MiR-328-3p promotes TGF-β1-induced proliferation, migration, and inflammation of airway smooth muscle cells by regulating the PTEN/Akt pathway

Main Article Content

Jie Gao
Xiao-Lei Wu


asthma, airway smooth -muscle cells, miR-328-3p, PTEN/Akt pathway


Background: Recent studies have shown that the up-regulation of microRNA miR-328-3p expression increases seasonal allergy and asthma symptoms in children, but the specific mechanism remains unclear. Therefore, the aim of this study was to explore the role and mechanism of -miR-328-3p in transforming growth factor (TGF)-β1-induced airway smooth muscle cells (ASMCs).

Methods: The effect of TGF-β1 on the expression of miR-328-3p in ASMCs was examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cells proliferation, migration, and inflammatory factors in TGF-β1-induced ASMCs were measured by cell counting kit-8 (CCK-8), transwell, and enzyme-linked immunosorbent assay (ELISA), respectively. Besides, TargetScan was used to predict phosphatase and tensin homolog (PTEN), the downstream target of miR-328-3p; double-luciferase reporter assay, western blot, and qRT-PCR were used to verify the targeting relationship between miR-328-3p and PTEN; western blot was also used to examine the effects of PTEN and miR-328-3p knockdown on the expression levels of PTEN, Akt, and p-Akt proteins.

Results: The expression of miR-328-3p was up-regulated in TGF-β1-induced ASMCs. Knockdown of miR-328-3p significantly inhibited proliferation, migration, and inflammation of ASMCs induced by TGF-β1 and decreased levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β. The dual--luciferase reporter assay results confirmed that PTEN was a target gene of miR-328-3p. Moreover, inhibition of PTEN expression reversed the inhibitory effect of low miR-328-3p expression on -TGF-β1-induced ASMC’s proliferation, migration, and inflammation. In comparison to the knockdown of miR-328-3p alone, the simultaneous knockdown of miR-328-3p with PTEN decreased PTEN protein expression levels and increased p-Akt/Akt ratio in TGF-β1-induced ASMCs.

Conclusion: Through regulating the expression of PTEN and the activity of Akt signaling pathway, miR-328-3p promotes TGF-β1-induced proliferation, migration, and inflammation of ASMCs.

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1. Backman H, Raisanen P, Hedman L, Stridsman C, Andersson M, Lindberg A, et al. Increased prevalence of allergic asthma from 1996 to 2006 and further to 2016-results from three population surveys. Clin Exp Allergy. 2017;47(11):1426–1435. 10.1111/cea.12963

2. Nunes C, Pereira AM, Morais-Almeida M. Asthma costs and social impact. Asthma Res Pract. 2017;3:1. 10.1186/s40733-016-0029-3

3. Diver S, Khalfaoui L, Emson C, Wenzel SE, Menzies-Gow A, Wechsler ME, et al. Effect of tezepelumab on airway inflammatory cells, remodelling, and hyperresponsiveness in patients with moderate-to-severe uncontrolled asthma (CASCADE): a double-blind, randomised, placebo-controlled, phase 2 trial. Lancet Respir Med. 2021;9(11):1299–1312. 10.1016/S2213-2600(21)00226-5

4. Hough KP, Curtiss ML, Blain TJ, Liu RM, Trevor J, Deshane JS, et al. Airway remodeling in asthma. Front Med (Lausanne). 2020;7:191. 10.3389/fmed.2020.00191

5. Lambrecht BN, Hammad H. The immunology of asthma. Nat Immunol. 2015;16(1):45–56. 10.1038/ni.3049

6. Hu L, Li L, Zhang H, Li Q, Jiang S, Qiu J, et al. Inhibition of airway remodeling and inflammatory response by Icariin in asthma. BMC Complement Altern Med. 2019;19(1):316. 10.1186/s12906-019-2743-x

7. Zhang J, Koussih L, Shan L, Halayko AJ, Chen BK, Gounni AS. TNF up-regulates Pentraxin3 expression in human-airway smooth muscle cells via JNK and ERK1/2 MAPK pathways. Allergy Asthma Clin Immunol. 2015;11:37. 10.1186/s13223-015-0104-y

8. Choby GW, Lee S. Pharmacotherapy for the treatment of asthma: current treatment options and future directions. Int Forum Allergy Rhinol. 2015;5(Suppl. 1):S35–S40. 10.1002/alr.21592

9. Deshpande DA, Dileepan M, Walseth TF, Subramanian S, Kannan MS. MicroRNA regulation of airway inflammation and airway smooth muscle function: relevance to asthma. Drug Dev Res. 2015;76(6):286–295. 10.1002/ddr.21267

10. Chen H, Guo SX, Zhang S, Li XD, Wang H, Li XW. MiRNA-620 promotes TGF-beta1-induced proliferation of airway smooth muscle cell through controlling PTEN/AKT signaling pathway. Kaohsiung J Med Sci. 2020;36(11):869–877. 10.1002/kjm2.12260

11. Qiu YY, Zhang YW, Qian XF, Bian T. miR-371, miR-138, miR-544, miR-145, and miR-214 could modulate Th1/Th2 balance in asthma through the combinatorial regulation of Runx3. Am J Transl Res. 2017;9(7):3184–3199.

12. Lacedonia D, Palladino GP, Foschino-Barbaro MP, Scioscia G, Carpagnano GE. Expression profiling of miRNA-145 and miRNA-338 in serum and sputum of patients with COPD, asthma, and asthma-COPD overlap syndrome phenotype. Int J Chron Obstruct Pulmon Dis. 2017;12:1811–1817. 10.2147/COPD.S130616

13. Di Meo A, Brown MD, Finelli A, Jewett MAS, Diamandis EP, Yousef GM. Prognostic urinary miRNAs for the assessment of small renal masses. Clin Biochem. 2020;75:15–22. 10.1016/j.clinbiochem.2019.10.002

14. Shi J, An G, Guan Y, Wei T, Peng Z, Liang M, et al. miR-328-3p mediates the anti-tumor effect in osteosarcoma via directly targeting MMP-16. Cancer Cell Int. 2019;19:104. 10.1186/s12935-019-0829-7

15. Pan S, Ren F, Li L, Liu D, Li Y, Wang A, et al. MiR-328-3p inhibits cell proliferation and metastasis in colorectal cancer by targeting Girdin and inhibiting the PI3K/Akt signaling pathway. Exp Cell Res. 2020;390(1):111939. 10.1016/j.yexcr.2020.111939

16. Gao Y, Xu W, Guo C, Huang T. GATA1 regulates the microRNA3283p/PIM1 axis via circular RNA ITGB1 to promote renal ischemia/reperfusion injury in HK2 cells. Int J Mol Med. 2022;50(2):100. 10.3892/ijmm.2022.5156

17. Tiwari A, Wang AL, Li J, Lutz SM, Kho AT, Weiss ST, et al. Seasonal variation in miR-328-3p and let-7d-3p are associated with seasonal allergies and asthma symptoms in children. Allergy Asthma Immunol Res. 2021;13(4):576–588. 10.4168/aair.2021.13.4.576

18. Saito A, Horie M, Micke P, Nagase T. The role of TGF-beta signaling in lung cancer associated with idiopathic pulmonary fibrosis. Int J Mol Sci. 2018;19(11):3611. 10.3390/ijms19113611

19. Evasovic JM, Singer CA. Regulation of IL-17A and implications for TGF-beta1 comodulation of airway smooth muscle remodeling in severe asthma. Am J Physiol Lung Cell Mol Physiol. 2019;316(5):L843–L868. 10.1152/ajplung.00416.2018

20. Liu L, Pan Y, Zhai C, Zhu Y, Ke R, Shi W, et al. Activation of peroxisome proliferation-activated receptor-γ inhibits transforming growth factor-β1-induced airway smooth muscle cell proliferation by suppressing Smad-miR-21 signaling. J Cell Physiol. 2018;234(1):669–681. 10.1002/jcp.26839

21. Agarwal V, Bell GW, Nam JW, Bartel DP. Predicting effective microRNA target sites in mammalian mRNAs. Elife. 2015;4:e05005 10.7554/eLife.05005

22. Zou P, Zhu M, Lian C, Wang J, Chen Z, Zhang X, et al. miR-192-5p suppresses the progression of lung cancer bone metastasis by targeting TRIM44. Sci Rep. 2019;9(1):19619. 10.1038/s41598-019-56018-5

23. Chen M, Shi JT, Lv ZQ, Huang LJ, Lin XL, Zhang W, et al. Triptolide inhibits transforming growth factor-β1-induced proliferation and migration of rat airway smooth muscle cells by suppressing nuclear factor-κB but not extracellular signal--regulated kinase 1/2. Immunology. 2015;144(3):486–494. 10.1111/imm.12396

24. Michalik M, Wojcik-Pszczola K, Paw M, Wnuk D, Koczurkiewicz P, Sanak M, et al. Fibroblast-to-myofibroblast transition in bronchial asthma. Cell Mol Life Sci. 2018;75(21):3943–3961. 10.1007/s00018-018-2899-4

25. Chen CY, Chen J, He L, Stiles BL. PTEN: tumor suppressor and metabolic regulator. Front Endocrinol (Lausanne). 2018;9:338. 10.3389/fendo.2018.00338

26. Bentley JK, Hershenson MB. Airway smooth muscle growth in asthma: proliferation, hypertrophy, and migration. Proc Am Thorac Soc. 2008;5(1):89–96. 10.1513/pats.200705-063VS

27. Dilasser F, Rose L, Hassoun D, Klein M, Rousselle M, Brosseau C, et al. Essential role of smooth muscle Rac1 in severe asthma-associated airway remodelling. Thorax. 2021;76(4): 326–334. 10.1136/thoraxjnl-2020-216271

28. Gao Y, Wang B, Luo H, Zhang Q, Xu M. miR-217 represses TGF-beta1-induced airway smooth muscle cell proliferation and migration through targeting ZEB1. Biomed Pharmacother. 2018;108:27–35. 10.1016/j.biopha.2018.09.030

29. Chen G, Tang J, Ni Z, Chen Q, Li Z, Yang W, et al. Antiasthmatic effects of resveratrol in ovalbumin-induced asthma model mice involved in the upregulation of PTEN. Biol Pharm Bull. 2015;38(4):507–513. 10.1248/bpb.b14-00610

30. Luongo F, Colonna F, Calapa F, Vitale S, Fiori ME, De Maria R. PTEN tumor-suppressor: the dam of stemness in cancer. Cancers (Basel). 2019;11(8):1076. 10.3390/cancers11081076

31. Wu Y, Lu Y, Zou F, Fan X, Li X, Zhang H, et al. PTEN participates in airway remodeling of asthma by regulating CD38/Ca(2+)/CREB signaling. Aging (Albany NY). 2020;12(16):16326–16340. 10.18632/aging.103664

32. Zhao J, Jiao X, Wu J, Wang J, Gong W, Liu F, et al. FIZZ1 promotes airway remodeling in asthma through the PTEN signaling pathway. Inflammation. 2015;38(4):1464–1472. 10.1007/s10753-015-0121-5

33. Lu XX, Cao LY, Chen X, Xiao J, Zou Y, Chen Q. PTEN inhibits cell proliferation, promotes cell apoptosis, and induces cell cycle arrest via downregulating the PI3K/AKT/hTERT pathway in lung adenocarcinoma A549 cells. Biomed Res Int. 2016;2016:2476842. 10.1155/2016/2476842

34. Song MS, Salmena L, Pandolfi PP. The functions and regulation of the PTEN tumour suppressor. Nat Rev Mol Cell Biol. 2012;13(5):283–296. 10.1038/nrm3330