LncRNA-TUG1 Affects 22Rv1 Cell Proliferation and Migration by Modulating miR-31-5p/YWHAE Axis
ZHAO Jia-Fu*, LI Yong, ZHOU Ming-Shuai, WEN Xiao-Yan, LU Qing-Mei, LIU Bin
Guizhou University College of Animal Science/Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education, Guizhou University, Guiyang 550025, China
Abstract：Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein ε (YWHAE) was highly expressed in prostate cancer, and miR-31-5p could target and regulate the expression of YWHAE to affect the proliferation of prostate cancer cells. However, it was not clear which lncRNAs were involved in the targeted regulation of miR-31-5p against YWHAE. In this study, 3 LncRNAs with long non-coding RNA tauronic upregulation gene (LncRNA-TUG1), LINC02604 and PDCD6IP-DT were screened to target and regulate the expression of miR-31-5p based on the results of previous studies and bioanalysis software. The expression of 3 LncRNAs in prostate cancer LNCaP, PC3, 22Rv1 and normal prostate stromal immortem cells (WPMY-1) was detected by RT-qPCR, and the optimal LncRNAs regulating the miR-31-5p/YWHAE axis were screened. The effects of interfering LncRNA-TUG1 and overexpression of miR-31-5p on the expression of LncRNA-TUG1, miR-31-5p and YWHAE in 22Rv1 cells were detected by RT-qPCR. Meanwhile, the effects of co-transfection of sh-TUG1 and miR-31-5p mimics on the proliferation and migration of 22Rv1 cells were detected by CCK-8 and cell scratch assay. Western blot was used to detect the expression of proliferating cell nuclear antigen (PCNA), B cell lymphoma-2 (BCL-2), Bcl-2 associated X protein (BAX) and cysteine proteinase-3 (Caspase-3). The results showed that LncRNA-TUG1, LINC02604 and PDCD6IP-DT were the 3 LncRNAs regulating the miR-31-5p/YWHAE axis, and compared with normal cells, LncRNA-TUG1 was highly expressed in all PCa cell lines, especially in 22Rv1 cells. Taking LncRNA-TUG1 as the research object, after transfecting sh-TUG1 and miR-31-5p mimics into 22Rv1 cells, respectively, qPCR results showed that the expression of miR-31-5p in the miR-31-5p mimics group was significantly up-regulated compared with the NC mimics group (P<0.01). The expression of LncRNA-TUG1 and YWHAE genes was significantly down-regulated (P<0.05). Compared with the sh-NC group, there was no significant change in the expression of miR-31-5p in the sh-TUG1 transfection group, and the expression of LncRNA-TUG1 and YWHAE were significantly down-regulated (P<0.05). CCK-8 and cell scratch experiments showed that co-transfection of sh-TUG1 and miR-31-5p mimics could significantly inhibit cell proliferation and migration. In addition, Western blot results showed that co-transfection of sh-TUG1 and miR-31-5p mimics could significantly inhibit the expression of PCNA and BCL-2/BAX protein, and promoted the expression of caspase-3 protein.These results indicated that LncRNA-TUG1 could regulate the expression of PCNA, BCL-2, BAX and caspase-3 proteins by regulating miR-31-5p/YWHAE, and further affected the proliferation of 22Rv1 cells. This study provides theoretical basis and experimental ideas for the study of targeted drugs for prostate cancer.
[1] 代江娜, 刘倩, 刘霄岩. 2023. TUG1在急性心肌梗死患者血清中的表达及其对心肌细胞凋亡的影响[J]. 南昌大学学报(医学版), 63(01):49-54. (Dai J N Liu Q, Liu X Y.2023.Expression of TUG1 in serum of patients with acute myocardial infarction and its effect on cardiomyocyte apoptosis[J]. Journal of Nanchang University (Medical Edition), 63(01): 49-54.) [2] 金益峰, 周勇, 王厚明, 等. 2023. lncRNA TUG1在结直肠癌组织和细胞中的表达及其对癌细胞增殖、侵袭能力的影响[J]. 现代肿瘤医学, 31(12): 2270-2274. (Jin Y F, Zhou Y, W H Met al.2023. The expression of lncRNA TUG1 in colorectal cancer tissues and cells and its effect on the proliferation and invasion of cancer cells[J]. Modern Oncology, 31(12): 2270-2274.) [3] 宁椿游, 李贵林, 杨羽晨, 等. 2017. MiR-26a靶向调节PTEN对3T3-L1前脂肪细胞分化的促进效应[J]. 农业生物技术学报, 25(08): 1314-1325. (Ning C Y, Li G L, Yang Y Cet al.2017.MiR-26a promotes the differentiation of 3T3-L1 preadipocytes by targeting PTEN[J]. Journal of Agricultural Biotechnology, 25(08): 1314-1325.) [4] 宋敬福, 陈国俊. 2019. 前列腺癌的致病因素、肿瘤标志物及前列腺穿刺活检的研究进展[J]. 世界最新医学信息文摘, 19(92):73. (Song J F, Chen G J.2019. Research progress on pathogenic factors, tumor markers and prostate biopsy of prostate cancer[J]. World 's Latest Medical Information Abstract, 19(92): 73.) [5] 孙殿钦, 雷林, 蔡颖, 等. 2020. 前列腺癌相关危险因素的研究进展[J]. 中国肿瘤, 29(04): 292-298. (Sun D Q, Lei L, Cai Y, et al.2020. Research progress on risk factors related to prostate cancer[J].Tumor in China, 29(04): 292-298.) [6] 孙静, 石丽媛, 康美丽. 2023. 冠心病病人血清lncRNA TUG1和miR-140-3p水平与血清炎性因子、斑块稳定性指标的相关性[J]. 中西医结合心脑血管病杂志, 21(08): 1483-1486. (Sun J, Shi L Y, Kang M L.2023. Correlation of serum lncRNA TUG1 and miR-140-3p levels with serum inflammatory factors and plaque stability indexes in patients with coronary heart disease[J]. Journal of Cardiovascular and Cerebrovascular Disease Combined with Traditional Chinese and Western Medicine, 21(08): 1483-1486.) [7] 王亚辉, 王阳, 严云勤, 等. 2014. mir-133b对牛骨骼肌卫星细胞增殖的影响[J]. 黑龙江畜牧兽医, (13): 14-18. (Wang Y H, Wang Y, Yan Y Q, et al. 2014 The effect of mir-133b on the proliferation of bovine skeletal muscle satellite cells[J]. Heilongjiang Animal Husbandry and Veterinary Medicine, (13): 14-18.) [8] 张殿宝, 郝吉庆, 张宪芬, 等. 2023. LncRNA TUG1调控miR-142-5p/PD-L1轴促进非小细胞肺癌细胞增殖、迁移与侵袭能力[J]. 临床与实验病理学杂志, 39(03): 297-304. (Zhang D B, Hao J Q, Zhang X F, et al.2023.LncRNA TUG1 regulates miR-142-5p/PD-L1 axis to promote proliferation, migration and invasion of non-small cell lung cancer cells[J]. Journal of Clinical and Experimental Pathology, 39(03): 297-304.) [9] 张恒. 2019. 前列腺癌的发生和肥胖、血脂的关系[D]. 硕士学位论文, 昆明医科大学. 导师: 张劲松, pp: 6-7. (Zhang H.2019. Relationship between the occurrence of prostate cancer and obesity, blood lipids[D]. Thesis for M.S., Kunming Medical University, Supervisor: Zhang J S, pp: 6-7.) [10] 赵广义, 赵健, 高杰, 等. 2010. mirRNA-17-5p抑制物对骨肉瘤细胞系SOSP_9607增殖和凋亡的影响[J]. 现代生物医学进展, 10(08): 1405-1407. (Zhao G Y, Zhao J, Gao J, et al.2010 The effect of mirRNA-17-5p inhibitor on the proliferation and apoptosis of osteosarcoma cell line SOSP_9607[J].Modern Biomedical Progress, 10(08): 1405-1407.) [11] 赵佳福. 2020. MicroRNA-31-5p调控14-3-3ε影响前列腺癌细胞增殖及凋亡的分子机制研究[D]. 博士学位论文, 贵州大学, 导师: 许厚强, pp: 102. (Zhao J F.2020. Study on the molecular mechanism of microRNA-31-5p regulating 14-3-3ε to affect the proliferation and apoptosis of prostate cancer cells[D]. Thesis for Ph.D., Guizhou University, Supervisor: Xu H Q, pp: 102.) [12] Ahn J, Lee H, Chung C H, et al.2011. High fat diet induced downregulation of microRNA-467b increased lipoprotein lipase in hepatic steatosis[J]. Biochemical and Biophysical Research Communications, 414(4): 664-669. [13] Arias N, Aguirre L, Fernandez-Quintela A, et al.2016. MicroRNAs involved in the browning process of adipocytes[J]. Journal of Physiology and Biochemistry, 72(3): 509-521. [14] Deng Y, Zhang L, Luo R.2021. LINC01783 facilitates cell proliferation, migration and invasion in non-small cell lung cancer by targeting miR-432-5p to activate the notch pathway[J]. Cancer Cell International, 21(1): 234. [15] Du L B, Li H Z, Wang X H, et al.2014. Analysis of cancer incidence in Zhejiang cancer registry in China during 2000 to 2009[J]. Asian Pacific Journal of Cancer Prevention, 15(14): 5839-5843. [16] Ewing C M, Ray A M, Lange E M, et al.2012. Germline mutations in HOXB13 and prostate-cancer risk[J]. New England Journal Of Medicine, 366(2): 141-149. [17] Filella X, Foj L.2017. miRNAs as novel biomarkers in the management of prostate cancer[J]. Clinical Chemistry and Laboratory Medicine, 55(5): 715-736. [18] Guo B H, Zhao Q, Li H Y.2019. TUG1 promotes the development of prostate cancer by regulating RLIM[J]. European Review for Medical and Pharmacological Sciences, 23(5): 1926-1933. [19] Gupta R A, Shah N, Wang K C, et al.2010. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis[J]. Nature, 464(7291): 1071-1076. [20] Han Y, Liu Y, Gui Y, et al.2013. Long intergenic non-coding RNA TUG1 is overexpressed in urothelial carcinoma of the bladder[J]. Journal of Surgical Oncology, 107(5): 555-559. [21] Hu Y, Sun X, Mao C, et al.2017. Upregulation of long noncoding RNA TUG1 promotes cervical cancer cell proliferation and migration[J]. Cancer Medicine, 6(2): 471-482. [22] Kuang D, Zhang X, Hua S, et al.2016. Long non-coding RNA TUG1 regulates ovarian cancer proliferation and metastasis via affecting epithelial-mesenchymal transition[J]. Experimental and Molecular Pathology, 101(2): 267-273. [23] Li Y, Quan J, Chen F, et al.2019. MiR-31-5p acts as a tumor suppressor in renal cell carcinoma by targeting cyclin-dependent kinase 1 (CDK1)[J]. Biomedicine & Pharmacotherapy, 111: 517-526. [24] Liang S, Zhang S, Wang P, et al.2017. LncRNA, TUG1 regulates the oral squamous cell carcinoma progression possibly via interacting with Wnt/beta-catenin signaling[J]. Gene, 608: 49-57. [25] Liu X, Yu C, Bi Y, et al.2019. Trends and age-period-cohort effect on incidence and mortality of prostate cancer from 1990 to 2017 in China[J]. Public Health, 172: 70-80. [26] Lv D, Xiang Y, Yang Q, et al.2020. Long non-coding RNA TUG1 promotes cell proliferation and inhibits cell apoptosis, autophagy in clear cell renal cell carcinoma via MiR-31-5p/FLOT1 axis[J]. Onco Targets and Therapy, 13: 5857-5868. [27] Nie W, Ge H J, Yang X Q, et al.2016. LncRNA-UCA1 exerts oncogenic functions in non-small cell lung cancer by targeting miR-193a-3p[J]. Cancer Letters, 371(1): 99-106. [28] Niu Y, Ma F, Huang W, et al.2017. Long non-coding RNA TUG1 is involved in cell growth and chemoresistance of small cell lung cancer by regulating LIMK2b via EZH2[J]. Molecular Cancer, 16(1): 5. [29] Ordonez-Mena J M, Schottker B, Mons U, et al.2016. Quantification of the smoking-associated cancer risk with rate advancement periods: Meta-analysis of individual participant data from cohorts of the CHANCES consortium[J]. Bmc Medicine, 14: 62. [30] Peng H, Wang L, Su Q, et al.2019. MiR-31-5p promotes the cell growth, migration and invasion of colorectal cancer cells by targeting NUMB[J]. Biomedicine & Pharmacotherapy, 109: 208-216. [31] Roddam A W, Allen N E, Appleby P, et al.2008. Endogenous sex hormones and prostate cancer: A collaborative analysis of 18 prospective studies[J]. Journal of the National Cancer Institute, 100(3): 170-183. [32] Song S, Sun K, Dong J, et al.2020. microRNA-29a regulates liver tumor-initiating cells expansion via Bcl-2 pathway[J]. Experimental Cell Research, 387(2): 111781. [33] Tse L A, Lee P, Ho W M, et al.2017. Bisphenol A and other environmental risk factors for prostate cancer in Hong Kong[J]. Environment International, 107: 1-7. [34] Vartolomei M D, Kimura S, Ferro M, et al.2018. The impact of moderate wine consumption on the risk of developing prostate cancer[J]. Clinical Epidemiology, 10: 431-444. [35] Wang L, Zhao Z, Feng W, et al.2016. Long non-coding RNA TUG1 promotes colorectal cancer metastasis via EMT pathway[J]. Oncotarget, 7(32): 51713-51719. [36] Wu D, Yang B, Chen J, et al.2018. Upregulation of long non-coding RNA RAB1A-2 induces FGF1 expression worsening lung cancer prognosis[J]. Cancer Letters, 438: 116-125. [37] Xiu D, Liu L, Cheng M, et al.2020. Knockdown of lncRNA TUG1 enhances radiosensitivity of prostate cancer via the TUG1/miR-139-5p/SMC1A axis[J]. Onco Targets and Therapy, 13: 2319-2331. [38] Zhao G, Han C, Zhang Z, et al.2017. Increased expression of microRNA-31-5p inhibits cell proliferation, migration, and invasion via regulating Sp1 transcription factor in HepG2 hepatocellular carcinoma cell line[J]. Biochemical and Biophysical Research Communications, 490(2): 371-377.
