目的 探究长链非编码RNA（long non-coding RNA，LncRNA）PVT1通过靶向miR-190对宫颈癌细胞生存及转移的影响。 方法 qRT-PCR鉴定宫颈癌细胞与正常宫颈细胞中lncRNA PVT1和miR-190的表达水平；生物信息学软件预测lncRNA PVT1和miR-190之间的靶向关系。MTT检测细胞增殖，克隆形成实验检测细胞生长，Hoechst染色和流式细胞术鉴定细胞凋亡，Transwell检测细胞侵袭，划痕实验检测细胞迁移；Western Blot验证体系中增殖、凋亡和上皮间质转化（epithelial to mesenchymal transition，EMT）相关分子表达。宫颈癌HeLa细胞经过sh-PVT1处理后进行裸鼠皮下移植瘤接种，检测肿瘤体积及肿瘤组织中增殖和凋亡相关分子表达。 结果 与正常宫颈细胞相比，lncRNA PVT1在各宫颈癌细胞中高表达，干扰PVT1后，miR-190表达量显著上调，经生物信息学和荧光素酶报告系统验证lncRNA PVT1和miR-190有较强结合性。sh-PVT1可有效抑制宫颈癌细胞增殖、侵袭和迁移，促进凋亡发生（P<0.01）；lncRNA PVT1可有效抑制EMT相关分子表达，降低宫颈癌细胞向EMT转化；miR-190抑制后可有效逆转上述现象（P<0.01）。体内实验证实PVT1敲降可以有效降低肿瘤细胞增殖，促进细胞凋亡（P<0.01）。 结论 lncRNA PVT1在宫颈癌中高表达，PVT1敲降后可有效解除PVT1对miR-190的抑制效果，从而降低宫颈癌细胞增殖、侵袭和迁移，并促进细胞凋亡。
Abstract
Objective To investigate the effects and mechanism of lncRNA PVT1 on the growth and migration of cervical cancer Hela cell via targeting miR-190. Methods The expression levels of lncRNA PVT1 and miR-190 in normal cervical cells and different cervical cancer cells were identified by qRT-PCR. The targeting relationship between lncRNA PVT1 and miR-190 was predicted by bioinformatics software. Cell proliferation was detected by MTT, cell growth was detected by clone formation assays, apoptosis was identified by Hoechst staining and flow cytometry, cell invasion was detected by Transwell, cell migration was detected by a scratch test; proliferation, apoptosis and epithelial-mesenchymal transition (EMT)-related molecular expression were verified by Western Blot. Cervical cancer HeLa cell was inoculated with subcutaneous xenografts of the nude mice after treated with sh-PVT1, and the related molecules expression of proliferation and apoptosis- in tumor tissues and tumor volume were detected. Results Compared with the normal cervical cells, lncRNA PVT1 was highly expressed in various cervical cancer cells. After interfering with PVT1, the expression level of miR-190 significantly up-regulated. LncRNA PVT1 and miR-190 had strong associativity, which was verified by bioinformatics and luciferase reporter system. Sh-PVT1 could effectively inhibit the proliferation, invasion and migration of cervical cancer cells and promote apoptosis (P<0.01). LncRNA PVT1 could effectively inhibit the related molecules expression of EMT- and reduce the transformation of cervical cancer cells into EMT. The above phenomenon was effectively reversed after the miR-190 inhibitor (P<0.01). In vivo experiments confirmed that PVT1 knockdown could effectively reduce tumor cell proliferation and promote cell apoptosis (P<0.01). Conclusions LncRNA PVT1 is highly expressed in cervical cancer. PVT1 knockdown can effectively relieve the inhibitory effect of PVT1 on miR-190, thereby reducing proliferation, invasion and migration of cervical cancer cells and promoting apoptosis.
关键词
长链非编码RNA PVT1 /
小RNA190 /
宫颈癌 /
上皮间质转化 /
增殖 /
凋亡
Key words
LncRNA PVT1 /
miR-190 /
Cervical cancer /
EMT /
Growth /
Apoptosis
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] 陈禹宏, 孟莹, 胡丽娜. 下调MARCH8基因的表达对宫颈癌Siha细胞侵袭、迁移与增殖的影响[J]. 中国免疫学杂志, 2018, 34(7): 13-17.
[2] Shi JF, Canfell K, Lew JB, et al. The burden of cervical cancer in china: synthesis of the evidence[J]. Int J Cancer, 2012, 130(3): 641-652.
[3] Sankaranarayanan R, Swaminathan R, Brenner H, et al. Cancer survival in africa, asia, and central america: a population-based study[J]. Lancet Oncol, 2010, 11(2): 165-173.
[4] Mattick JS, Makunin IV. Non-coding rna[J]. Hum Mol Genet, 2006, 15(1): R17-29.
[5] Chen YT, Bali D, Sullivan J. Prenatal diagnosis in glycogen storage diseases[J]. Prenat Diagn, 2002, 22(5): 357-359.
[6] Fatica A, Bozzoni I. Long non-coding rnas: New players in cell differentiation and development[J]. Nat Rev Genet, 2014, 15(1): 7-21.
[7] Heward JA, Lindsay MA. Long non-coding rnas in the regulation of the immune response[J]. Trends Immunol, 2014, 35(9): 408-419.
[8] Rossi MN, Antonangeli F. Lncrnas: new players in apoptosis control[J]. Int J Cell Biol, 2014, 5740(2014): 473857.
[9] De Coninck B, Carron D, Tavormina P, et al. Mining the genome of arabidopsis thaliana as a basis for the identification of novel bioactive peptides involved in oxidative stress tolerance [J]. J Exp Bot, 2013, 64(17): 5297-5307.
[10] 徐健, 江跃全, 蔡华荣, 等. miR-217调控lncRNA MALAT1影响食管鳞癌细胞的生物学行为[J]. 中国临床解剖学杂志, 2018, 36(4): 408-413.
[11] Yin J, Wen J, Hang D, et al. Expression quantitative trait loci for card8 contributes to risk of two infection-related cancers--hepatocellular carcinoma and cervical cancer[J]. PloS one, 2015, 10(7): e0132352.
[12] Wang XL, Wang GC, Zhang LJ, et al. Lncrna pvt1 promotes the growth of hpv positive and negative cervical squamous cell carcinoma by inhibiting tgf-beta1[J]. Cancer Cell Int, 2018, 18(1): 70.
[13]Shang CL, Wang W, Liao YD, et al. Lnmicc promotes nodal metastasis of cervical cancer by reprogramming fatty acid metabolism[J]. Cancer Res, 2018, 78(4): 877-890.
[14]Xue WS, Chen JJ, Liu XB, et al. PVT1 regulates the malignant behaviors of human glioma cells by targeting miR-190a-5p and miR-488-3p[J]. Biochim Biophys Acta Mol Basis Dis, 2018,1864(5): 173-1794.
[15]Chen W, Zheng R, Baade PD, et al. Cancer statistics in china, 2015[J]. CA Cancer J Clin 2016, 66(2): 115-132.
[16]Park S, Eom K, Kim J, et al. Mir-9, mir-21, and mir-155 as potential biomarkers for hpv positive and negative cervical cancer[J]. BMC cancer, 2017, 17(1): 658.
[17]Rui XH, Xu Y, Huang YQ, et al. Lncrna dlg1-as1 promotes cell proliferation by competitively binding with mir-107 and up-regulating zhx1 expression in cervical cancer[J]. Cell Physiol Biochem, 2018, 49(5): 1792-1803.
[18] Zhu H, Zheng T, Yu J, et al. Lncrna xist accelerates cervical cancer progression via upregulating fus through competitively binding with mir-200a[J]. Biomed Pharmacother, 2018, 105(1): 789-797.
[19] Cheng RJ, Li N, Yang SY, et al. Long non-coding rna zeb1-as1 promotes cell invasion and epithelial to mesenchymal transition through inducing zeb1 expression in cervical cancer[J]. Onco Targets Ther, 2018, 11(8): 7245-7253.
[20] Ma SY, Deng XH, Yang Y, et al. The lncrna linc00675 regulates cell proliferation, migration, and invasion by affecting wnt/beta-catenin signaling in cervical cancer[J]. Biomed Pharmacother, 2018, 108(2): 1686-1693.
[21] Liang HL, Zhang CY, Guan HY. Lncrna dancr promotes cervical cancer progression by upregulating rock1 via sponging mir-335-5p[J]. J Cell Physiol, 2019, 234(5): 7266-7278.
[22] Gao YL, Zhao ZS, Zhang MY, et al. Long noncoding rna pvt1 facilitates cervical cancer progression via negative regulating of mir-424[J]. Oncol Res, 2017, 25(8): 1391-1398.
[23] Shen CJ, Cheng YM, Wang CL. Lncrna pvt1 epigenetically silences mir-195 and modulates emt and chemoresistance in cervical cancer cells[J]. J Drug Target, 2017, 25(7): 637-644.
[24] Jia WZ, Yu T, An Q, et al. Microrna-190 regulates foxp2 genes in human gastric cancer[J]. Onco Targets Ther, 2016, 9(2): 3643-3651.
京ICP备08002354号-3
