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Microrna-488 Inhibits Ovarian Cancer Cell Metastasis Through Regulating CCNG1 and P53 Expression

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Microrna-488 Inhibits Ovarian Cancer Cell Metastasis Through Regulating CCNG1 and P53 Expression European Review for Medical and Pharmacological Sciences 2020; 24: 2902-2910 MicroRNA-488 inhibits ovarian cancer cell metastasis through regulating CCNG1 and p53 expression J.-Y. GUO, X.-Q. WANG, L.-F. SUN Department of Obstetrics and Gynecology, Beijing Jishuitan Hospital, Beijing, China Jiayi Guo and Xueqing Wang contributed equally to this work Abstract. – OBJECTIVE: The roles of mi- serious threat to women’s lives2. The reason for croRNAs (miRNAs) have been widely exploit- the high mortality rate is that the growth site of ed in cancer. MiRNAs have become a poten- OC is concealed and it is difficult to find it ear- tial breakthrough in cancer diagnosis and treat- ly3. In addition, the symptoms of early OC are ment. Here, the regulatory mechanism of mi- not significant, and there is still a lack of simple croRNA-488 (miR-488) was investigated in ovar- 4 ian cancer (OC). and practical diagnostic methods . Most OC pa- PATIENTS AND METHODS: The expression tients have advanced in the first diagnosis, with levels of miR-488 and CCNG1 (Cyclin G1) were a 5-year survival rate of approximately 25-50%. detected by quantitative Real Time-Polymerase Cell metastasis is the main cause of poor prog- Chain Reaction (qRT-PCR) and Western blot as- nosis in patients with OC5. There is an urgent says. Transwell assay and epithelial-mesenchy- need to investigate the molecular mechanism of mal transition (EMT) markers were used to clar- ify the effect of miR-488 on cell metastasis. The cell metastasis to find effective biomarkers for dual-luciferase reporter assay was used to ver- early diagnosis of OC patients. ify the relation between miR-488 and CCNG1. As a research hotspot, microRNA (miRNA) RESULTS: The expression of miR-488 was re- has been widely explored in the pathogenesis duced in OC, which was associated with poor of human cancer. MiRNAs play an important clinical outcomes and prognosis in OC pa- role in gene regulation and tumorigenesis6. Some tients. MiR-488 inhibited cell metastasis in OC by blocking EMT and promoting tumor suppres- miRNAs have been reported to have the poten- sor p53 expression. In addition, CCNG1 was tial to diagnose and treat OC. Of note, miR-423 confirmed as a direct target of miR-488. Upreg- served as a diagnostic indicator and inhibited the ulation of CCNG1 impaired the inhibitory effect proliferation and invasion of OC cells7. MiR-532 of miR-488 in OC. was a prognostic marker that suppressed cell pro- CONCLUSIONS: MiR-488 serves as a tumor liferation and invasion by targeting TWIST1 in inhibitor in OC by suppressing cell metastasis, epithelial OC8. In contrast, miR-590 was found to indicating that miR-488 has a great potential in the diagnosis and treatment of OC. promote tumor growth and metastasis in OC via the novel FOXA2-versican pathway9. Among the Key Words: numerous miRNAs, an abnormal regulation of Ovarian cancer, MiR-488, CCNG1, p53, Cell me- miR-488 has been found in various cancers and it tastasis. is actively involved in the pathogenesis of human cancers. Downregulation of miR-488 has been identified in glioma, malignant melanoma, and Introduction prostate carcinoma10-12. Moreover, it was report- ed13 that a low miR-488 expression was associ- Ovarian cancer (OC) accounts for 2.4% to ated with tumor node metastasis (TNM) stage in 6.5% of common malignant tumors in women, patients with gastric cancer and acted as a tumor ranking third in female reproductive system suppressor. However, aberrant expression and carcinomas, followed by cervical cancer and biological functions of miR-488 have not been re- endometrial cancer1. Epithelial cancer is the ported in OC, which still needs to be investigated. most common in OC and its mortality is the Cyclin G1 (CCNG1) has been identified as first in various gynecological tumors, posing a a new member of the cyclin family that shares 2902 Corresponding Auth-or: Jiayi Guo, MD; e-mail: [email protected] MicroRNA-488 inhibits ovarian cancer cell metastasis homology with c-src14. CCNG1 has contrast tis- Quantitative Real Time-Polymerase sue specificity and can regulate cell cycle15. In Chain Reaction (qRT-PCR) addition, CCNG1 was reported to be upregulated The total RNA was extracted from tissue sam- in nasopharyngeal carcinoma and was correlated ples or cell lines using TRIzol reagent (Jining with tumor progression16. Besides that, CCNG1 is Shiye, Shanghai, China). The complementary a transcriptional target of p53 tumor suppressor deoxyribose nucleic acid (cDNA) was obtained protein and negatively regulates p53 expression17. using the iScript cDNA Synthesis kit (Bio-Rad, It was found that CCNG1 induced expansion of Hercules, CA, USA). The temperature conditions liver tumor-initiating cells by Sox2 via protein of the reverse transcription were as follows: 37°C kinase B (AKT)/mammalian target of rapamycin for 15 min and 85°C for 5 sec. The mixture of the (mTOR) signaling18. Moreover, miR-122/CCNG1 qRT-PCR standard reaction system was placed interaction has been identified to modulate p53 on ABI7300 real-time PCR machine (Applied activity in hepatocellular carcinoma cells19. In Biosystems, Foster City, CA, USA) using the ABI particular, miR-1271 inhibited OC tumor growth Power SYBR Green PCR Master Mix (Unique, by targeting CCNG120. However, as far as we Beijing, China). The thermocycling conditions know, the interaction between CCNG1 and miR- for PCR amplification were as follows: 5 min 488 in OC has not been elucidated. at 95°C, followed by 38 cycles of 95°C for 30 Therefore, the relation between miR-488 and sec, and 60°C for 45 sec. U6 and glyceraldehyde CCNG1 and their effect on OC cell metastasis 3-phosphate dehydrogenase (GAPDH) were used were investigated. At the same time, the associ- as controls for miR-488 and CCNG1, which were ation between miR-488 and clinical outcomes or quantified by the −∆∆ct2 method. The primers prognosis in OC patients was analyzed. In addi- used in our work were as follows: miR-488, tion, it was explored how miR-488 regulates p53 forward primer: 5’-TTATTGCGATGTGTTCCT- expression. Our results will provide new ideas for TATG-3’, reverse primer: 5’-ATGCATGCCAC- the therapeutic targets of human OC. GGGCATATACACT-3’; U6, forward prim- er: 5’-CTCGCTTCGGCAGCACA-3’, reverse primer: 5’-AACGCTTCACGAATTTGCGT-3’; Patients and Methods CCNG1 forward primer: 5’-GTTACCGCTGAG- GAGCTGCAGTC-3’, reverse primer: 5’-GCAG- Clinical Tissues CCATCCTGGATGGATTCAG-3’; GAPDH for- Experiment tissues were obtained from 58 ward, 5’-ACATCGCTCAGACACCATG-3’, re- patients with OC at the Beijing Jishuitan Hospi- verse, 5’-TGTAGTTGAGGTCAATGAAGGG-3’. tal. All OC patients enrolled in this research did not receive any other treatment prior to surgery. Transwell Assay Before the study began, participants provided The upper transwell chamber (8-μm pore size written informed consent and the Human Eth- membranes) surface of the bottom membrane ics Committee of Beijing Jishuitan Hospital ap- was coated with Matrigel (BD, Franklin Lakes, proved the investigation. NJ, USA). 3×104 SKOV3 cells were added to the transwell chamber. Then, a medium containing Cell Culture and Transfection 20% FBS (600 μL) was added to the 24-well plate Human OC cell lines A2780, OVCAR3, in the lower chamber. After 24 h of routine incu- SKOV3, and normal human ovarian epithelium bation, the cells were fixed and stained. Fields of IOSE80 cells were purchased from the Ameri- x200 magnification of each insert were randomly can Type Culture Collection (ATCC; Manassas, selected and counted under a light microscope VA, USA). These cells were incubated at 37°C (Olympus, Tokyo, Japan). Transwell cell migra- with 5% CO2 in Roswell Park Memorial Insti- tion assay was performed without Matrigel and tute-1640 (RPMI-1640) medium (HyClone; South the other procedure was essentially consistent Logan, UT, USA) with 10% fetal bovine serum with the transwell invasion assay. (FBS; Gibco, Rockville, MD, USA). Lipofect- amine 2000 (Invitrogen, Carlsbad, CA, USA) was Western Blot Analysis used to transfer miR-488 mimics or inhibitors, Radioimmunoprecipitation assay (RIPA) buf- CCNG1 plasmid and unexpressed sequence (NC, fer (Beyotime, Shanghai, China) was used to lyse Generay Biotech, Shanghai, China) to SKOV3 the protein samples. Equal amounts of protein cells, respectively. were loaded for 10% sodium dodecyl sulfate-poly- 2903 J.-Y. Guo, X.-Q. Wang, L.-F. Sun acrylamide gel electrophoresis (SDS-PAGE) on (ANOVA) test followed by Post-Hoc Test (Least polyacrylamide gels, followed by transfer to poly- Significant Difference). Survival differences vinylidene difluoride (PVDF) membranes (Mil- were calculated by Kaplan-Meier analysis with lipore, Billerica, MA, USA). The membranes the log-rank test. Data are shown as mean ± SD were blocked in 5% dried skimmed milk for 2 h (standard deviation). When p<0.05, the difference at room temperature. Next, the protein was incu- is considered significant. bated with the corresponding primary antibodies overnight at 4°C, including E-cadherin, N-cad- herin, Vimentin, p53, CCNG1, and GAPDH. Af- Results ter washing, PVDF membranes were incubated with the diluted secondary antibodies for 1 h at MiR-488 was Downregulated in room temperature. Finally, proteins were detected OC Tissues using the enhanced chemiluminescence (ECL) The mRNA expression of miR-488 was ob- protein detection kit (ECL; Millipore, Billerica, served in OC tissues. The qRT-PCR assay showed MA, USA). The signals from target proteins were that miR-488 was downregulated in OC tissues normalized to those of GAPDH. compared to normal tissues (Figure 1A). Fur- thermore, the imbalance in miR-488 expression Dual-Luciferase Reporter Gene Assay was closely related to FIGO stage or lymph node The 3ʹ-untranslated region (3ʹ-UTR) of wild- metastasis in OC patients (Table I). In addition, type or mutant CCNG1 was inserted into the OC patients with low expression of miR-488 pRL-SV40 Renilla plasmid (Promega, Madison, had shorter overall survival, suggesting that the WI, USA).
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