cell growth, proliferation, apoptosis, invasion and migration(Chen et al., 2018; Guanen et al., 2018; Tian et al., 2018; Wang et al., 2018; Zhang et al., 2018). Various studies showed significantly decreased miR-214 expression in cervical cancer tissues, indicating its potential tumor-suppressing role in cervical cancer pathogenesis(Peng et al., 2017; Wang et al., 2017). The mitogen-activated protein kinase kinase 3 (MKK3), localized on chromosome 17q11.2, can specifically phosphorylate p38 MAPK to activate the mitogen-activated protein kinase (MAPK) signaling pathway, affecting the cell differentiation, motility, division, and death(Derijard et al., 1995). Bioinformatics analysis revealed the existence of complementary binding sites between miR-214 and 3’UTR of MKK3 mRNA. This work investigated whether miR-214 played a role in mediating MKK3 expression to regulate the growth, proliferation, migration and invasion of cervical cancer. 2. Material and methods 2.1. Prediction of the targets of miR-214 The potential targets of miR-214 were predicted using three public databases: PicTar, TargetScan, and miRBase Targets. 2.2. Construction of plasmids Construction of the pcDNA3/pri-miR-214 and ASO-miR-214 was as previously described (Peng et al., 2017). The 3’UTR fragment of MKK3 with the miR-214 putative binding site predicted above and binding site mutated fragment were PCR amplified (Table 1), and inserted into the pcDNA3/EGFP plasmid (Hind III/BamH I sites) to generate pcDNA3/EGFP-MKK3 3’UTR 3
and pcDNA3/EGFP-MKK3 3’UTR mut, which were sequenced to confirm the correction of the sequence. A 70-bp double-strand fragment which expresses MKK3 siRNA was generated by annealing two single-strand DNAs (Table 1) and inserted into the pSilencer2.1/neo vector (Ambion), and named as pSilencer/shRNA-MKK3 (siR-MKK3) A PCR amplified full-length human MKK3 cDNA (NM_145109) was cloned into the pcDNA3 vector (XhoI/XbaI sites) and named as pcDNA3/MKK3. 2.3. Cell culture and transfection HeLa and C33A, were maintained in RPMI-1640 medium containing FBS (10%), penicillin (100 IU/ml) and streptomycin (100 μg/ml) in a incubator with 5% CO2 at 37 ℃. For transfection, Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA) was applied. 2.4. RNA isolation from the human tissue samples The preparation of the 30 pairs of human cervical cancer tissues and adjacent normal tissues were approved by the by the Ethics Committee of Tianjin Medical University General Hospital. All of the patients were provided with informed consents. The obtained tissues were used for RNA isolation using the mirVana miRNA Isolation Kit (Ambion, USA). 2.5. Quantitative real-time polymerase chain reaction To quantify the expression of miR-214, a stem-loop qRT-PCR was conducted. Briefly, the cDNA was generated using small RNA (2 μg), stem-loop RT primer, and M-MLV reverse transcriptase (Promega, USA). For MKK3 gene quantification, cDNA was generated with large RNA (5 μg). The resulted products were then applied for qPCR on an Bio-Rad iQ5 apparatus with SYBR Kit (TaKaRa, Japan). To amplify miR-214 and U6 snRNA (as an endogenous control), thermo cycles were as follows: 6 min at 94 ℃, followed by 40 cycles of 30 s at 94 ℃, 30 s at 56 ℃ and 30 s at 72 ℃. To amplify the MKK3 gene and β-actin (as an endogenous control gene, the thermo cycles were 5 min at 94 ℃, followed by 40 cycles of 30 s at 94 ℃, 30 s at 58 ℃ and 30 s at 72 ℃. The 2–ΔΔCt method was used for calculating the relative expression of miR-214 or MKK3. All primers are listed in Table 1, which were provided from AuGCT, Inc. (Beijing, China). 2.6. Western blot Protein samples were separated with 10% SDS-PAGE, followed by transferring to a nitrocellulose membranes, which were incubated with 3% skim-milk for 60 min at room temperature and MKK3 antibody (Saier Biotech, Tianjin, China) or GAPDH antibody (Saier Biotech, Tianjin, China) at 4 ℃ overnight, followed by washing the membranes for 3 times at a 5 min interval and incubating secondary antibody labelled with HRP (Saier Biotech, Tianjin, China). The signals were captured on a chemiluminescent film via the enhanced chemiluminescence and quantified via the Lab Works image acquisition and analysis software (Version 3.0, UVP, Upland, CA). 2.7. Fluorescent reporter assay
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After seeding HeLa cells into a 24-well plate for 24 h, the cells were co-transfecting with plasmids which were divided into 8 groups: pcDNA3/EGFP-MKK3 3’UTR+pcDNA3, pcDNA3/EGFP-MKK3 3’UTR+pri-miR-214, pcDNA3/EGFP-MKK3 3’UTR+ASO-NC, pcDNA3/EGFP-MKK3 3’UTR+ASO-miR-214, pcDNA3/EGFP-MKK3 3’UTR mutant+pcDNA3, pcDNA3/EGFP-MKK3 3’UTR mut+pri-miR-214, pcDNA3/EGFP-MKK3 3’UTR mut+ASO-NC, pcDNA3/EGFP-MKK3 3’UTR mut+ASO-miR-214. pDsRed2-N1 (Clontech, USA) was co-transfected into each group as an internal control. After 48 h, proteins were obtained via cells lysis, followed by detection of the EGFP and RFP fluorescent protein intensity with an F-4500 Fluorescence Spectrophotometer (Hitachi, Tokyo, Japan). 2.8. Cell viability assays HeLa cells (6,000 cells/well) or C33A cells (10,000 cells/well) were seeded in 96-well plates. MTT was used to analyze the cell viability at the indicated time points. 2.9. Colony formation assay HeLa (200 cells/well) or C33A (300 cells/well) cells were seeded in 12-well plates in triplicate, and stained with crystal violet for 12 or 15 days, respectively, the colony formation ability of the cells were assessed via counting the colonies (with more than 50 cells) of each group. 2.10. Transwell assays After coating the chamber membranes (pore size of 8 μm; Corning, USA) with (for invasion assay) or without (for migration assay) 40 μl Matrigel (2 mg/ml, Growth Factor Reduced BD Matrigel TM Matrix) for 1 h, the upper chambers were added with 200 μl FBS-free RPMI-1640 containing HeLa (6×104 cells/well) or C33A (1.2×105 cells/well). The lower part of the chambers were added with 600 μl RPMI 1640 with 20%, which were incubated with 24 h and 48 h for invasion assay and 18 h and 30 h for migration assay, followed by treating the membranes with 2% crystal violet. After 10 min, the membranes were then cut and placed on glass slides, followed by counting the cell numbers with a light microscope in five random visual fields. All assays were conducted with three independent times in triplicate. 2.11. Xenograft tumor formation assay Female nude mice were purchased from the Laboratory Animal Research Institute of the Beijing Chinese Academy of Medical Sciences and raised in specific pathogen-free conditions. Each of the 24 mice was randomly assigned to one of four cages (6 mice per cage). HeLa cells were stably transfected with pcDNA3 or pri-miR-214, siR-Ctrl or siR-MKK3. About 2×106 transfected cells, resuspended in 100 μl serum-free RPMI 1640 culture medium, were subcutaneously injected into the oxter of the 5 week-old nude mice. Mouse tumor sizes were measured every 2 days after 9 days of injection. The tumor volume was calculated as follows: length×width2×1/2. All the mice were sacrificed at day 21 after injection. The tumors were isolated from the mice and stored at -80 °C. All animal experiments were approved by the Animal Ethics Committees of Tianjin Medical University General Hospital. 5
2.12. Statistical analysis All the data were performed Student’s t test analysis, and presented as means ± standard deviation (SD). Differences were considered statistically significant with p < 0.05 (*p < 0.05, **p < 0.01). 3 Results 3.1. miR-214 represses the expression of MKK3 via directly binding its 3’UTR To investigate whether the 3’UTR of MKK3 was regulated by miR-214, algorithm programs was used for prediction, which harbors a potential target site (nucleotides 1521– 1545) of miR-214 (Fig. 1A). Then the transfection efficiency was conducted, and showed that pri-miR-214 increased the expression of miR-214 by 11.7- and 8.8-fold in HeLa and C33A cells, while ASO-miR-214 decreased its expression by nearly 67% and 70% (Fig. 1B). Furthermore, a significant reduction of the intensity of EGFP fluorescence was mediate by the pri-miR-214, while the intensity of EGFP fluorescence was increased by ASO-miR-214. However, the fluorescence activity of pcDNA3/EGFP-MKK3 3’UTR mut was not affected by either pri-miR-214 or ASO-miR-214 (Fig. 1C), indicating that miR-214 repressed the expression of MKK3 via binding directly to its 3’UTR. We then performed qRT-PCR and western blot to confirm whether the endogenous expression of MKK3 can be regulated by miR-214, the results illustrated that ectopic expression of miR-214 decreased 56% mRNA and 66% protein of MKK3, whereas blocking miR-214 led to an increase of 5.1-fold mRNA and 2.1-fold protein of MKK3 in HeLa cells (Fig. 1D, E). In C33A cells, the over-expression of miR-214 led to a decrease of 64% mRNA and 72% protein levels of MKK3, conversely, blocking miR-214 led to an increase of 3.6-fold mRNA and 0.9-fold protein levels of MKK3 (Fig. 1D, E). These results indicated that miR-214 can inhibited the level of MKK3 via binding to its 3’UTR directly in HeLa and C33A cells. Moreover, we performed qRT-PCR to analyze the transcripts level of MKK3 and miR-214 with 30 pairs of human cervical cancer tissues and adjacent normal tissues to analyze, which showed that the expression of miR-214 was lower (Fig. 1F), while MKK3 was higher in human cervical cancer tissues (Fig. 1G). Therefore, these results further provide evidence for the previous results that MKK3 was negatively regulated by miR-214. 3.2.
miR-214 suppresses the malignant phenotypes in cervical cancer in vitro and in vivo
We further detected whether the malignant phenotypes can be regulated by miR-214 in cervical cancer cells. The viability of HeLa cells was repressed in the cells over-expressed with miR-214 by 19%, while increased with ASO-miR-214 by 37% (Fig. 2A). The pri-miR-214 decreased the colony numbers by 52% and 54%, while ASO-miR-214 led to the increase of colony numbers by 1.1-fold and 0.78-fold in HeLa and C33A cells, respectively (Fig. 2B). Furthermore, pri-miR-214 gave rise to a reduction of the migration and invasion abilities by 53% and 59%, while ASO-miR-214 increased the migration and invasion abilities by 1.74- and 1.5-fold in HeLa cells. We also observed similar results in C33A cells (Fig. 2C, D). 6
To further examine the effect of miR-214 on tumor growth in vivo, miR-214 over-expressed-HeLa cells and control cells were injected into the oxter of nude mice. At 21 days after injection, the nude mice were sacrificed. The volume of tumors for the miR-214 group was decreased compared with the control group (Fig 2E). Together, these results demonstrated that the malignant phenotypes of cervical cancer can be inhibited by miR-214 in vitro and in vivo. 3.3. Knockdown of MKK3 inhibits the malignant phenotypes of in cervical cancer in vitro and in vivo To detect the effect of MKK3 in cervical cancer cells, pSilencer/shRNA-MKK3 (siR-MKK3) was constructed, which decreased the HeLa cells’ MKK3 by 68% and C33A cells’ MKK3 by 66% (Fig. 3A), and inhibited viability (Fig. 3B), colony formation (Fig. 3C), the rate of cell migration (Fig. 3D) and invasion (Fig. 3E). The in vivo results were consistent with the in vitro results (Fig. 3F). The results demonstrated that the effects of MKK3 down-regulation on malignant phenotypes were consistent with the effects of over-expression of miR-214 in vitro and in vivo. 3.4. MKK3 mediates the downstream function of miR-214 in vitro We further confirmed whether MKK3 mediates the function of miR-214, the results showed that over-expression of MKK3 by pcDNA3/MKK3 partly restored the decreased endogenous MKK3 caused by over-expression of miR-214 (Fig. 4A). Furthermore, pri-miR-214 induced inhibition of cell growth (Fig. 4B), migration (Fig. 4C) and invasion (Fig. 4D) were abrogated by the over-expression of MKK3. Taken together, these data demonstrated that the up-regulation of MKK3 countered the effect of miR-214 on malignant phenotypes of cervical cancer cells. 4 Discussion Cervical cancer has relatively insidious disease onset, as no obvious symptom can be observed at early stage. It is frequently being misdiagnosed, leading to lower early diagnostic rate. Although combined therapy, including surgical resection and chemo-/radio-therapy, have achieved a big progress, the treatment efficacy is still not satisfactory for patients at the terminal stage. Therefore, investigation and identification of critical regulatory factors are of critical importance for early diagnosis, treatment efficacy and prognosis. Our results indicated that down-regulation of miR-214 exerted the facilitating effects on MKK3 expression and malignant phenotypes of cervical cancer in vitro and in vivo. Recent findings(Peng et al., 2017) found lower level of miR-214 was expressed in cervical cancer tissues and up-regulation of miR-214 inhibited proliferation of HeLa or C33A, and decreased their migration or invasion potency via suppression of ARL2 gene expression. Chandrasekaran et al(Chandrasekaran et al., 2016)found cervical cancer tissues had lower level of miR-214 and higher level of its target gene HMGA1, and miR-214 up-regulation could target HMGA1 to decrease the malignant phenotypes of cervical cancer cells. Qiang et al(Qiang et al., 2011)found that, compared to normal cervical tissues, cervical cancer tissues 7
had abnormally lower expression of miR-214, which was correlated with peripheral tissue infiltration and distal metastasis and showed that the expression of Plexin-B1 was inhibited by up-regulation of miR-214, which inhibited in vivo growth of HeLa cells inside BALB/c nude mice. Wang et al(Wang et al., 2013) also showed that miR-214 over-expression inhibited Bcl2 expression, proliferation or survival of cervical cancer cells, induced cell apoptosis, as well as enhanced their sensitivity against chemotherapy drugs cisplatin. Wen et al(Wen et al., 2014) showed a correlation between miR-214 down-regulation and enhanced malignant biological features of cervical cancer cells. MKK3, a member of MAPK signaling pathway, play a key function in a plethora of cellular programs(Johnson and Lapadat, 2002). Recent study indicated that MKK3 can promote the tumor progression of gliomas and breast tumors(Demuth et al., 2007). Baldariet al(Baldari et al., 2015)found that depletion of MKK3 inhibited the proliferation of cancer cell and increased the response of tumor cell chemotherapeutic drugs in vivo(Baldari et al., 2015). Another report observed increased MKK3 mRNA expression in NSCLC(MacNeil et al., 2014). Gabriely et al(Gabriely et al., 2008)showed that Alpha-4 can bind to MKK3, which directed its site-specific dephosphorylation of Thr193, leading to the activation of p38 MAPK. In this study, we obtained the results that up-regulation of MKK3 promoted cell viability, proliferation, migration, and invasion of cervical cancer cells. In sum, the data here confirmed that as a new negative regulator of MKK3, miR-214 targets MKK3’s 3’UTR in cervical cancer cells. miR-214-inhibited malignant phenotypes of cervical cancer cells can be rescued by up-regulation of MKK3. These findings illustrated novel molecular mechanism of cervical cancer and may provide new diagnosis approach of cervical cancer carcinogenesis.
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Acknowledgements This work was supported by the Youth Incubation Foundation of TMUGH (grant number ZYYFY2015021) and the Science and Technology Foundation of Tianjin Municipal Health Bureau (grant number 2015KZ115).
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References Baldari, S., Ubertini, V., Garufi, A., D'Orazi, G. and Bossi, G., 2015. Targeting MKK3 as a novel anticancer strategy: molecular mechanisms and therapeutical implications. Cell Death Dis 6, e1621. Chandrasekaran, K.S., Sathyanarayanan, A. and Karunagaran, D., 2016. MicroRNA-214 suppresses growth, migration and invasion through a novel target, high mobility group AT-hook 1, in human cervical and colorectal cancer cells. Br J Cancer 115, 741-51. Chen, X., Du, J., Jiang, R. and Li, L., 2018. MicroRNA-214 inhibits the proliferation and invasion of lung carcinoma cells by targeting JAK1. Am J Transl Res 10, 1164-1171. Dandan, W., Jianliang, C., Haiyan, H., Hang, M. and Xuedong, L., 2019. Long noncoding RNA MIR31HG is activated by SP1 and promotes cell migration and invasion by sponging miR-214 in NSCLC. Gene. Demuth, T., Reavie, L.B., Rennert, J.L., Nakada, M., Nakada, S., Hoelzinger, D.B., Beaudry, C.E., Henrichs, A.N., Anderson, E.M. and Berens, M.E., 2007. MAP-ing glioma invasion: mitogen-activated protein kinase kinase 3 and p38 drive glioma invasion and progression and predict patient survival. Mol Cancer Ther 6, 1212-22. Derijard, B., Raingeaud, J., Barrett, T., Wu, I.H., Han, J., Ulevitch, R.J. and Davis, R.J., 1995. Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267, 682-5. Duenas-Gonzalez, A. and Campbell, S., 2016. Global strategies for the treatment of early-stage and advanced cervical cancer. Curr Opin Obstet Gynecol 28, 11-7. Gabriely, G., Wurdinger, T., Kesari, S., Esau, C.C., Burchard, J., Linsley, P.S. and Krichevsky, A.M., 2008. MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol Cell Biol 28, 5369-80. Guanen, Q., Junjie, S., Baolin, W., Chaoyang, W., Yajuan, Y., Jing, L., Junpeng, L., Gaili, N., Zhongping, W. and Jun, W., 2018. MiR-214 promotes cell meastasis and inhibites apoptosis of esophageal squamous cell carcinoma via PI3K/AKT/mTOR signaling pathway. Biomed Pharmacother 105, 350-361. Gui, T. and Shen, K., 2012. miRNA-101: a potential target for tumor therapy. Cancer Epidemiol 36, 537-40. Johnson, G.L. and Lapadat, R., 2002. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298, 1911-2. Karlidag, T., Cobanoglu, B., Keles, E., Alpay, H.C., Ozercan, I., Kaygusuz, I., Yalcin, S. and Sakallioglu, O., 2007. Expression of Bax, p53, and p27/kip in patients with papillary thyroid carcinoma with or without cervical nodal metastasis. Am J Otolaryngol 28, 31-6. Li, S., Li, C. and Fang, Z., 2019. MicroRNA 214 inhibits adipocyte enhancer-binding protein 1 activity and increases the sensitivity of chemotherapy in colorectal cancer. Oncol Lett 17, 55-62. Liu, F., Lou, K., Zhao, X., Zhang, J., Chen, W., Qian, Y., Zhao, Y., Zhu, Y. and Zhang, Y., 2018. miR-214 regulates papillary thyroid carcinoma cell proliferation and metastasis by targeting PSMD10. Int J Mol Med 42, 3027-3036. MacNeil, A.J., Jiao, S.C., McEachern, L.A., Yang, Y.J., Dennis, A., Yu, H., Xu, Z., Marshall, J.S. and Lin, T.J., 2014. MAPK kinase 3 is a tumor suppressor with reduced copy number in breast cancer. Cancer Res 74, 162-72.
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Peng, R., Men, J., Ma, R., Wang, Q., Wang, Y., Sun, Y. and Ren, J., 2017. miR-214 down-regulates ARL2 and suppresses growth and invasion of cervical cancer cells. Biochem Biophys Res Commun 484, 623-630. Qiang, R., Wang, F., Shi, L.Y., Liu, M., Chen, S., Wan, H.Y., Li, Y.X., Li, X., Gao, S.Y., Sun, B.C. and Tang, H., 2011. Plexin-B1 is a target of miR-214 in cervical cancer and promotes the growth and invasion of HeLa cells. Int J Biochem Cell Biol 43, 632-41. Rehei, A.L., Zhang, L., Fu, Y.X., Mu, W.B., Yang, D.S., Liu, Y., Zhou, S.J. and Younusi, A., 2018. MicroRNA-214 functions as an oncogene in human osteosarcoma by targeting TRAF3. Eur Rev Med Pharmacol Sci 22, 5156-5164. Schiffman, M. and Solomon, D., 2013. Clinical practice. Cervical-cancer screening with human papillomavirus and cytologic cotesting. N Engl J Med 369, 2324-31. Tian, C., Wu, H., Li, C., Tian, X., Sun, Y., Liu, E., Liao, X. and Song, W., 2018. Downreguation of FoxM1 by miR-214 inhibits proliferation and migration in hepatocellular carcinoma. Gene Ther 25, 312-319. Wang, F., Liu, M., Li, X. and Tang, H., 2013. MiR-214 reduces cell survival and enhances cisplatin-induced cytotoxicity via down-regulation of Bcl2l2 in cervical cancer cells. FEBS Lett 587, 488-95. Wang, F., Tan, W.H., Liu, W., Jin, Y.X., Dong, D.D., Zhao, X.J. and Liu, Q., 2018. Effects of miR-214 on cervical cancer cell proliferation, apoptosis and invasion via modulating PI3K/AKT/mTOR signal pathway. Eur Rev Med Pharmacol Sci 22, 1891-1898. Wang, J.M., Ju, B.H., Pan, C.J., Gu, Y., Li, M.Q., Sun, L., Xu, Y.Y. and Yin, L.R., 2017. MiR-214 inhibits cell migration, invasion and promotes the drug sensitivity in human cervical cancer by targeting FOXM1. Am J Transl Res 9, 3541-3557. Wen, Z., Lei, Z., Jin-An, M., Xue-Zhen, L., Xing-Nan, Z. and Xiu-Wen, D., 2014. The inhibitory role of miR-214 in cervical cancer cells through directly targeting mitochondrial transcription factor A (TFAM). Eur J Gynaecol Oncol 35, 676-82. Xu, M., Chen, X., Lin, K., Zeng, K., Liu, X., Xu, X., Pan, B., Xu, T., Sun, L., He, B., Pan, Y., Sun, H. and Wang, S., 2019. lncRNA SNHG6 regulates EZH2 expression by sponging miR-26a/b and miR-214 in colorectal cancer. J Hematol Oncol 12, 3. Zhang, K., Zhang, M., Jiang, H., Liu, F., Liu, H. and Li, Y., 2018. Down-regulation of miR-214 inhibits proliferation and glycolysis in non-small-cell lung cancer cells via down-regulating the expression of hexokinase 2 and pyruvate kinase isozyme M2. Biomed Pharmacother 105, 545-552.
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Table 1 Primers and oligonucleotides used in this work. Name
Sequence (5’–3’)
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Figures
Fig.1. MKK3 expression is inhibited by miR-214 directly. (A) miR-214 potentially targets the 3’UTR of MKK3 and its mutated sequence. pri-miR-214 or ASO-miR-214 were transfected into HeLa and C33A cells, and (B) qRT-PCR was performed to detect the expression of miR-214. U6 snRNA was used for normalization. (C) HeLa cells were co-transfected with MKK3 3’UTR or 3’UTR mut, and detected the EGFP intensity. (D, E) The level of MMK3 was analyzed by qRT-PCR and western blot. (F, G) qRT-PCR analysis of the transcripts of miR-214 and MKK3 in the 30 pairs of cervical cancer tissues and matched normal tissues. The results are given as mean ± SD values for three experiments. *p < 0.05, ** p < 0.01.
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Fig.2. miR-214 represses the malignant phenotypes of cervical cancer. pri-miR-214 or ASO-miR-214 were transfected into HeLa and C33A cells, followed by the MTT analysis after 48 h (A), colony formation (B), migration (C) and invasion assays (D). The volume of the tumors in the nude mice xenograft experiment showed the effect of miR-214 on tumor growth in vivo (E). Tumor size were measured every 2 days after 9 days of injection. The tumor volume was calculated as follows: length×width2×1/2. All the mice were sacrificed 21 days after injection. Representative images are shown. The results are given as mean ± SD values. *p < 0.05, ** p < 0.01.
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Fig.3. Repression of MKK3 suppresses the malignant phenotypes of cervical cancer. Cells were transfected with siR-MKK3, followed by the western blot analysis of MKK3 expression after 48 h (A), MTT analysis (B), colony formation (C), migration (D) and invasion assays (E). The volume of the tumors in nude mice xenograft experiment (F) showed the effect of siR-MKK3 on tumor growth in vivo. Representative images are shown. The results are given as mean ± SD values. *p < 0.05, ** p < 0.01.
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Fig.4. miR-214-repressed malignant phenotypes of HeLa cells were rescued by MKK3 over-expression. Cells were co-transfected with the pcDNA3/MKK3 vector with or without pri-miR-214 vector, followed by the western blot detection of MKK3 protein level at 48 h post-transfection (A), MTT (B), migration (C) and invasion assays (D). Representative images of the randomly selected fields are shown. The results are given as mean ± SD values for three experiments. *p < 0.05, ** p < 0.01.
Abstract: miRNA mediated genetic regulation is widely involved in carcinogenesis of cervical cancer. In this study, miR-214 was confirmed to directly regulate MKK3 via imperfect base-pairing to its 3’UTR, resulting down-regulation of its expression level. Compared to normal tissues, a down-regulated level of miR-214 was observed in cervical cancer, while MKK3 was up-regulated. Next, we demonstrated that over-expression of miR-214 or knockdown of MKK3 can inhibit the growth, proliferation, invasion and migration of cervical cancer in vitro and in vivo. Moreover, the effects of miR-214 in HeLa cells were rescued by the restoration of MKK3. In conclusion, our results laid new foundations for investigating the pathogenesis and diagnosis of cervical cancer.
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1miR-214 targets MKK3 2 MKK3 maybe an oncogene in cervical cancer 3 MKK3 rescues miR-214 1
2 3 4 5
miRNAs: microRNAs 3’UTR: 3’-untranslated region miR-214: microRNA-214 MAPK: mitogen-activated protein kinase MKK3: mitogen-activated protein kinase kinase3
Conflict of interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
Ruiqing Peng, Xiangshan Cheng, Yue Zhang, Xin Lu, Zhidong Hu
Declaration of Interest Statement: We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Ruiqing Peng, Xiangshan Cheng, Yue Zhang, Xin Lu, Zhidong Hu
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