RETRACTED ARTICLE: Formaldehyde inhibits proliferation of bronchial epithelial cells by down-regulating miR-375

Abstract We, the Editors and Publisher of the journal Artificial Cells, Nanomedicine, and Biotechnology, have retracted the following article: Lei Yu & ChunWen Li (2019) Formaldehyde inhibits proliferation of bronchial epithelial cells by down-regulating miR-375. Artificial Cells, Nanomedicine, and Biotechnology, 47:1, 2293–2297, DOI: 10.1080/21691401.2019.1624369 It has come to our attention that the full authorship list and affiliations for this manuscript were changed after the article was submitted. We have contacted the corresponding author for an explanation, but we have not received a satisfactory explanation. As determining authorship is core to the integrity of published work, we are therefore retracting the article. The corresponding author listed in this publication has been informed. We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.


Introduction
MicroRNA is a small non-coding RNA with a length of about 20-25 nucleotides. It partially or completely complements the target gene 3 0 UTR by base complementation and regulates the target gene expression after transcription [1]. In recent years, more and more studies have confirmed that miRNA is involved in a variety of diseases and pathophysiological processes of tumors. Studies have reported that miR-375 plays an important role in myocardial infarction repair and testicular cell proliferation [2,3], but its role in bronchial epithelial cells is not yet clear. Kr€ uppel-like transcription factors (KLFs) form a subclass of $17 zinc fingers, which contain a DNA-binding transcription factor expressed in humans and belong to the specific protein 1 (Sp1)/KLF zinc finger-binding transcription factor family [4][5][6]. KLF4 is expressed in different cell types, such as endothelial cells, epithelial cells and bone marrow cells [7][8][9][10]. KLF4 contains activation domain and inhibition domain, so it can induce and inhibit the transcription of different genes, including molecules involved in regulating immune response [11]. However, the mechanism of KLF4 in bronchial epithelial cells has not been studied. In this study, human bronchial epithelial cells 16HBE were used as the research object to detect the effects of formaldehyde, overexpression of miR-375 and knockdown of KLF4 on the proliferation of 16HBE cells, revealing that the mechanism may be related to the inhibition of miR-375 expression targeting KLF4 by formaldehyde, which will provide a basis for the application of formaldehyde in respiratory diseases.

Materials
Human bronchial epithelial cells 16HBE were purchased from ATCC; DMEM medium, fetal bovine serum, MTT reagent, trypsin, LipofectamineTM2000, BCA protein quantification kit and reverse transcription kit were purchased from Takara Company (Dalian, China); The SDS-PAGE kit, ECL luminescent solution and RIPA protein lysate were purchased from Beyotime Biotechnology Co., Ltd.; the dual luciferase reporter assay kit was purchased from Promega, USA.

Cell culture
Human bronchial epithelial cells 16HBE were cultured in DMEM medium containing 10% fetal bovine serum, and cultured in an incubator at 37 C, 5% CO 2 .

MTT experiment
An appropriate amount of cells in each group were taken and 20 lL 5 g/L MTT solution was added. The solution was cultured for 3.5/4 h and then the supernatant was discarded. 150 lL DMSO was added per well with shaking and the crystal was dissolved. The cell absorbance (A) was measured at 490 nm wavelength. Cell proliferation ability was positively correlated with absorbance.

qRT-PCR experiment
An appropriate amount of cells in the logarithmic growth phase were taken following the instructions of the RNA extraction kit to extract RNA, quantify and then, cDNA was synthesized according to the instructions of the reverse transcription kit. Finally, the miR-375 detection was performed according to the instructions of the qRT-PCR kit. The expression of miR-375 was calculated using 2 ÀDDCt .

Dual luciferase reporter gene assay
The luciferase reporter vector (psiCHECK2-KLF4-WT, psiCHECK2-KLF4-MUT) was transfected into 16HBE cells with miR-375 mimics and miR-NC, respectively by liposome method. After 6 h of culture, fresh medium was replaced and then continued to be cultured for 48 h, then operated according to the instructions of the dual luciferase reporter assay kit. The results showed that the binding intensity of miR-375 to KLF4 was reflected by the ratio of the luminescence intensity of sea cucumber luciferase to the luminescence intensity of firefly luciferase.

Western blot experiment
The cells of each group, at the logarithmic growth phase, were taken for quantitative analysis by BCA after RIPA lysis, and the supernatant was taken for protein sampling after denaturation and centrifugation. Electrophoresis-transmembrane-blocking-I anti-incubation-II anti-incubation-development exposure was performed according to the routine procedure of Western blot experiment. Image J analyzed the grey value of the target band and expressed the expression of the target protein by the ratio of the grey value of the target band to the grey value of the beta-actin.

Statistical analysis
All data in the experiment were analyzed using SPSS 21.0 software (Armonk, NY). Measurement data were expressed as mean ± standard deviation x6s: Data among groups were compared by one-way analysis of variance. Pairwise comparisons were performed using SNK-q test. p < .05 was considered statistically significant.

Results
Effects of different concentrations of formaldehyde on the proliferation of human bronchial epithelial cells The activity of 16HBE in human bronchial epithelial cells treated with formaldehyde (0, 50, 100, 150, 200, 250, 300, 350 lmol/L) was detected by MTT assay. Results as shown in Figure 1, the activity of 16HBE cells was negatively correlated with formaldehyde in a concentration-dependent manner, and the IC50 of formaldehyde in 16HBE cells was 172.53 ± 5.35 lmol/L. Therefore, formaldehyde with a concentration of 200 lmol/L was selected for subsequent experiments.

Effect of formaldehyde on the expression of miR-375 in 16HBE cells
The 200 HBE cells treated with formaldehyde 200 lmol/L were labelled as FCOH group, the untreated cells were labelled as Control group, and the expression of miR-375 in each group was detected by qRT-PCR. The results are shown in Figure 2.
Compared with the Control group, the expression of miR-375 in the FCOH group was significantly decreased (p < .05).
Expression of miR-375 reverses the inhibitory effect of formaldehyde on the proliferation of human bronchial epithelial cells The miR-375 mimics and miR-con were transfected into 16HBE cells and treated with formaldehyde 200 lmol/L, and labelled as FCOH þ miR-375 group and FCOH þ miR-con group. The results are shown in Figure 3. Compared with the Control group, the miR-375 was significantly decreased and the cell viability was significantly decreased in the

R E T R A C T E D
FCOH þ miR-con group; compared with the FCOH þ miR-con group, MiR-375 was significantly increased and cell viability was significantly increased in the FCOH þ miR-375 group, both of which were statistically significant (p < .05).

MiR-375 targets KLF4
Target scan was used to predict the possible binding of SUZ12 to microRNA-195. It was found that the binding of miR-195 to SUZ12 was highly possible (Figure 4(A)). The luciferase reporter gene assay was used to detect the fluorescence activity of 16HBE cells. Compared with the miR-con group, the fluorescence activity of WT cells was significantly decreased, and the fluorescence activity of MUT cells was not affected (Figure 4(B)), which was statistically significant (p < .05).

Knockdown of KLF4 affects the inhibitory effect of formaldehyde on the proliferation of human bronchial epithelial cells
Si-KLF4 and si-con were transfected into 16HBE cells and treated with formaldehyde 200 lmol/L, They were labeled as FCOH þ si-KLF4 group and FCOH þ si-con group. Results as shown in Figure 5, Compared with the Control group, the expression of KLF4 protein in FCOH þ si-con group was significantly increased ( Figure 5(A,B)), and cell viability was significantly decreased (Figure 5(C)). Compared with the FCOH þ si-con group, the expression of KLF4 protein in the FCOH þ si-KLF4 group was significantly decreased ( Figure  5(A,B)), and the cell viability was significantly increased ( Figure 5(C)), both of which were statistically significant (p < .05).
Overexpression of KLF4 can reverse the overexpression of miR-375 to promote the proliferation of formaldehyde-treated 16HBE cells Results, as shown in Figure 6, compared with the miR-con group, the expression of KLF4 protein in the miR-375 group treated with formaldehyde, was significantly decreased (Figure 6(A)), and the cell viability was significantly increased (Figure 6(B)). Compared with the miR-375 þ pcDNA group, the expression of KLF4 protein was significantly decreased in the formaldehyde-treated cells of the miR-375 þ pcDNA-KLF4 group ( Figure 6(A)), and the cell viability was significantly increased (Figure 6(B)), both of which were statistically significant (p < .05).

Discussion
MicroRNAs (miRNA) are small RNA molecules that play a role in gene silencing and translational inhibition by binding to target mRNAs [12]. In recent years, the regulation of miRNAs has received widespread attention due to its role in biological processes and the development of various human diseases including retinal diseases, neurodegenerative diseases, cardiovascular diseases, and cancer [13][14][15]. Bleck et al. [16] found in human bronchial epithelial cells that environmental particulate matter (PM) and diesel exhaust particles (DEP) upregulate thymic stromal lymphopoietin TSLP mRNA and human miR-375 in primary human bronchial epithelial cells (pHBEC), the aromatic hydrocarbon receptor (AhR) is a target of miR-375, and inhibition of miR-375 could up-regulate AhR mRNA expression in DEEP-treated pHBEC. It is suggested that both DEP and ambient PM can up-regulate TSLP in human bronchial epithelial cells through hsa-miR-375 and other

R E T R
A C T E D mechanisms, which have complex regulatory effects on AhR mRNA. Lu et al. [17] reported that in the differential miRNA of human esophageal squamous and bronchial columnar epithelial cells induced by interleukin-13, the expression of miR-375 was conservatively down-regulated, and the expression of miR-375 was abnormally down-regulated in human diseases characterized by overproduction of interleukin-13. In this study, the expression of miR-375 in formaldehyde-treated 16HBE cells was detected by qRT-PCR. It was found that formaldehyde could significantly inhibit the expression of miR-375 in 16HBE cells. Dual luciferase reporter gene assay confirmed that miR-375 targets KLF4. It was found that over-expression of microRNA-375 could reverse the inhibition of formaldehyde on the proliferation of 16HBE cells, and inhibit the expression of KLF4 protein and promote cell proliferation in 16HBE cells treated with formaldehyde. KLF participates in various pathophysiological processes such as cell proliferation, differentiation and the occurrence and development of tumors in the body. KLF4 is one of them and has similar functions with KLF [18]. In the study of diabetic pulmonary fibrosis, Zou et al. [19] found that the levels of LOX-1, TGF-b1 and kr€ uppel-like factor 6 (KLF6) in lung tissue of STZ-induced diabetic rats increased significantly. LOX-1 siRNA inhibits high glucose-induced EMT in human bronchial epithelial cells HBECs and human lung adenocarcinoma cells A549. TGF-b1 siRNA can decrease the expression of LOX-1 and KLF6, suggesting that EMT is involved in the pathological process of diabetic pulmonary fibrosis, which is related to the activation of LOX-1/TGF-b1/KLF6 signal transduction pathway. Zahlten et al. [20] reported that pneumococcus could induce KLF4 expression by activation of tyrosine kinase in lung epithelial cells BEAS-2B. Overexpression of KLF4 inhibits the activation and release of NF-jB and IL-8 reporter genes induced by Streptococcus pneumoniae, while silencing of KLF4 or yes1 kinase mediated by small interfering RNA increases IL-8 release. KLF4-dependent downregulation of NF-jB luciferase activity can be rescued by overexpression of histone acetylase p300/ cAMP response element binding protein-related factors. It is revealed that KLF4 acts as a counter-regulatory transcription factor in the pneumococcal-related proinflammatory activation of lung epithelial cells, possibly preventing organ failure caused by excessive inflammation of the lung. In this study, Western blot was used to detect the protein expression of KLF4 in formaldehyde-treated 16HBE cells. It was found that formaldehyde can promote the expression of KLF4 in cells and inhibit cell

R E T R A C T E D
proliferation, while KLF4 knockdown can promote the proliferation of 16HBE cells. Further in-depth studies found that overexpression of KLF4 could reverse the promotion of miR-375 on the proliferation of formaldehyde-treated 16HBE cells.
In conclusion, formaldehyde can inhibit the proliferation of human bronchial epithelial cells, and its mechanism is related to the down-regulation of miR-375 by formaldehyde, and then to the negative regulation of KLF4, providing experimental evidence for the clinical use of formaldehyde.

Disclosure statement
No potential conflict of interest was reported by the authors. Figure 6. KLF4 could reverse the promotion of miR-375 on the proliferation of formaldehyde-treated 16HBE cells. Compared with the miR-con group, Ã p < .05; Compared with the miR-375 þ pcDNA group, # p < .05.