Effects of targeted silencing of PRL-3 gene on proliferation, migration, invasion and epithelial-mesenchymal transition of lung cancer cells

Abstract

Abstract:

ObjectiveTo observe the effects of lentivirus-mediated shRNA silencing of PRL-3 gene on the proliferation, migration and invasion of lung cancer A549 cells and regulation of epithelial-mesenchymal transition (EMT) signaling pathway.MethodsLung cancer A549 cells were transfected with lentiviral interference vector carrying PRL-3 shRNA to build a stable PRL-3-silencing cell line. The cells were divided into blank control group, NC shRNA group (negative control group) and PRL-3 shRNA group (PRL-3 inhibiting RNAi lentivirus group). CCK-8 method, colony formation assay, Transwell and invasion chamber assay were performed to detect the proliferation, migration and invasion ability of A549 cells respectively. The expressions of E-cadherin and Snail mRNA were detected by real-time fluorescent quantitative PCR.ResultsThe stable PRL-3-silencing cell line was successfully constructed. The knockdown efficiency of PRL-3 gene in the PRL-3 shRNA group reached 83.5%. CCK-8 method detected the proliferation ability of A549 cells, and the results showed the 24 h absorbance (A) values of A549 cells in the blank control group, NC shRNA group and PRL-3 shRNA group were 0.296±0.008, 0.342±0.007 and 0.292±0.004, with a statistically significant diffe-rence (F=106.300,P<0.001), and the PRL-3 shRNA group was significantly lower than the NC shRNA group (P<0.001); at 48, 72, 96 h after transfection, the cell proliferation abilities of the PRL-3 shRNA group were also significantly inhibited. Colony formation assay showed that the numbers of colony formation in the blank control group, NC shRNA group and PRL-3 shRNA group were 166.7± 6.7, 158.0±6.1 and 119.7±1.5 (F=67.290,P<0.001). The ability of colony formation of the PRL-3 shRNA group was significantly lower than that of the NC shRNA group (P<0.001). The numbers of migrated cells in the blank control group, NC shRNA group and PRL-3 shRNA group were 100.0±1.9, 98.8±1.9 and 44.6±7.6 (F=430.300,P<0.001). The migration ability of the PRL-3 shRNA group was significantly lower than that of the NC shRNA group (P<0.001). The numbers of invaded cells in the three groups were 117.7±4.1, 113.1±6.6 and 55.6±8.4 (F=247.200,P<0.001). The invasion ability of the PRL-3shRNA group was significantly lower than that of the NC shRNA group (P<0.001). Real-time fluorescent quantitative PCR detection results showed that after silencing the expression of PRL-3, the relative expression level of E-cadherin mRNA in A549 cells was significantly up-regulated, and the level of Snail mRNA was significantly down-regulated.ConclusionPRL-3 silencing can inhibit the proliferation, migration and invasion of A549 cells effectively. PRL-3 may affect the invasion of lung cancer cells through the EMT pathway.

Key words:Lung neoplasms,Phosphatase of regenerating liver-3,Invasion,Epithelial-mesenchymal transition

Wang Ningju, Chen Dongmei, Zhang Heng, Hu Ping, Wang Yan. Effects of targeted silencing of PRL-3 gene on proliferation, migration, invasion and epithelial-mesenchymal transition of lung cancer cells[J]. Journal of International Oncology, 2021, 48(1): 18-23.

Figures/Tables6

基因	引物序列(5'→3')
Snail	正向引物:CGGAAGCCTAACTACAGCGA
	反向引物:ACAGAGTCCCAGATGAGCATT
上皮钙黏素	正向引物:GGTTAAGCACAACAGCAACAG
	反向引物:GGCATCAGCATCAGTCACTT
GAPDH	正向引物:GTCTTCACCACCATGGAGAA
	反向引物:TAAGCAGTTGGTGGTGCAG

组别	24 h	48 h	72 h	96 h
空白对照组	0.296±0.008	0.391±0.006	0.949±0.030	1.555±0.025
NC shRNA组	0.342±0.007^a	0.395±0.011	0.957±0.030	1.540±0.020
PRL-3 shRNA组	0.292±0.004^b	0.347±0.003^b	0.660±0.025^b	1.121±0.054^b
F值	106.300	76.210	213.000	280.300
P值	<0.001	<0.001	<0.001	<0.001

References14

[1]	王宁, 刘硕, 杨雷, 等. 2018全球癌症统计报告解读[J]. 肿瘤综合治疗电子杂志, 2019,5(1):87-97. DOI: 10.12151/JMCM.2019.01-10.
[2]	Kim NW, Chu CW, Ahn TS, et al. Correlation between liver metastases and the level of PRL-3 mRNA expression in patients with primary colorectal cancer[J]. J Korean Soc Coloproctol, 2011,27(5):231-236. DOI: 10.3393/jksc.2011.27.5.231. doi:10.3393/jksc.2011.27.5.231pmid:22102972
[3]	Vandsemb EN, Bertilsson H, Abdollahi P, et al. Phosphatase of regenerating liver 3 (PRL-3) is overexpressed in human prostate cancer tissue and promotes growth and migration[J]. J Transl Med, 2016,14:71. DOI: 10.1186/s12967-016-0830-z. doi:10.1186/s12967-016-0830-zpmid:26975394
[4]	Gari HH, DeGala GD, Ray R, et al. PRL-3 engages the focal adhesion pathway in triple-negative breast cancer cells to alter actin structure and substrate adhesion properties critical for cell migration and invasion[J]. Cancer Lett, 2016,380(2):505-512. DOI: 10.1016/j.canlet.2016.07.017. pmid:27452906
[5]	张平, 张志培, 李香敏, 等. 非小细胞肺癌中PRL-3与RhoC的表达及相关性意义[J]. 中国肺癌杂志, 2010,13(6):598-601. DOI: 10.3779/j.issn.1009-3419.2010.06.006.
[6]	刘楠, 王力宁, 姜奕, 等. PRL-3对肺癌细胞迁移侵袭及RhoA活性调控的研究[J]. 中华临床医师杂志(电子版), 2013,7(7):2995-3000. DOI: 10.3877/cma.j.issn.1674-0785.2013.07.103.
[7]	Lai W, Liu L, Zeng Y, et al. KCNN4 channels participate in the EMT induced by PRL-3 in colorectal cancer[J]. Med Oncol, 2013,30(2):566. DOI: 10.1007/s12032-013-0566-z. doi:10.1007/s12032-013-0566-zpmid:23572150
[8]	Ming J, Liu N, Gu Y, et al. PRL-3 facilitates angiogenesis and metastasis by increasing ERK phosphorylation and up-regulating the le-vels and activities of Rho-A/C in lung cancer[J]. Pathology, 2009,41(2):118-126. DOI: 10.1080/00313020802579268. doi:10.1080/00313020802579268pmid:19152186
[9]	张建龙, 张萦斐, 张育超, 等. PRL-3调节PI3K信号通路在促进结肠癌细胞增殖侵袭中的作用[J]. 中山大学学报(医学科学版), 2013,34(1):16-21.
[10]	Ye Z, Al-Aidaroos AQ, Park JE, et al. PRL-3 activates mTORC1 in cancer progression[J]. Sci Rep, 2015,5:17046. DOI: 10.1038/srep17046. pmid:26597054
[11]	李文清, 候劲松. 上皮间质转化的表观遗传调控[J]. 国际肿瘤学杂志, 2017,44(12):918-921. DOI: 10.3760/cma.j.issn.1673422x.2017.12.009.
[12]	隋小芳, 朱英丽, 范巧菊, 等. PRL-3与E-cad在非小细胞肺癌中的表达及临床意义[J]. 黑龙江医药科学, 2014,37(6):38-40. DOI: 10.3969/j.issn.1008-0104.2014.06.020.
[13]	Zhu LF, Hu Y, Yang CC, et al. Snail overexpression induces an epithelial to mesenchymal transition and cancer stem cell like properties in SCC 9 cells[J]. Lab Invest, 2012,92(5):744-752. DOI: 10.1038/labinvest.2012.8. doi:10.1038/labinvest.2012.8pmid:22349639
[14]	郝鸿泽, 刘言, 陆平. 胸苷激酶1和锌指转录因子Snail在非小细胞肺癌组织中的表达及其生物学行为的关系[J]. 中华实验外科, 2016,33(5):1378-1380. DOI: 10.3760/cma.j.issn.1001-9030.2016.05.065.

[1]	Liu Na, Kou Jieli, Yang Feng, Liu Taotao, Li Danping, Han Junrui, Yang Lizhou.Clinical value of serum miR-106b-5p and miR-760 combined with low-dose spiral CT in the diagnosis of early lung cancer[J]. Journal of International Oncology, 2024, 51(6): 321-325.
[2]	He Jiahui, Hu Qinyong.Comparative analysis of lung cancer incidence and mortality trends and risk factors in China and the United States based on GBD data[J]. Journal of International Oncology, 2024, 51(1): 29-36.
[3]	Xu Fang, Zhu Wentian.Progress in adjuvant therapy of hepatocellular carcinoma complicated with microvascular invasion[J]. Journal of International Oncology, 2023, 50(5): 304-309.
[4]	Zuo Xiaoping, Liu Xiaochuan, Wu Xiqiang, Li Zhou, Xia Tian, Liu Guofeng.Risk factors and prediction model construction of arrhythmia in elderly patients with early lung cancer after thoracoscopic pulmonary resection[J]. Journal of International Oncology, 2023, 50(12): 711-716.
[5]	Chen Yu, Xu Hua, Liu Hai, Chen Shixin.Construction of pathological classification prediction model for malignant pulmonary pure ground-glass nodule patients based on CT imaging[J]. Journal of International Oncology, 2023, 50(11): 655-660.
[6]	Yang Sha, Yang Xiaohua, Wang Suhua, Xue Xiaoyan, Xu Jun.Analysis of risk factors for deep vein thrombosis of lower extremity after thoracoscopic surgery for elderly lung cancer and establishment and validation of prediction model[J]. Journal of International Oncology, 2022, 49(9): 532-536.
[7]	Chen Huangjing, Zhu Pengfei, Zhang Qing, Chen Guifang, Yang Chunlin, He Ying.Comparative study on the clinical value of contrast-enhanced ultrasound- and CT-guided percutaneous puncture biopsy in peripheral lung masses[J]. Journal of International Oncology, 2022, 49(8): 459-463.
[8]	Zhang Zishu, Wu Xinlin.Mechanism of action of lactic acid in tumor microenvironment and related treatment[J]. Journal of International Oncology, 2022, 49(6): 349-352.
[9]	Cai Gangxiang, Li Jing, Xu Bin.Advances in neoadjuvant immunotherapy for lung cancer[J]. Journal of International Oncology, 2022, 49(6): 366-370.
[10]	Zhang Jingxian, Yi Dan, Li Xiaojiang.Application of antibody-drug conjugates in the treatment of non-small cell lung cancer[J]. Journal of International Oncology, 2022, 49(5): 296-301.
[11]	Gao Min, Feng Jing, Wang Li, Zhong Hai, Wen Yuting, Wan Bing, Zhang Xiuwei.Application of microbiota in the early diagnosis and adjunctive treatment of lung cancer[J]. Journal of International Oncology, 2022, 49(4): 247-251.
[12]	Shi Yu, Chen Xi, Xu Mengqi, Zhang Yingying, Ji Honghai, Jiang Yingying.Effects of PSIP1 gene silencing on migration and invasion of oral squamous cell carcinoma cells[J]. Journal of International Oncology, 2022, 49(3): 129-133.
[13]	Gao Shile, Lu Donghui, Liu Meiqin, Xu Xingjun, Ma Huan, Zhang Yu.Clinical efficacy and optimal dose of apatinib combined with chemotherapy in patients with advanced non-small cell lung cancer[J]. Journal of International Oncology, 2022, 49(3): 140-145.
[14]	Xie Hongxia, Zuo Jinhui, Liao Dongying, Deng Renfen, Yao Yang, Jia Yingjie, Li Xiaojiang, Kong Fanming.Application of PD-L1 inhibitors in the treatment of non-small cell lung cancer[J]. Journal of International Oncology, 2022, 49(2): 111-115.
[15]	Huang Huayu, Song Qibin, Gong Hongyun, Song Jia.Analysis on the incidence and risk factors of pneumonia in patients with lung cancer receiving thoracic radiotherapy and immunotherapy[J]. Journal of International Oncology, 2022, 49(12): 718-723.