Inhibition of proliferation and migration and induction of apoptosis in glioma cells by silencing TLR4 expression levels via RNA interference

Liu,Yingzi; Ju,Yingchao; Liu,Jianghui; Chen,Yuetong; Huo,Xiangran; Liu,Liang

doi:10.3892/ol.2020.12274

Inhibition of proliferation and migration and induction of apoptosis in glioma cells by silencing TLR4 expression levels via RNA interference

Authors:
- Yingzi Liu
- Yingchao Ju
- Jianghui Liu
- Yuetong Chen
- Xiangran Huo
- Liang Liu
View Affiliations

Affiliations: Department of Neurosurgery, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China, Animal Experimental Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China, Tumor Institute, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China
Published online on: November 6, 2020 https://doi.org/10.3892/ol.2020.12274
Article Number: 13
Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The objective of the present study was to investigate the expression levels of toll‑like receptor 4 (TLR4) in glioma cells and the mechanisms underlying its regulatory effects on proliferation, migration and apoptosis of glioma cells. A total of three TLR4 silencing short hairpin (sh)RNA plasmids were established, and Lipofectamine® was used to the transfect the human glioma cell line U‑87MG. Transfection efficiency was measured via flow cytometry. The interference plasmid exhibiting the largest silencing effect on TLR4 was screened for subsequent experiments using puromycin. Reverse transcription‑quantitative PCR and western blot analysis were used to detect the TLR4 gene and protein expression levels, respectively, in stably transfected cells. Flow cytometry measured cell cycle and apoptosis and a wound healing assay was employed to assess the migration ability of transfected cells. The proliferation of transfected cells was detected using Cell Counting Kit‑8 assay. TLR4‑sh2 exhibited the highest transfection efficiency. Following transfection of U‑87MG cells with TLR4‑sh2 and negative control (NC) plasmids for 48 h and screening by puromycin, stable transfected cells were named U‑87MG‑Sh and U‑87MG‑NC cells respectively. The TLR4 gene and protein expression levels in the U‑87MG‑Sh cells were significantly lower than in U‑87MG and U‑87MG‑NC cells. The apoptosis rate and the percentage of G0/1 cells were significantly higher, whereas the cell proliferation rate was notably lower, in U‑87MG‑Sh cells than in the U‑87MG‑NC and U‑87MG cells. The proliferation rate and the cell migration ability of U‑87MG‑Sh cells were significantly lower than those of U‑87MG‑NC and U‑87MG cells. TLR4 is associated with the proliferation of glioma cells. Inhibition of TLR4 expression levels significantly inhibited proliferation of glioma cells and induced apoptosis. The present study provided insights into the mechanisms associated with the development, progression and invasion ability of glioma cells.

View Figures

View References

1	Davis ME: Epidemiology and overview of gliomas. Semin Oncol Nurs. 34:420–429. 2018. View Article : Google Scholar : PubMed/NCBI
2	Ostrom QT, Gittleman H, Stetson L, Virk SM and Barnholtz-Sloan JS: Epidemiology of gliomas. Cancer Treat Res. 163:1–14. 2015. View Article : Google Scholar : PubMed/NCBI
3	Ostrom QT, Gittleman H, Liao P, Rouse C, Chen Y, Dowling J, Wolinsky Y, Kruchko C and Barnholtz-Sloan J: CBTRUS statistical report: Primary brain and central nervous system tumors diagnosed in the United States in 2007–2011. Neuro Oncol. 16 (Suppl 4):iv1–iv63. 2014. View Article : Google Scholar : PubMed/NCBI
4	Rasmussen BK, Hansen S, Laursen RJ, Kosteljanetz M, Schultz H, Nørgård BM, Guldberg R and Gradel KO: Epidemiology of glioma: Clinical characteristics, symptoms, and predictors of glioma patients grade I–IV in the the Danish neuro-oncology registry. J Neurooncol. 135:571–579. 2017. View Article : Google Scholar : PubMed/NCBI
5	Kawai T and Akira S: Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity. 34:637–650. 2011. View Article : Google Scholar : PubMed/NCBI
6	Kumar H, Kawai T and Akira S: Toll-like receptors and innate immunity. Biochem Biophys Res Commun. 388:621–625. 2009. View Article : Google Scholar : PubMed/NCBI
7	Hua Z and Hou B: TLR signaling in B-cell development and activation. Cell Mol Immunol. 10:103–106. 2013. View Article : Google Scholar : PubMed/NCBI
8	Kawai T and Akira S: TLR signaling. Semin Immunol. 19:24–32. 2007. View Article : Google Scholar : PubMed/NCBI
9	Seydoux E, Liang H, Dubois Cauwelaert N, Archer M, Rintala ND, Kramer R, Carter D, Fox CB and Orr MT: Effective combination adjuvants engage both TLR and inflammasome pathways to promote potent adaptive immune responses. J Immunol. 201:98–112. 2018. View Article : Google Scholar : PubMed/NCBI
10	Tian S, Wang M, Liu C, Zhao H and Zhao B: Mulberry leaf reduces inflammation and insulin resistance in type 2 diabetic mice by TLRs and insulin signalling pathway. BMC Complement Altern Med. 19:3262019. View Article : Google Scholar : PubMed/NCBI
11	Dickinson SE and Wondrak GT: TLR4-directed molecular strategies targeting skin photodamage and carcinogenesis. Curr Med Chem. 25:5487–5502. 2018. View Article : Google Scholar : PubMed/NCBI
12	Wang YH, Cao YW, Yang XC, Niu HT, Sun LJ, Wang XS and Liu J: Effect of TLR4 and B7-H1 on immune escape of urothelial bladder cancer and its clinical significance. Asian Pac J Cancer Prev. 15:1321–1326. 2014. View Article : Google Scholar : PubMed/NCBI
13	Monlish DA, Bhatt ST and Schuettpelz LG: The role of toll-like receptors in hematopoietic malignancies. Front Immunol. 7:3902016. View Article : Google Scholar : PubMed/NCBI
14	Dajon M, Iribarren K and Cremer I: Toll-like receptor stimulation in cancer: A pro- and anti-tumor double-edged sword. Immunobiology. 222:89–100. 2017. View Article : Google Scholar : PubMed/NCBI
15	Makkar S, Riehl TE, Chen B, Yan Y, Alvarado DM, Ciorba MA and Stenson WF: Hyaluronic acid binding to TLR4 promotes proliferation and blocks apoptosis in colon cancer. Mol Cancer Ther. 18:2446–2456. 2019. View Article : Google Scholar : PubMed/NCBI
16	Sun NK, Huang SL, Chang TC and Chao CC: TLR4 and NFκB signaling is critical for taxol resistance in ovarian carcinoma cells. J Cell Physiol. 233:2489–2501. 2018. View Article : Google Scholar : PubMed/NCBI
17	Shetab Boushehri MA and Lamprecht A: TLR4-based immunotherapeutics in cancer: A review of the achievements and shortcomings. Mol Pharm. 15:4777–4800. 2018. View Article : Google Scholar : PubMed/NCBI
18	Dapito DH, Mencin A, Gwak GY, Pradere JP, Jang MK, Mederacke I, Caviglia JM, Khiabanian H, Adeyemi A, Bataller R, et al: Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4. Cancer Cell. 21:504–516. 2012. View Article : Google Scholar : PubMed/NCBI
19	Allen M, Bjerke M, Edlund H, Nelander S and Westermark B: Origin of the U87MG glioma cell line: Good news and bad news. Sci Transl Med. 8:354re32016. View Article : Google Scholar : PubMed/NCBI
20	Zhou T, Li Y, Yang L, Liu L, Ju Y and Li C: Silencing of ANXA3 expression by RNA interference inhibits the proliferation and invasion of breast cancer cells. Oncol Rep. 37:388–398. 2017. View Article : Google Scholar : PubMed/NCBI
21	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
22	Ostrom QT, Bauchet L, Davis FG, Deltour I, Fisher JL, Langer CE, Pekmezci M, Schwartzbaum JA, Turner MC, Walsh KM, et al: The epidemiology of glioma in adults: A ‘state of the science’ review. Neuro Oncol. 16:896–913. 2014. View Article : Google Scholar : PubMed/NCBI
23	Lim M, Xia Y, Bettegowda C and Weller M: Current state of immunotherapy for glioblastoma. Nat Rev Clin Oncol. 15:422–442. 2018. View Article : Google Scholar : PubMed/NCBI
24	Satoh T and Akira S: Toll-like receptor signaling and its inducible proteins. Microbiol Spectr. 4:2016.PubMed/NCBI
25	Steinhagen F, Kinjo T, Bode C and Klinman DM: TLR-based immune adjuvants. Vaccine. 29:3341–3355. 2011. View Article : Google Scholar : PubMed/NCBI
26	Kumar V: Toll-like receptors in the pathogenesis of neuroinflammation. J Neuroimmunol. 332:16–30. 2019. View Article : Google Scholar : PubMed/NCBI
27	Medzhitov R, Preston-Hurlburt P and Janeway CA Jr: A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 388:394–397. 1997. View Article : Google Scholar : PubMed/NCBI
28	Vijay K: Toll-like receptors in immunity and inflammatory diseases: Past, present, and future. Int Immunopharmacol. 59:391–412. 2018. View Article : Google Scholar : PubMed/NCBI
29	Mikulic J, Longet S, Favre L, Benyacoub J and Corthesy B: Secretory IgA in complex with Lactobacillus rhamnosus potentiates mucosal dendritic cell-mediated Treg cell differentiation via TLR regulatory proteins, RALDH2 and secretion of IL-10 and TGF-β. Cell Mol Immunol. 14:546–556. 2017. View Article : Google Scholar : PubMed/NCBI
30	Tang AT, Choi JP, Kotzin JJ, Yang Y, Hong CC, Hobson N, Girard R, Zeineddine HA, Lightle R, Moore T, et al: Endothelial TLR4 and the microbiome drive cerebral cavernous malformations. Nature. 545:305–310. 2017. View Article : Google Scholar : PubMed/NCBI
31	Yin Q, Jiang D, Li L, Yang Y, Wu P, Luo Y, Yang R and Li D: LPS promotes vascular smooth muscle cells proliferation through the TLR4/Rac1/Akt signalling pathway. Cell Physiol Biochem. 44:2189–2200. 2017. View Article : Google Scholar : PubMed/NCBI
32	Fu HY, Li C, Yang W, Gai XD, Jia T, Lei YM and Li Y: FOXP3 and TLR4 protein expression are correlated in non-small cell lung cancer: Implications for tumor progression and escape. Acta Histochem. 115:151–157. 2013. View Article : Google Scholar : PubMed/NCBI
33	Grimmig T, Matthes N, Hoeland K, Tripathi S, Chandraker A, Grimm M, Moench R, Moll EM, Friess H, Tsaur I, et al: TLR7 and TLR8 expression increases tumor cell proliferation and promotes chemoresistance in human pancreatic cancer. Int J Oncol. 47:857–866. 2015. View Article : Google Scholar : PubMed/NCBI
34	Xiao T, Wu S, Yan C, Zhao C, Jin H, Yan N, Xu J, Wu Y, Li C, Shao Q and Xia S: Butyrate upregulates the TLR4 expression and the phosphorylation of MAPKs and NK-κB in colon cancer cell in vitro. Oncol Lett. 16:4439–4447. 2018.PubMed/NCBI
35	Li C, Li H, Jiang K, Li J and Gai X: TLR4 signaling pathway in mouse Lewis lung cancer cells promotes the expression of TGF-β1 and IL-10 and tumor cells migration. Biomed Mater Eng. 24:869–875. 2014.PubMed/NCBI
36	Ren WH, Zhang LM, Liu HQ, Gao L, Chen C, Qiang C, Wang XL, Liu CY, Li SM, Huang C, et al: Protein overexpression of CIRP and TLR4 in oral squamous cell carcinoma: An immunohistochemical and clinical correlation analysis. Med Oncol. 31:1202014. View Article : Google Scholar : PubMed/NCBI
37	Pandey N, Chauhan A and Jain N: TLR4 polymorphisms and expression in solid cancers. Mol Diagn Ther. 22:683–702. 2018. View Article : Google Scholar : PubMed/NCBI
38	Chen CL, Tsukamoto H, Liu JC, Kashiwabara C, Feldman D, Sher L, Dooley S, French SW, Mishra L, Petrovic L, et al: Reciprocal regulation by TLR4 and TGF-β in tumor-initiating stem-like cells. J Clin Invest. 123:2832–2849. 2013. View Article : Google Scholar : PubMed/NCBI
39	Yao RR, Li JH, Zhang R, Chen RX and Wang YH: M2-polarized tumor-associated macrophages facilitated migration and epithelial-mesenchymal transition of HCC cells via the TLR4/STAT3 signaling pathway. World J Surg Oncol. 16:92018. View Article : Google Scholar : PubMed/NCBI
40	Szajnik M, Szczepanski MJ, Czystowska M, Elishaev E, Mandapathil M, Nowak-Markwitz E, Spaczynski M and Whiteside TL: TLR4 signaling induced by lipopolysaccharide or paclitaxel regulates tumor survival and chemoresistance in ovarian cancer. Oncogene. 28:4353–4363. 2009. View Article : Google Scholar : PubMed/NCBI
41	Zhang H and Zhang S: The expression of Foxp3 and TLR4 in cervical cancer: Association with immune escape and clinical pathology. Arch Gynecol Obstet. 295:705–712. 2017. View Article : Google Scholar : PubMed/NCBI
42	Zhan Z, Xie X, Cao H, Zhou X, Zhang XD, Fan H and Liu Z: Autophagy facilitates TLR4- and TLR3-triggered migration and invasion of lung cancer cells through the promotion of TRAF6 ubiquitination. Autophagy. 10:257–268. 2014. View Article : Google Scholar : PubMed/NCBI
43	Li J, Yin J, Shen W, Gao R, Liu Y, Chen Y, Li X, Liu C, Xiang R and Luo N: TLR4 promotes breast cancer metastasis via Akt/GSK3β/β-catenin pathway upon LPS stimulation. Anat Rec (Hoboken). 300:1219–1229. 2017. View Article : Google Scholar : PubMed/NCBI
44	Kim KH, Jo MS, Suh DS, Yoon MS, Shin DH, Lee JH and Choi KU: Expression and significance of the TLR4/MyD88 signaling pathway in ovarian epithelial cancers. World J Surg Oncol. 10:1932012. View Article : Google Scholar : PubMed/NCBI
45	Zhou Q, Wang C, Wang X, Wu X, Zhu Z, Liu B and Su L: Association between TLR4 (+896A/G and +1196C/T) polymorphisms and gastric cancer risk: An updated meta-analysis. PLoS One. 9:e1096052014. View Article : Google Scholar : PubMed/NCBI