miR-142-5p Regulates the Proliferation and Metastasis of Gallbladder Carcinoma Cells through CCND1
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摘要:
目的 研究miR-142-5p调控CCND1对胆囊癌细胞增殖和转移的作用，并阐明其具体作用机制。 方法 采用RT-qPCR鉴定miR-142-5p在人胆囊上皮细胞和人胆囊细胞系中的表达差异。双荧光素酶报告基因实验用于验证miR-142-5p与CCND1的靶向关系。CCK-8用于检测人胆囊细胞增殖。Transwell和划痕实验检测胆囊细胞迁移。Western blot检测上皮间充质转化标志蛋白的表达水平。 结果 miR-142-5p在胆囊癌细胞系中显著低表达，且在GBC-SD细胞中表达最低。双荧光素酶报告基因实验显示，miR-142-5p能够抑制野生型CCND1的荧光素酶活性。 结论 功能试验表明，过表达miR-142-5p抑制GBC-SD细胞的增殖和转移，同时过表达miR-142-5p和CCDN1能够部分逆转过表达CCDN1对GBC-SD细胞增殖和转移的影响。证实miR-142-5p通过抑制CCND1表达，进而抑制胆囊癌细胞的增殖和转移。 -
关键词:
- 胆囊癌 /
- miR-142-5p /
- CCND1 /
- 增殖 /
- 转移
Abstract:Objective To explore the effect of miR-142-5p on the proliferation and metastasis of gallbladder cancer through CCND1 and to clarify the special mechanism of action. Methods RT-qPCR was performed to identify the differential expression of miR-142-5p in human gallbladder epithelial cells HGBEC and human gallbladder cell lines. Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-142-5p and CCND1. CCK-8 was used for detecting human gallbladder cell proliferation. Transwell and wound healing assays were carried out to detect gallbladder cell migration. Western blot was performed to detect the marker expression of epithelial mesenchymal transition. Results miR-142-5p was significantly low expressed in gallbladder cancer cell lines, and lowest expressed in GBC-SD cells. Dual luciferase reporter gene assays revealed that miR-142-5p was able to inhibit the luciferase activity of wild-type CCND1. Conclusion Overexpressing of miR-142-5p inhibited the proliferation and metastasis of GBC-SD cells, simultaneous overexpression of miR-142-5p and CCDN1 partially reversed the effect of overexpressing CCDN1 on the proliferation and metastasis of GBC-SD cells. We demonstrate that miR-142-5p can inhibit the proliferation and metastasis of gallbladder cancer cells by suppressing CCND1 expression. -
Key words:
- Gallbladder /
- miR-142-5 p /
- CCND1 /
- Proliferation /
- Metastasis
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图 1 miR-142-5p在胆囊癌细胞系中表达降低
与HGBEC组相比，**P < 0.01，***P < 0.001；与GBC-SD组相比，△P < 0.05。
Figure 1. The expression of miR-142-5p decreased in gallbladder cancer cell line.
图 2 过表达miR-142-5p抑制GBC-SD细胞的增殖和转移
A：RT-qPCR检测miR-142-5p mimics转染效率；B：CCK-8检测细胞增殖活力；C和E：Transwell检测细胞侵袭能力；D和F：划痕实验检测细胞迁移能力；G：Western blot检测细胞EMT标志物E-cadherin、N-cadherin、Vimentin的表达；与NC mimics组相比，*P < 0.05，**P < 0.01，***P < 0.001。
Figure 2. Overexpression of miR-142-5p inhibited the proliferation and metastasis of GBC-SD cells
图 3 miR-142-5p靶向CCND1
A：starBase生信数据库预测miR-142-5p与CCND1的靶向结合序列；B：双荧光素酶报告基因实验验证miR-142-5p和CCND1的靶向关系，与NC mimics组相比，**P < 0.01；C和E：Western blot检测HGBEC和GBC-SD细胞中CCND1的表达，与HGBEC组相比，**P < 0.01；D和F：Western blot检测过表达miR-142-5p对CCND1表达的影响，与NC mimics组相比，**P < 0.01。
Figure 3. miR-142-5p targeted CCND1
图 4 miR-142-5p靶向CCND1抑制GBC-SD细胞的增殖和转移
A：Western blot检测CCND1的表达；B：CCK-8检测细胞增殖活力；C和D：Transwell检测细胞侵袭；E和F：划痕实验检测细胞迁移；G：Western blot检测细胞EMT标志物的表达；与pcDNA-NC+NC mimics组相比，*P < 0.05，**P < 0.01；与pcDNA-CCND1+NC mimics组相比，△P < 0.05，△△P < 0.01，△△△P < 0.001。
Figure 4. miR-142-5p inhibited cell proliferation and metastasis of GBC-SD cells by targeting CCND1.
表 1 引物序列
Table 1. Primer sequences
基因 引物序列（F：Forward primer；R：Reversed primer；5′ -3′ ） miR-142-5p F: AACTCCAGCTGGTCCTTAG R: TCTTGAACCCTCATCCTGT U6 F: CTCGCTTCGGCAGCACA R: AACGCTTCACGAATTTGCGT 
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[1] Ganeshan D,Kambadakone A,Nikolaidis P,et al. Current update on gallbladder carcinoma[J]. Abdom Radiol (NY),2021,46(6):2474-2489. doi: 10.1007/s00261-020-02871-2 [2] Singh B P,Khan W F,Rathore Y S,et al. Incidental Carcinoma Gallbladder:Incidence,Risk Factors,and Factors Affecting Survival-5-Year Experience from a Tertiary Care Institute[J]. J Gastrointest Cancer,2020,51(3):980-987. doi: 10.1007/s12029-019-00347-1 [3] Hundal R,Shaffer E A. Gallbladder cancer:epidemiology and outcome[J]. Clin Epidemiol,2014,6:99-109. [4] Valihrach L,Androvic P,Kubista M. Circulating miRNA analysis for cancer diagnostics and therapy[J]. Mol Aspects Med,2020,72:100825. doi: 10.1016/j.mam.2019.10.002 [5] Troschel F M, Böhly N, Borrmann K, et al. miR-142-3p attenuates breast cancer stem cell characteristics and decreases radioresistance in vitro[J]. Tumour Biol, 2018, 40（8）: 1010428318791887. [6] Li Z,Lan Y,Zhao K,et al. miR-142-5p Disrupts Neuronal Morphogenesis Underlying Porcine Hemagglutinating Encephalomyelitis Virus Infection by Targeting Ulk1[J]. Front Cell Infect Microbiol,2017,7:155. doi: 10.3389/fcimb.2017.00155 [7] Zhang D,Wang Y,Xia Y,et al. Repression of miR-142-3p alleviates psoriasis-like inflammation by repressing proliferation and promoting apoptosis of keratinocytes via targeting Sema3A[J]. Mol Cell Probes,2020,52:101573. doi: 10.1016/j.mcp.2020.101573 [8] Anandagoda N,Willis J C,Hertweck A,et al. microRNA-142-mediated repression of phosphodiesterase 3B critically regulates peripheral immune tolerance[J]. J Clin Invest,2019,129(3):1257-1271. doi: 10.1172/JCI124725 [9] Wang J, Jin Y, Li S, et al. Identification of microRNAs associated with the survival of patients with gallbladder carcinoma[J]. J Int Med Res, 2020, 48（5）: 300060520918061. [10] Chen C,Geng Z,Shen H,et al. Long-Term Outcomes and Prognostic Factors in Advanced Gallbladder Cancer:Focus on the Advanced T Stage[J]. PLoS One,2016,11(11):e0166361. doi: 10.1371/journal.pone.0166361 [11] Zhang X,Zhang L,Chen M,et al. miR-324-5p inhibits gallbladder carcinoma cell metastatic behaviours by downregulation of transforming growth factor beta 2 expression[J]. Artif Cells Nanomed Biotechnol,2020,48(1):315-324. doi: 10.1080/21691401.2019.1703724 [12] Lu W,Hu Y,Ma Q,et al. miR-223 increases gallbladder cancer cell sensitivity to docetaxel by downregulating STMN1[J]. Oncotarget,2016,7(38):62364-62376. doi: 10.18632/oncotarget.11634 [13] Zong H,Zhou H,Xiang Y,et al. miR-125b suppresses cellular proliferation by targeting c-FLIP in gallbladder carcinoma[J]. Oncol Lett,2019,18(6):6822-6828. [14] Qin Y,Mi W,Huang C,et al. Downregulation of miR-575 Inhibits the Tumorigenesis of Gallbladder Cancer via Targeting p27 Kip1[J]. Onco Targets Ther,2020,13:3667-3676. doi: 10.2147/OTT.S229614 [15] Lu W,Zhang Y,Zhou L,et al. miR-122 inhibits cancer cell malignancy by targeting PKM2 in gallbladder carcinoma[J]. Tumour Biol,2015:15615-15625. [16] Gong Y Q,Ni J L,Fang Q,et al. MiR-1231 enhances docetaxel sensitivity to gallbladder carcinoma cells by downregulating FOXC2[J]. Eur Rev Med Pharmacol Sci,2020,24(23):12116-12123. [17] Li X,Chen W,Jin Y,et al. miR-142-5p enhances cisplatin-induced apoptosis in ovarian cancer cells by targeting multiple anti-apoptotic genes[J]. Biochem Pharmacol,2019,161:98-112. doi: 10.1016/j.bcp.2019.01.009 [18] Wang Z,Liu Z,Fang X,et al. MiR-142-5p Suppresses Tumorigenesis by Targeting PIK3CA in Non-Small Cell Lung Cancer[J]. Cell Physiol Biochem,2017,43(6):2505-2515. doi: 10.1159/000484459 [19] Chen G,Hu M,Qu X,et al. MicroRNA-584 directly targets CCND1 and inhibits cell proliferation and invasion in pancreatic cancer[J]. Mol Med Rep,2019,19(1):719-726. [20] Long B,Li N,Xu X X,et al. Long noncoding RNA LOXL1-AS1 regulates prostate cancer cell proliferation and cell cycle progression through miR-541-3p and CCND1[J]. Biochem Biophys Res Commun,2018,505(2):561-568. doi: 10.1016/j.bbrc.2018.09.160 -
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