特异性小干扰RNA下调中期因子基因表达对黑素瘤细胞黏附和侵袭的影响

Abstract

Abstract:

Objective To study the effects of midkine（MK） gene-targeting small interfering RNA （siRNA） on the invasion of melanoma cells. Methods Three MK gene-targeting siRNAs（S1, S2 and S3） were designed, constructed, and transfected into human A375 melanoma cells. Real-time PCR was performed to measure the expression of MK gene and to screen the siRNA with best efficacy. Then, A375 cells were transfected with the optimal siRNA of various doses （3.125, 6.25 and 12.5 nmol/L） followed by additional culture of various durations （24, 48, 72 hours）. Some A375 cells remaining untreated served as the blank control group, and some transfected only with liposomes served as the vector control group. Reverse transcription （RT）-PCR and Western blot were conducted to detect the mRNA and protein expression of MK, respectively, MTT assay to observe the adhesion of A375 cells, and Boyden chamber was used to evaluate cell invasion. Results The expression of MK mRNA was downregulated by all the three siRNAs, especially by the siRNA S3, which was used in the following transfection experiment. Real-time quantitative PCR revealed that the MK mRNA expression was reduced by the siRNA in a dose- （r 24 hours = -0.906, r 48 hours = -0.922, r 72 hours = -0.939, all P < 0.01） and time-dependent （r 3.125 nmol/L = -0.889, r 6.25 nmol/L = -0.935, r 12.5 nmol/L = -0.928, all P < 0.01） manner. MTT assay showed that the percentage of adhesing cells was 73.66% ± 2.25%, 49.36% ± 2.16% and 28.35% ± 1.68% in A375 cells transfected with the siRNA of 3.125, 6.25 and 12.5 nmol/L, respectively. The number of cells migrating across the chamber filter was 23.9 ± 1.6, 12.1 ± 1.5, 5.6 ± 1.2 among A375 cells transfected with the siRNA of 3.125, 6.25 and 12.5 nmol/L, respectively, significantly lower than that in the blank control group （36.8 ± 1.5）. The percentage of adhesing cells and number of migrating cells decreased with the dose of siRNA （r = -0.936, -0.915, P < 0.01, 0.05, respectively）. Conclusions MK gene might play an important role in the adhesion and invasion of melanoma cells. To down-regulate the expression of MK gene by siRNA may suppress the adhesion and invasion of melanoma cells.

Key words: invasion

References

[1] Tsao H,Atkins MB,Sober AJ.Management of cutaneous melanoma.N Engl J Med,2004,351:998-1012. [2]Takada T, Toriyama K, Muramatsu H, et al. Midkine, a retinoic acid-inducible heparin-binding cytokine in inflammatory responses: chemotactic activity to neutrophils and association with inflammatory synovitis. J Biochem,1997,122: 453-458. [3]Shimada H, Nabeya Y, Tagawa M,et al. Preoperative serum midkine concentration is a prognostic marker for esophageal squamous cell carcinoma. Cancer Sci,2003;94:628-632. [4] Barthlen W, Flaadt D, Girgert R,et al. Significance of heparin-binding growth factor expression on cells of solid pediatric tumors. J Pediatr Surg,2003,38:1296-304. [5] Kadomatsu K, Muramatsu T. Midkine and pleiotrophin in neural development and cancer. Cancer Lett 2004;204:127-143. [6] Obata Y, Kikuchi S, Lin Y,et al. Tokyo Research Group on Prevention of Gastric Cancer. Serum midkine concentrations and gastric cancer. Cancer Sci,2005,96:54-56. [7] Maehara H, Kaname T, Yanagi K,et al. Midkine as a novel target for antibody therapy in osteosarcoma. Biochem Biophys Res Commun,2007,358:757-762. [8] Krzystek-Korpacka M, Matusiewicz M, Diakowska D, et al. Serum midkine depends on lymph node involvement and correlates with circulating VEGF-C in oesophageal squamous cell carcinoma. Biomarkers,2007,12:403-413. [9] Maeda S, Shinchi H, Kurahara H,et al. Clinical significance of midkine expression in pancreatic head carcinoma. Br J Cancer,2007,6;97:405-411. [10]Tanabe K, Matsumoto M, Ikematsu S, et al. Midkine and its clinical significance in endometrial carcinoma. Cancer Sci,2008,99:1125-1130. [11] Friedrich C, Holtkamp N, Cinatl J Jr, et al. Overexpression of Midkine in malignant peripheral nerve sheath tumor cells inhibits apoptosis and increases angiogenic potency. Int J Oncol. 2005,27:1433-1440. [12] Tong Y, Mentlein R, Buhl R, et al. Overexpression of midkine contributes to anti-apoptotic effects in human meningiomas. J Neurochem,2007,100:1097-1107. [13] 范钰，郑树，丁佳逸. 脂质体介导的cripto-1反义寡核苷酸抑制黑色素瘤细胞端粒酶活性. 中国病理生理杂志，2006，22：761-765. [14] Fan Y,Zhang YL,Wu Y,et al. Inhibition of STAT3 expression by RNA interference suppresses invasion through inducing anoikis in human colon cancer cell. World J Gastroenterol, 2008,14:428-434. [15] Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature,2001,411: 494-498. [16]Woo MM, Salamanca CM, Minor A,et al. An improved assay to quantitate the invasiveness of cells in modified Boyden chambers. In Vitro Cell Dev Biol Anim,2007,43:7-9.

[1]	WANG Hong-wei, WANG Xiu-li, GUO Ming-xia, LIU Fang, LE Jia-yu. Topical imiquimod in the treatment of bowenoid papulosis:a retrospective study[J]. Chinese Journal of Dermatology, 2007, 40(2): 80 -82 .
[2]	HUANG Jin-mei, ZHENG He-ping, PAN Hui-qing, ZENG Wei-ying, WU Xing-zhong, LI Mei-ling, XUE Yao-hua. External quality assessment of STD laboratories in Guangdong Province[J]. Chinese Journal of Dermatology, 2007, 40(5): 260 -262 .
[3]	CHEN Xian-zhen, CHENG Hao, TANG Xiao-yan, ZHANG Xing, ZHU Ke-jian, YE Jun, LIN Ai-hua, CEN Jian-ping, JIN Na. Silencing of HPV11ET gene expression by different RNA interference technology[J]. Chinese Journal of Dermatology, 2007, 40(5): 267 -270 .
[4]	. [J]. Chinese Journal of Dermatology, 2007, 40(6): 374 -375 .
[5]	. [J]. Chinese Journal of Dermatology, 2007, 40(6): 382 .
[6]	. [J]. Chinese Journal of Dermatology, 2007, 40(6): 370 .
[7]	. [J]. Chinese Journal of Dermatology, 2008, 41(1): 29 .
[8]	. [J]. Chinese Journal of Dermatology, 2008, 41(1): 36 .
[9]	. Effect of antimicrobial barrier dressing on delayed wound repair[J]. Chinese Journal of Dermatology, 2008, 41(2): 127 .
[10]	. [J]. Chinese Journal of Dermatology, 2008, 41(3): 160 .

Down-regulation of midkine gene expression by small interfering RNA affects melanoma cell adhesion and invasion

PDF

Like

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 3

Metrics

Comments

Recommended 10

[1]	Li Jing, Yu Yin, Chen Jing, Zhang Xin, Liu Dongyang, Li Zhi, Diao Qingchun, Liu Sutao, Jin Jing, Wang Can. Mechanism underlying the inhibitory effect of overexpression of tumor suppressor gene DKK3 on migration and invasion of human cutaneous malignant melanoma cells [J]. Chinese Journal of Dermatology, 2018, 51(12): 874-878.
[2]	. Effects of metformin on transforming growth factor?beta1?induced epithelial?mesenchymal transition and invasion in the human melanoma cell line 1205Lu [J]. Chinese Journal of Dermatology, 2017, 50(6): 426-430.
[3]	. Inhibitory effects of siRNA targeting survivin on the growth of a human melanoma cell line, M14 [J]. Chinese Journal of Dermatology, 2009, 42(3): 189-192.