LncRNA–ENST00000446135 is a novel biomarker of cadmium toxicity in 16HBE cells, rats, and Cd-exposed workers and regulates DNA damage and repair

Abstract

Cadmium (Cd) and its compounds are well-known human carcinogens, but the mechanisms underlying the carcinogenesis are not well understood. This study aimed to investigate whether long noncoding RNA (LncRNA)–ENST00000446135 could serve as a novel biomarker of Cd toxicity in cells, animals, and Cd-exposed workers and regulate DNA damage and repair. LncRNA–ENST00000446135 expression increased gradually in cadmium chloride-transformed 16HBE cells. Small interfering RNA-mediated knockdown of LncRNA–ENST00000446135 inhibited the growth of DNA-damaged cells and decreased the expressions of DNA damage-related genes (ATM, ATR, and ATRIP), whereas increased the expressions of DNA repair-related genes (DDB1, DDB2, OGG1, ERCC1, MSH2, XRCC1, and BARD1). Chromatin immunoprecipitation-sequencing showed that MSH2 is a direct transcriptional target of lncRNA–ENST00000446135. Cadmium increased lncRNA–ENST00000446135 expression in the lung of Cd-exposed rats in a dose-dependent manner. A significant positive correlation was observed between blood ENST00000446135 expression and urinary/blood Cd concentrations, and there were significant correlations of LncRNA–ENST00000446135 expression with the DNA damage cell and the expressions of target genes in the lung of Cd-exposed rats and the blood of Cd-exposed workers and significantly correlated with liver and renal function in Cd-exposed workers. These results indicate that the expression of LncRNA–ENST00000446135 is upregulated and may serve as a signature for DNA damage and repair related to the epigenetic mechanisms underlying the cadmium toxicity and become a novel biomarker of cadmium toxicity.

Introduction

Metal cadmium (Cd) and its compounds classified as human carcinogens in 1993 by the International Agency for Research on Cancer and ranked seventh on the 2017 agency for Toxic Substances and Disease Registry [1, 2]. Experimental and epidemiological studies have shown that cadmium and its compounds are carcinogenic to animals and humans. Studies have indicated that long-term exposure to Cd can result in the occurrence and development of many diseases including tumors, osteoporosis and bone fractures, hypertension, atherosclerosis, diabetes, and kidney diseases [3–8]. Although some of the molecules involved in Cd tolerance have been identified, the regulatory mechanisms involved are still largely unknown. Reports suggest that Cd may lead to cell epigenetic changes including aberrant DNA methylation, different microRNAs, and long noncoding RNAs (LncRNAs) expression profiles. Among of these mechanisms, noncoding RNA is the front edge of Cd toxicity research and has opened up a new field for Cd-associated disease study [9–13]. We previously found that there were aberrant expression profiles of LncRNAs in Cd-treated 16HBE cells, and some could modulate DNA damage and repair in cadmium toxicology [14]. However, the studies about the role of LncRNAs in the cadmium-induced toxicity and carcinogenicity were limited.

LncRNAs are a class of transcripts that are longer than 200 nucleotides but lack protein-coding sequence [15]. It has been evidenced that LncRNAs are transcribed from the intergenic and intronic regions of genome primarily by polymerase II, 5′-methyl-capped and polyadenylated in manner similar to that of mRNAs [15]. It is believed that the genome encodes at least as many LncRNAs as known protein-coding genes [16]. Thousands of LncRNAs have been found to be evolutionarily conserved [17] and exhibit expression patterns correlating with various cellular processes [18]. It is now considered that these LncRNAs represent a significant feature of normal cellular networks. Specifically, increasing evidence suggests that LncRNAs play a critical role in the regulation of diverse cellular processes such as stem cell pluripotency, development, cell growth, and apoptosis [19]. Given their abundance and regulatory potential, it is likely that some LncRNAs are involved in tumor initiation and progression. Recent studies suggest a number of modes of action for LncRNAs [20], most notably the regulation of epigenetic marks and gene expression [21]. Also, LncRNAs may function as decoy, scaffold, and guide molecules. Some LncRNAs act in cis or trans to regulate transcription of nearby genes and regulate the expression of both local and distal genes [22, 23]. Rapicavoli et al. [24] found that LncRNA Six3OS acts in trans to regulate retinal development by modulating Six3 activity and demonstrated that LncRNA can modulate the activity of their associated protein coding genes. Vance  et al.’s study [25] showed that LncRNA can function in trans at transcriptional regulatory elements distinct from its site of synthesis to control large-scale transcriptional programs.

We previously established a model of morphological cell transformation with cadmium chloride (CdCl₂) in human bronchial epithelial cells (16HBE) [26]: 16HBE cells were treated by different concentrations of CdCl₂ (0.1, 1.0, 5.0, 10.0, and 15.0 μmol/l) for 14 weeks. Tumorigenic potential of transformed cells was identified by assays for anchorage-independent growth in soft agar and for tumorigenicity in nude mice after the 35th passage. A cadmium exposure rat model was established [27]. These models are helpful to examine the molecular events occurring during Cd toxicity and carcinogenesis. In our previous study, results showed some LncRNAs were aberrantly expressed in Cd-treated 16HBE cells and LncRNAs could modify cell proliferation, apoptosis, and DNA damage in cadmium toxicity [28, 29]. However, mechanisms of Cd toxicity regulated by LncRNAs are unknown. This study aimed to investigate whether LncRNA–ENST00000446135 could regulate DNA damage and repair in Cd toxicity. Moreover, we validated LncRNA–ENST00000446135 as a novel biomarker of Cd toxicity in cells, animals, and workers with Cd exposure.

Materials and methods

Cell culture and treatments

16HBE cells were morphologically transformed using CdCl_2, as previously described [26]. Untransformed 16HBE cells (control group); Cd-transformed cells of the 5th (5 μmol/l Cd for 2 weeks), 15th (5 μmol/l Cd for 6 weeks), and 35th (5 μmol/l Cd for 14 weeks) passages were maintained in RPMI-1640 containing L-glutamine, 10% fetal bovine serum, and 1% penicillin/streptomycin (Life Technologies) at 37°C in a 5% CO₂ humidified atmosphere. The cells were passaged twice weekly and cells in logarithmic growth phase (2–5 × 10⁵ cells/ml) were harvested for following experiments.

Animals and cadmium exposure

Specific pathogen-free Sprague–Dawley (SD) rats (90 ± 10 g) were purchased from Guangdong Medical Laboratory Animal Center (license No: SCXK 2008-0002, Guangdong, China) and maintained under a pathogen-free condition in Laboratory Animal Center of Guangzhou Army General Hospital [license No: SYXK (Military) 2007-33, 2008C1230034834, Guangdong, China]. Ninety-six SD rats (half male and half female) were randomly divided into four groups. Rats were chronically exposed to Cd by intraperitoneal injection of CdCl₂ (Sigma, St. Louis, MO, USA) in normal saline at different concentrations (high dose: 1.225 mg/kg; mid dose: 0.612 mg/kg, and low dose: 0.306 mg/kg). Rats in control group were intraperitoneally injected with 0.5 ml of normal saline. Cd treatment was performed five times weekly. After 14 weeks, 24-hour urine samples were collected. On the second day, rats were anesthetized and blood was collected from the heart and stored at 4°C. The lung were harvested and stored in liquid nitrogen [25].

Study population

A total of 186 workers were recruited from a Cd refinery factory with the assistance of Center for Disease Control and Prevention, Institute for Health Supervision in Shenzhen, P.R. China. The workers included production workers, machine maintenance workers, product development personnel, management personnel, and other personnel engaged in cleaning, service, security, and so on. Detailed information including the age, marital status, smoking habits, alcohol consumption, professional and medical history was collected from each subject and evaluated by well-trained interviewers. In addition, the workers were asked to receive a comprehensive physical examination. The physical examination included detection of blood pressure and pulse rate, examination of the throat and pharynx, detection of lung function, electrocardiography, liver and kidney ultrasonography, cardiopulmonary X-ray, and detection of blood cells, serum alanine aminotransferase (ALT), urinary Cd (UCd), and creatinine (Cr). In this study, subjects who could not provide reliable information on the smoking history, had a smoking history, or had a history of kidney or liver diseases were excluded. Finally, 186 nonsmoking subjects (109 males and 77 females) with the age ranging from 23 to 50 years were included for analysis.

Bioinformatics analysis of LncRNA–ENST00000446135

We previously detected the LncRNA and mRNA aberrant expression profiles were in Cd-induced 35th cells by microarray assay [14]. To further analyze the lncRNA–ENST00000446135 in this study, the lncRNA–ENST00000446135–mRNA regulatory network was constructed. Pearson correlation coefficient and P-value of lncRNA–ENST00000446135–mRNA were calculated using false discovery rate (FDR) correction. LncRNA–ENST00000446135–mRNA regulatory network was drawn using the cytoscape (http://cytoscape.org/). The predicted target genes were input into the database Annotation Visualization and Intergrated Discovery (http://david,abcc,ncifcrf,gov/). Gene Ontology (GO) was used to identify the molecular function represented in the gene profile. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome, ad, jp/kegg) and BioCarta (http://www.biocarta,com) were used to analyze the roles of these target genes in the pathways.

Quantitative real-time polymerase chain reaction

Total RNA was isolated using the Trizol reagent. Reverse transcription was performed using a TITANIUM real-time polymerase chain reaction (RT-PCR) kit (Clontech, Mountain View, CA) according to the manufacturer’s instructions. The gene expression was quantified using a fluorescence-based quantitative real-time polymerase chain reaction (RT-qPCR) according to manufacturer’s instructions (Bio-Rad Laboratories). The sequences of primers used for RT-qPCR are shown in Table 1.

Table 1.

Primers used for RT-PCR and the sequences of LncRNA–ENST00000446135–siRNA and scramble control siRNA

LncRNAs	Primers
ENST00000446135	Forward: 5′-GGGACAAGCAGCACAGAACT-3′
	Reverse:5′-CAGCAGAATAACGGCACAAG-3′
mRNAs	Primers
ATM	Forward:5′-TGCCAGACAGCCGTGACTTAC-3′
	Reverse: 5′-ACCTCCACCTGCTCATACACAAG -3′
ATR	Forward: 5′-GCCGTTCTCCAGGAATACAG-3′
	Reverse: 5′-GAGCAACCGAGCTTGAGAGT-3′
ATRIP	Forward: 5′-CAGCTGGAGACAGAGATCAA-3′
	Reverse: 5′-GACATTCCAGCCAAGGTACT-3′
DDB1	Forward: 5′-TGGTTGCCAAGCACCTACTA-3′
	Reverse: 5′-ACTGCGATCACCATGGAAGC-3′
DDB2	Forward: 5′-ATCCTGTCAACGCAGCTTGT-3′
	Reverse: 5′-GATGCCAGGCTGCCTTGAT-3′
OGG1	Forward: 5′-CCGAGCCATCCTGGAAGAAC-3′
	Reverse: 5′-CCATCAGGCAGATGCAGTCA-3′
ERCC1	Forward: 5′-CTTGTCCAGGTGGATGTGAA-3′
	Reverse: 5′-GCCTTGTAGGTCTCCAGGTA-3′
MSH2	Forward: 5′-CATCCAGGCATGCTTGTGTTGA-3′
	Reverse: 5′- GCAGTCCACAATGGACACTTC-3′
RAD50	Forward: 5′-GGGTTTCCAAGGCTGTGCTAA-3′
	Reverse: 5′-TCTGACGTACCTGCCGAAGT-3′
XRCC1	Forward: 5′-CAGCCGGATCAACAAGACAT-3′
	Reverse: 5′-CTGAGGAGGCAGCACTAGAA-3′
β-Actin	Forward: 5′-ACAGAGCCTCGCCTTTGCCGAT-3′
	Reverse: 5′-CTTGCACATGCCGGAGCCGTT-3′
siRNA	Sequence
silncRNA–ENST00	Sense: 5′-GAGGUGUAAAGGGAUUUAUTT-3′
000446135-4311	Antisense: 5′-AUAAAUCCCUUUACACCUCTT-3′
silncRNA–ENST00000446135-4312	Sense: 5′-GAGGUGUAAAGGGAUUUAUTT-3′
	Antisense: 5′-AUAAAUCCCUUUACACCUCTT-3′
silncRNA–ENST00	Sense: 5′-GGCAUUUGCAUCUUUAAAUTT-3′
000446135-4313	Antisense: 5′-AUUUAAAGAUGCAAAUGCCTT-3’
Control siRNA	Sense: 5′-UUCUCCGAACGUGUCACGUTT-3′
	Antisense: 5′- ACGUGACACGUUCGGAGAATT-3′

RNA interference

To inhibit lncRNA–ENST00000446135, 50 nM of small interfering RNA (siRNA) (siRNA–ENST00000446135-4311, siRNA–ENST00000446135-4312, siRNA–ENST00000446135-4313)， Shanghai Genepharma, China) were transfected into untreated 16HBE cells，Cd-transformed cells of the 15th passage, and Cd-transformed cells of the 35th passage using Lipofectamine 2000 reagent according to the manufacturer’s instructions. Cells transfected with scramble-control siRNA (negative control) were used as controls. Cells were harvested 72 hours after transfection. Compared with controls, siRNA–ENST00000446135-4311 and siRNA–ENST00000446135-4312 successfully decreased the expression of LncRNA–ENST00000446135. The sequences of LncRNA–ENST00000446135 siRNAs and scramble control siRNA were listed in Table 1.

Comet assay

DNA damage was investigated using the comet assay. 16HBE cells at different stages of Cd-induced malignant transformation, siRNA/ENST00000446135 transfected 35th passage cells, negative control 35th passage cells, and white blood cells form Cd-exposed workers were diluted in phosphate-buffered saline (PBS) so that three or four cells could be observed in a single field at ×400. Comet assay was done according to the published protocol with a minor modification. Briefly, 100 μl of low-melting agarose [1% (v/v) in PBS (pH 7.4)] at 37°C was mixed with 10 μl of PBS containing lymphocytes and then transferred onto a precoated [0.5% (v/v) normal melting agarose in PBS (pH 7.4)] slide. Electrophoresis was conducted for 20 minutes at 25 V. DNA damage was measured using an image analysis system (version 1.0, IMI Comet Analysis Software, China; ref.). Fifty cells were analyzed per slide, and the Olive tail moment (Olive TM) value was used as a measurement of DNA damage level as recommended.

Chromatin immunoprecipitation sequencing

The Cd-transformed 35th passages cells and siRNA/ENST00000446135-transfected 35th passage cells were fixed with formaldehyde at a 1% final concentration by incubation at 37°C for 10 minutes. Then, glycine was added to a final concentration of 0.14 mol/l, and the cells were incubated at room temperature for 30 minutes. The cells were harvested into a fresh tube and lysed with 400 ml of lysis buffer with 8 ml of protease inhibitor, with ice vortexing every 2 minutes. The cell lysate was sonicated to shear cross-linked DNA and was then mixed with Dynal beads and washed three times with PBS/bovine serum albumin (BSA). The beads were resuspended with 1 ml of PBS/BSA with the tube against a magnet. Then, 5 μg of antibody (control IgG or，anti-MSH2, anti-DDB1) (Santa Cruz) were added to the bead slurry， for a total volume of 1 ml. This mixture was then incubated for 6 hours on a rotating platform at 4°C. The antibody–bead slurry was washed three times with PBS/BSA. Sheared chromatin was added to the antibody–bead slurry and incubated at 4°C overnight on a rotating platform. Then, 30 ml of chromatin were saved for input. The antibody–bead slurry was washed five times with RIPA buffer (50 mmol/l HEPES pH 8.0, 1% NP-40, 0.7% DOC, 0.5 M LiCl, and 1× proteinase inhibitor cocktail), resuspended with 100 ml of elution buffer (10 mmol/l Tris pH 8.0. 1 mmol/l EDTA, and 1% sodium dodecyl sulfate), and incubated at 65°C for 10 minutes. Reverse cross-linking was performed at 65°C overnight. Then, a 0.4 mg/ml final concentration of proteinase K was added to the elution, and the sample was incubated at 55°C for 1 hour. Genomic DNA was isolated using a Tiangen DNA purification kit and then incubated with 1 ml of RNAse (20 mg/ml) at 37°C for 1 hour. Finally, the DNA was reisolated using AMpure XP beads. The chromatin immunoprecipitation sequencing (Chip-seq) library was generated with a TD503 kit (Vazyme). Sequencing was carried out using Illumina HiSeq X Ten. Chip-seq peakfinding was performed with the MACS algorithm.

Cadmium determination and functional and pathological examinations of organs in cadmium-exposed rats

The cadmium level was determined using the cadmium standard solution (BZ/WJ/GB101/2009-1, Guangdong Occupational Health Inspection Center, Guangdong, China) by atomic absorption spectrometry (ZEENIT700, Analytik Jena, Jena, German). The concentration of UCd was normalized by urinary creatinine (Cr). Tissue samples were fixed in 10% formalin and the pathological features were examined following hematoxylin and eosin staining. Serum ALT and aspartate aminotransferase (AST) were used as biochemical markers of liver function. Blood urea nitrogen (BUN), serum creatinine (Scr), and 24-hour urine protein (24-h Pro) were used to evaluate the renal function. ALT, AST, BUN, SCR, and 24-h Pro were measured using corresponding kits according to the manufacturer’s instructions with an automatic biochemistry analyzer (Hitachi 7600-020/7170A; Tokyo, Japan). All animal experiments were performed in accordance with the principals of the Declaration of Helsinki. All experimental protocols were approved by Research Ethic Committee of Guangzhou Medical University.

Collection and treatment of biological samples from cadmium-exposed workers

Venous blood was collected after fasting for 10–12 hours and transferred into anticoagulant and metal-free tube for blood cadmium (BCd) detection, blood routine examination, blood biochemical examination (ALT, AST, Cr, and BUN), and detection of blood lncRNA–ENST00000446135 and its target genes. BCd concentrations were measured by atomic absorption spectrometry (ZEENIT700; Analytik Jena, Jena, Germany). Blood biochemistry was done with an automatic biochemical analyzer (HITACHI7600-020/7170A; Hitachi, Tokyo, Japan). The expression of lncRNA–ENST00000446135 and its target genes was measured by RT-qPCR.

Urine samples were collected from all participants and transferred into a metal-free polyethylene bottle as per the guidelines of clinical chemistry division of International Union of Pure and Applied Chemistry. These samples were diluted with equal volume of 0.3 mol/l HNO₃ and stored at 4°C until further analysis. Urine Cd concentration was measured by atomic absorption spectrometry (ZEENIT700; Analytik Jena, Jena, Germany). Cd standard curve was linear up to 25 μg/l and the detection limit was 0.33 μg/l. The internal standard of Cd was added to urine and analyzed, and a recovery rate of 98.2% was found.

Ethical statement

All experimental protocols were approved by Research Ethic Committee of Shenzhen Futian Second People’s Hospital. All the experiments were performed in accordance with the principals of the Declaration of Helsinki and relevant laws or guidelines. All experiments followed institutional guidelines. Informed consent was obtained from each participant. Participants were assured of their right to refuse to participate or to withdraw from the study at any time. Anonymity and confidentiality of the participants were assured. Participants were presented with a small gift (valued 10.0 USD) on completion of the survey. The personal information of samples involved in the study was not opened.

Statistical analysis

All the data are represented as rate (%) or mean ± standard deviation (SD; ± s) of three or more independent experiments. Comparisons were done using the chi-square test for rate (%) from several independent experiments and Student’s t-test or analysis of variance (ANOVA) followed by Dunnett’s test for mean ± SD. The correlation of two groups was tested by Pearson or Spearman’s correlation analysis. Statistical analysis was performed with SPSS version 20.0 software. A value of P < 0.05 was considered statistically significant.

Results

Abnormally high LncRNA–ENST00000446135 expression in CdCl₂ transformed 16HBE cells

RT-qPCR was performed to detect the lncRNA–ENST00000446135 expression in CdCl₂-transformed 16HBE cells at different stages. Results showed the lncRNA–ENST00000446135 expression increased over time in CdCl₂-transformed 16HBE cells: the LncRNA–ENST00000446135 expression in untransformed 16HBE cells (control group); Cd-transformed cells of the 5th, 15th, and 35th passages cell were 1.0 ± 0.15, 1.49 ± 0.24, 5.60 ± 0.46, and 7.90 ± 0.82, respectively. The lncRNA–ENST00000446135 expression in 16HBE cells of 15th passage, and 16HBE cells of 35th passage was 5.6 and 7.9 times that in control group (P < 0.05). These suggested that there is abnormally high lncRNA–ENST00000446135 expression in CdCl₂-transformed 16HBE cells.

Bioinformatics analysis of LncRNA–ENST00000446135

LncRNA–ENST00000446135–mRNAs coexpression network was constructed based on the correlation analysis between differentially expressed LncRNAs and mRNAs profiles (Supplementary Tables S1–S4). As shown in Figure 1A, LncRNA–ENST00000446135 and their associated mRNAs were identified, with most of the pairs showing a positive correlation. According to the GO-Pathway analysis of differentially expressed LncRNA–ENST00000446135/mRNAs, the neighbor gene function upregulated LncRNA–ENST00000446135 mainly involved the following pathway to the target genes: DNA damage and repair, biological cycle, cell cycle progression, metabolism, molecular transducer activity, etc. (Fig. 1B). There were 33 potential target mRNAs regulated by ENST00000446135 (Supplementary Table S5), and most of the target mRNAs have been reported to be related to cadmium toxicity and cancers.

Figure 1.

Bioinformatics analysis of LncRNA–ENST00000446135. (A) Coexpression network of LncRNA–ENST00000446135 and mRNA was constructed with cystoscope software (http://www.cytoscape.org/) based on the correlation analysis between LncRNA–ENST00000446135 and differentially expressed mRNAs in Cd-induced 35th 16HBE cells as compared with untreated 16HBE cells. (B) GO and signaling pathway analysis of LncRNA–ENST00000446135. Pathway analysis was predominantly based on the KEGG database, and the mRNAs were annotated and classified according to the GO database.

Silencing of ENST00000446135 decreased DNA damage in HBEs with Cd-induced malignant transformation

During the Cd-induced malignant transformation of 16HBE cells, the tail lengths of the DNA comets were significantly longer than those in the untransformed 16HBE cells (P < 0.05), and the tail lengths of the DNA comets in Cd-treated 35th passage cells were significantly longer than those in siRNA–ENST00000446135-transfected cells (P < 0.05). The Cd-induced DNA damage rates in 5th, 15th, and 35th passage cells were 10.45%, 22.00%, and 46.75%, respectively, as compared with untreated 16HBE cells (4.75%; P < 0.05). However, the DNA damage rate in Cd-induced 35th passage cell after siRNA/ENST00000446135-4311 and siRNA/ENST00000446135-4312 transfection were 28.93% and 29.13%. The tail lengths of Cd-induced 35th passage cell after siRNA/ENST00000446135-4311 and siRNA/ENST00000446135-4312 were shorter than negative control group, which were significantly lower than that in negative control group (P < 0.05) (Table 2). These results suggest that siRNA–ENST00000446135 inhibits the growth of DNA-damaged cells during the Cd-induced malignant transformation of 16HBE cells.

Table 2.

DNA damage and its suppression during Cd-induced malignant transformation of 16HBE cells and siRNA/ENST00000446135-transfected 35th passage cells determined by comet assay

Cell type	DNA damage rate (%)	Tail length (μm)
Untransformed 16HBE cells	4.75	12.8 ± 1.76
5th passage	10.45#	15.2 ± 3.54#
15th passage	22.00*#	31.6 ± 2.80*#
35th passage	46.75*	47.8 ± 2.36*
siRNA/ENST00000446135-4311 1-transfected 35th passage cells	28.93*#	31.14 ± 3.86*#
siRNA/ENST00000446135-4312-transfected 35th passage cells	29.13*#	32.02 ± 4.02*#
Negative control transfected 35th passage cells	41.14*	44.56 ± 5.17*

The tail lengths of cells in which DNA damage was induced and suppressed were determined by comet assay in untransformed 16HBE cells, 5th, 15th, 35th passage-transformed cells, siRNA/ENST00000446135-transfected 35th passage cells, and negative control-transfected 35th passage cells. *P < 0.05, vs. untransformed controls, ^#P < 0.05, vs. 35th passage cells.

Silencing of ENST00000446135 regulated the expression of DNA damage- and repair-related genes in 16HBE cells with Cd-induced malignant transformation

The mRNA expression of genes related to DNA damage and repair was detected by RT-PCR. Results showed the mRNA expression of ATM, ATR, and ATRIP progressively increased, but that of MSH2, OGG1, ERCC1, DDB1, DDB2, and XRCC1 progressively reduced during the Cd-induced malignant transformation in HBEs. Furthermore, the expression of these genes in 35th passage-transformed cells was significantly different from that in untreated 16HBE cells (P < 0.05) (Fig. 2A). Transfection with siRNA–ENST00000446135-4311 and siRNA–ENST00000446135-4312 in Cd-treated 35th passage-transformed cells (Fig. 2B) resulted in a significant decrease in the mRNA expression of DNA damage-related genes ATM, ATR ,and ATRIP, and a marked increase in the mRNA expression of DNA repair-related genes, which are a negative regulator of DNA repair signaling pathway (Fig. 2C and D). These findings suggest that siRNA–ENST00000446135 activates DNA damage and repair signaling pathway.

Figure 2.

mRNA expression of DNA damage- and repair-related genes in 16HBE cells with Cd-induced malignant transformation and siRNA/LncRNA–ENST00000446135-transfected cells. (A) mRNA expression of DNA damage- and repair-related genes in 16HBE cells with Cd-induced malignant transformation. The gene expression was validated in untreated controls 16HBE cells, Cd-induced 5th-, 15th-treated cells, 35th passage-transformed cells by qPCR and normalized to that of β-actin. The fold change in expression of experiment group was normalized to that of untreated control 16HBE cells. Data are expressed as mean ± SD. *P < 0.05 vs. untreated 16HBE (one-way ANOVA). (B) Untreated control 16HBE cells and Cd-transformed 35th passage cells were treated with LncRNA–ENST00000446135–siRNA, and the LncRNA–ENST00000446135 expression was detected after 72 hours by qPCR. *P < 0.05 vs. control cells (one-way ANOVA). (C) Untreated 16HBE cells and Cd-transformed 35th passage cells were independently treated with LncRNA–ENST00000446135–siRNA4311 and siRNA4312, and the mRNA expression of DNA damage-related genes was detected after 48 hours by qPCR. (D) Untreated 16HBE cells and Cd-transformed 35th passage cells were independently treated with LncRNA-ENST00000446135–siRNA531 and siRNA532, and the mRNA expression of DNA repair-related genes was detected after 72 hours by qPCR. *P < 0.05 vs. control cells (one-way ANOVA).

Chip-seq results for silencing of ENST00000446135 in Cd-treated 35th passage-transformed cells

Msh2 and DDB1were selected to further research the mechanism of ENST00000446135 regulating DNA repair genes. Chip was carried out and Chip nucleic acid quantitative results were following: DDB1: 5 μg/ml; MSH2: 14 μg/ml; IgG: 14 μg/ml; input: 43 μg/ml. Therefore, we performed Chip-seq for LncRNA–ENST00000446135 and the Msh2-binding regions were identified with the peak-calling program MACS, which also identified the Msh2 target genes harboring the peak regions in their approximate promoters. Interestingly, the Msh2 target gene set exhibited a strong enrichment in a gene set enrichment analysis using the differential mRNA expression data upon LncRNA–ENST00000446135 silencing (Fig. 3A). We identified a Chip-seq peak in the promoter region of the LncRNA–ENST00000446135 (total peaks = 359, peak size = 180, total tags = 3063699.0, total tags in peaks = 31502.0, approximate immunoprecipitation (IP) efficiency = 1.03%) (Fig. 3B, Supplementary Table S6). DNA sequence of No. 4 motifs was searched for enriching in Chip-seq datasets (Supplementary Table S7), and the sequence of the peak corresponded to a consensus Msh2-binding motif (AGTTTCTGAGAA), which was found a match in Msh2 sequence. The location was in 1989-1995. The length is 7 (Fig. 3C). This result again supported our finding that Msh2 activity depended on LncRNA–ENST00000446135 expression.

Figure 3.

Chip-seq results for silencing of LncRNA–ENST00000446135 in Cd-treated 35th passage-transformed cells. (A) Chromosomal location (red rectangle; top) and integrated genomics viewer screenshots for Chip-seq data. It is the bedGraph of MSH2, and the peak in the picture was the position of the readings. (B) Peaks. The double peaks were the location where the data gathering, which represented the position of protein binding. (C) Motif enrichment analysis of LncRNA–ENST00000446135 binding regions identified motifs recognized by Msh2. It was the matching sequence in the fourth motif from 25 motifs.

LncRNA–ENST00000446135 expression in rats with chronic Cd exposure

The expression of LncRNA–ENST00000446135 in Cd-exposed rats was confirmed by qPCR. The expression of LncRNA–ENST00000446135 in the lung of low-dose, mid-dose, and high-dose Cd-exposed rats was 3.135 ± 0.538-fold, 7.22 ± 0.753-fold, and 12.912 ± 0.769-fold, respectively, as compared with that in control rats. Significantly upregulated lncRNA–ENST00000446135 expression was found in the lung of Cd-exposed rats (P < 0.05). Additionally, Cd increased LncRNA–ENST00000446135 expression in the lung in a dose-dependent manner (P < 0.05).

LncRNA–ENST00000446135 expression was correlated with target gene expression in Cd-exposed rats

As shown in Table 3, a significant positive correlation of ENST00000446135 expression with the expression of ATM, ATR, and ATRIP was observed, whereas there was a significant negative correlation between ENST00000446135 expression and expression of DDB1, DDB2, OGG1, ERCC1, MSH2, and XRCC1 in Cd-exposed rats (P < 0.05). These findings indicate that ENST00000446135 expression correlates well with the expression of its target genes in Cd-exposed rats.

Table 3.

Correlation between lncRNA–ENST00000446135 expression and its target gene expression in Cd-exposed rats

Target genes	Correlation coefficient (r)	P	Target genes	Correlation coefficient (r)	P
ATM	0.581	0.002	ERCC1	−0.468	<0.001
ATR	0.636	<0.001	DDB1	−0.622	<0.001
ATRIP	0.617	<0.001	DDB2	−0.654	<0.0001
MSH2	−0.629	0.002	XRCC1	−0.529	<0.0001
OGG1	−0.612	<0.0001

Health status of the workers exposed to Cd

The subjects (median age, 31 years) were directly or indirectly exposed to Cd for <2 years with no history of exposures to other toxins. Only nonsmokers were included in the present study. Urine Cd concentration normalized to the urine creatinine (Cr) showed a positive skewness distribution. The median, maximum, and minimum UCd concentrations were 1.61, 113.86, and 0.31 μg/g Cr, respectively. The 25 and 75 percentiles of UCd concentrations were 0.69 and 9.54 μg/g Cr, respectively. According to UCd concentration, subjects were categorized into three groups: 0–2, 2–5, and >5 μg/g Cr (Table 4). The age, gender, and duration of employment were comparable among three groups, suggesting that our results were not confounded by these factors.

Table 4.

Blood LncRNA–ENST00000446135 expression at different Cd exposure levels in Cd-exposed workers (n = 186)

Exposure to Cd at different levels	n	lncRNA–ENST00000446135	F	P-value
UCd levels
≤2 μg/g Cr	153	0.876 ± 0.831	74.297	P total < 0.001
②2-5 μg/g Cr	19	3.104 ± 2.579		P ①② = < 0.001
③>5 μg/g Cr	14	7.565 ± 5.748		P ①③ < 0.001
				P ②③ < 0.001
Urine β2-MG
≤500 μg/g Cr	152	0.955 ± 1.490	78.099	P total < 0.001
500-1000μg/g Cr	19	2.500 ± 1.087		P ①② = 0.002
				P ①③ < 0.001
>1000 μg/g Cr	15	8.148 ± 5.672		P ②③ < 0.001
BCd level
≤2 μg/l	150	0.893 ± 1.189	32.506	P total < 0.001
2–5 μg/l	20	2.178 ± 3.601		P ①② = 0.003
>5 μg/l	16	5.685 ± 4.588		P ①③ < 0.001
				P ②③ < 0.001

LncRNA–ENST00000446135 expression was correlated with Cd exposure in Cd-exposed workers

In order to evaluate whether ENST00000446135 serves as a biomarker of Cd exposure, the expression of ENST00000446135 in the blood of Cd-exposed workers was detected by RT-qPCR. According to the UCd concentration and BCd concentration, these works were divided into three groups (Table 4). The blood LncRNA–ENST00000446135 expression increased with the increase in UCd concentration and BCd concentration. The ENST00000446135 expression was significantly higher in workers with UCd concentration at 2–5 and >5 μg/g Cr (3.104 ± 2.579-fold and 7.565 ± 5.748-fold, respectively), when compared with control group (UCd concentration: 0–2 μg/g Cr) (P < 0.05). A similar finding was identified in blood LncRNA–ENST00000446135 expression in workers with different BCd concentrations (2.178 ± 3.601-fold and 5.685 ± 4.588-fold, respectively) when compared with control group (0–2 μg/l) (P < 0.05). There was a significant positive correlation between ENST00000446135 expression and BCd concentration (r = 0.554, P < 0.001), UCd concentration (r = 0.756, P < 0.001), and urine β₂-MG concentration (r = 0.725, P < 0.001) (Fig. 4). These findings indicate that ENST00000446135 expression is correlated with Cd exposure in Cd-exposed workers.

Figure 4.

Correlation analysis between LncRNA–ENST00000446135 expression and Cd concentration in Cd-exposed workers. Correlation analysis between LncRNA–ENST00000446135 expression and BCd concentration (A), UCd concentration (B), and urine β₂-MG concentration (C). Blood LncRNA–ENST00000446135 expression was calculated by the ratio of its expression to that of β-actin. The UCd concentration was normalized by urine creatinine (μg/g Cr) and urine β₂-MG (μg/g Cr). The linear relationship was analyzed by Pearson correlation analysis.

LncRNA–ENST00000446135 expression was correlated with liver and renal function in Cd-exposed workers

To evaluate the liver and renal function in Cd-exposed workers, their venous blood was collected. ALT, AST, γ-glutamyl transpeptidase (γ-GT), lactate dehydrogenase (LDH), Cr, BUN were detected. A dose-dependent increase of the liver and renal function indictors including AST, ALT, γ-GT, LDH, Cr, and BUN with accumulation of Cadmium in Cd-workers (P < 0.05) (Table 5). Furthermore, a positive correlation of the LncRNA–ENST00000446135 expression level was observed with the liver and renal function indictors (P < 0.01). These findings indicated that LncRNA–ENST00000446135 expression correlate with Cd-induced organ damages in Cd-exposed workers.

Table 5.

Liver and renal function in Cd-exposed workers (n = 186)

Exposure levels of Cd	N	LncRNA–ENST00000446135	ALT	AST	γ-GT	LDH	Cr	BUN
UCd levels
≤2 μg/g Cr	153	0.876 ± 0.831	12.63 ± 6.29	18.69 ± 8.11	13.01 ± 7.98	205.76 ± 44.77	63.12 ± 11.54	3.65 ± 1.01
2–5 μg/g Cr	19	3.104 ± 2.579	13.21 ± 11.41	20.42 ± 7.87	15.91 ± 9.66	214.89 ± 39.21	65.96 ± 12.45	3.98 ± 1.11
>5 μg/g Cr	14	7.565 ± 5.748	19.31 ± 11.43	21.27 ± 6.66	19.24 ± 7.33	225.46 ± 70.32	68.87 ± 15.77	4.69 ± 1.02
Urine β2-MG
≤500 μg/g Cr	152	0.955 ± 1.490	12.45 ± 10.83	18.32 ± 7.98	13.54 ± 6.89	206.45 ± 47.99	62.32 ± 13.85	3.53 ± 1.05
500–1000 μg/g Cr	19	2.500 ± 1.087	14.21 ± 10.16	19.49 ± 8.44	17.34 ± 10.78	203.26 ± 60.33	6611 ± 14.23	4.19 ± 1.64
>1000 μg/g Cr	15	8.148 ± 5.672	18.121 ± 8.21	21.65 ± 7.98	18.96 ± 9.56	238.21 ± 71.34	70.29 ± 11.21	4.95 ± 1.12
BCd level								4.11 ± 1.23
≤2 μg/l	150	0.893 ± 1.189	13.13 ± 6.93	18.54 ± 8.87	13.12 ± 7.87	209.76 ± 56.22	60.23 ± 13.44	3.61 ± 1.14
2–5 μg/l	20	2.178 ± 3.601	14.13 ± 10.17	20.07 ± 6.88	15.21 ± 10.24	210.54 ± 44.65	69.26 ± 13.55	4.11 ± 1.23
>5 μg/l	16	5.685 ± 4.588	18.00 ± 8.59	19.98 ± 8.94	17.94 ± 10.72	200.12 ± 57.87	76.45 ± 12.58	4.91 ± 1.24

LncRNA–ENST00000446135 expression was correlated with DNA damage of blood cells in Cd-exposed workers

A significant correlation was found between blood LncRNA–ENST00000446135 expression and DNA damage (r = 0.650, P < 0.001). The blood ENST00000414355 expression was moderately related to mean tail moment (TM) of 50 comets (r = 0.582, P = 0.002). The partial correlation (excluding UCd and BCd) analysis also found significant relationship between blood ENST00000414355 expression with TM and DNA damage rate (control UCd: r = 0.681, P = 0.002; control BCd: r = 0.649, P = 0.001).

LncRNA–ENST00000446135 expression was correlated with target gene expression in Cd-exposed workers

There was a significant positive correlation between LncRNA–ENST00000446135 expression and mRNA expression of ATM, ATR, and ATRIP, whereas there was a significant negative correlation between ENST00000446135 expression and mRNA expression of DDB1, DDB2, OGG1, ERCC1, MSH2, and XRCC1 in Cd-exposed workers. In addition, the associations between ENST00000446135 expression and target gene expression were further evaluated after adjustment for UCd concentration and BCd concentration that might affect ENST00000446135 expression and target gene expression, respectively, in Cd-exposed workers. A significant correlation between ENST00000414355 expression and target gene expression was still observed (Table 6). These findings indicate that ENST00000446135 expression correlate very well with target gene expression in Cd-exposed workers.

Table 6.

Correlation Analysis between LncRNA–ENST00000446135 expression and target gene expression in Cd-exposed workers

Target genes	Correlation coefficient (r)	P	Target genes	Correlation coefficient (r)	P
ATM	0.417	0.008	ERCC1	−0.544	0.062
ATR	0.581	<0.001	DDB1	−0.498	<0.001
ATRIP	0.487	<0.001	DDB2	−0.566	<0.001
MSH2	−0.712	0.016	XRCC1	−0.425	<0.001
OGG1	−0.634	<0.001

Discussion

Cadmium is a heavy metal that has been widely used in industrial processes such as during the fabrication of nickel cadmium batteries [28]. Human exposure to cadmium primarily occurs through eating wheat and rice from cadmium-contaminated soil, as well as inhalation and cigarette smoking. Cadmium has a wide spectrum of toxic effects including nephrotoxicity, neurotoxicity, carcinogenicity, teratogenicity, endocrine and reproductive disorders, interference with DNA repair mechanisms, generation of oxidative stress, and induction of apoptosis [30]. However, the toxicity mechanisms of Cd are not yet elucidated. Studied have showed that oxidative stress, apoptosis, autophagy, DNA methylation, and abnormal level of noncoding RNA are all involved in the Cd toxicity [9–11]. In our previous study, results showed some LncRNAs are aberrantly expressed in Cd-treated 16HBE cells. Cd could increase DNA damage and decrease DNA repair capacity. Cadmium upregulates the LncRNA ENST00000414355 and MALT1 in 16HBE cells and in the lungs of Cd-treated rats in a dose-dependent manner, whereas blood LncRNA ENST00000414355 and MALT1 positively correlates with UCd of exposed workers [14, 30]. However, few research teams investigated the roles of LncRNAs in cadmium toxicity. Therefore, we speculate that LncRNA–ENST00000446135 might play an important biological role in cadmium toxicity. In this study, we found that the expression level of LncRNA–ENST00000446135 was elevated in CdCl₂-transformed 16HBE cells, CdCl₂-treated rats, and Cd-exposed workers. Moreover, LncRNA–ENST00000446135 can regulate the DNA damage and DNA repair capacity in CdCl₂-transformed 16HBE cells.

The gene symbol of LncRNA–ENST00000446135 is AC073464.10, which located at chromosome 2 and its size is 2716 bp. It is an intergenic LncRNA, and is 95480609 bp at 3′ end of gene encoding apoptosis inhibitor 5 and 95522992 bp at 5′ end of gene encoding. GO-pathway analysis showed the upregulated ENST00000446135 mainly involved the following pathways: DNA damage and repair, biological cycle, etc. There were 33 potential target mRNAs regulated by ENST00000446135. Most of the targets mentioned here have been reported to be linked to cadmium toxicity and cancers [31–33]. These findings suggest that LncRNA–ENST00000446135 might play an important role in cadmium toxicity and Cd-induced carcinogenesis.

To verify the role of ENST00000446135 in cadmium toxicity, the expression of ENST00000446135 in untreated 16HBE cells and Cd-induced 35th cells was knocked down via siRNA. Results showed siRNA ENST00000446135 significantly inhibited the growth of DNA-damaged 16HBE cells during the Cd-induced malignant transformation. Moreover, ENST00000446135 knockdown also decreased the mRNA expression of DNA damage-related genes ATM, ATR, and ATRIP, but increased that of DNA repair-related genes in Cd-induced 35th cells. These findings suggested that siRNA–ENST00000446135 activated DNA repair signaling pathway in Cd-induced carcinogenesis. Then, the RNA–protein interaction of LncRNA and corresponding trans-acting factors (TFS) was further analyzed based on the Chip-Seq. Results showed that there was highly enriched region between ENST00000446135 and Msh2. The Chip-Seq results again supported that Msh2 activity depends on lncRNA–ENST00000446135 expression, which is a mechanism of ENST00000446135 modulating the expression of DNA repair-related genes MSH2 in cadmium toxicity and Cd-induced carcinogenesis.

Many cellular and molecular events are involved in the toxic effects of chemical carcinogens [34, 35]. Cartularo et al. [36] found the mechanisms of malignant transformation by low-dose cadmium in BEAS-2B cells including upregulation of SATB2, downregulation of MGMT, and increased oxidative stress and found HepG2 cells treated with cadmium for 24 hours indicated a reduction in global levels of histone methylation and acetylation that persisted 72 hours after treatment [37]. Jordan et al. [38] found that there were slightly different effects on decreased stem-loop binding protein (SLBP) mRNA and protein as well as increased poly A H3.1 in nickel-exposed cells and saw that the depletion of SLBP protein was reversed by inhibiting the proteosome. The author considered that SLBP depletion is mechanism exploited by several epigenetic modulating metals has not been fully supported, as cadmium did not have all the consistent changes found with As, Ni and yet other carcinogenic metals (chromium, vanadium, etc.) have yet to be tested. However, few studies have been conducted to investigate LncRNAs as new biomarkers of Cd exposure [39]. The present study was undertaken to investigate the biomarkers role of LncRNAs in Cd toxicity of animal model and Cd-exposed workers. The animal model of chronic Cd exposure used in this study was established by continuous intraperitoneal injection of CdCl₂ for 14 weeks. The cadmium toxicity was evaluated by the weight coefficient, histopathological examination, and liver and renal function (ALT, AST, SCR, BUN, and 24-h Pro) detection. The metal concentration of the blood reflects the recent exposure and that of the urine reflects the body burden after a long-term exposure, whereas that of tissues reflects the metal accumulation and organ damage [40, 41]. In the present study, the expression of LncRNA–ENST00000446135 in the lung of Cd-treated rats was positively correlated with the Cd exposure, suggesting that LncRNA-ENST00000446135 reflects the accumulation of cadmium in the body. LncRNA–ENST00000446135 expression in the body is useful in predicting the Cd-induced toxicity.

In addition, the expression of LncRNA–ENST00000446135 was also detected in the blood of workers exposed to Cd. Results showed a positive correlation between blood LncRNA–ENST00000446135 and BCd concentration, UCd concentration, and urine β₂-MG concentration, and expression of target genes, suggesting that blood LncRNA–ENST00000446135 is a potentially novel biomarker of Cd exposure in humans [40]. Subjects with UCd exhibited significantly higher blood LncRNA–ENST00000446135 expression than those without UCd, suggesting that, even at a lower range of UCd concentration, the change in blood LncRNA–ENST00000446135 may reflect the alteration in Cd accumulation.

In conclusion, our study for the first time determines the role of LncRNA–ENST00000446135 in the Cd toxicity. This study suggested that the expression of LncRNA–ENST00000446135 was upregulated and regulated DNA damage and DNA repair in Cd toxicity. LncRNA–ENST00000446135 may serve as a novel valuable biomarker of cadmium exposure and cadmium toxicity and may become a significant biomarker for field investigations and risk assessment in humans exposed to occupational and environmental cadmium.

Author contributions

Z.Z., Z.H., Q.L., and B.C. conceived and designed the study. Z.Z. and W.C. wrote the manuscript. Z.H., Q.L., B.C., L.C. performed the experiments. B.C. performed the statistical analysis. Q.L. contributed reagents, materials, and analysis tools.

Funding

National Natural Science Foundation of China (81473001 to Z.Z.); Key project for universities of Guangdong Province (2019GZDXM017 to Z.Z.); Guangdong Medical Research Fund (A2019128 to Z.Z.); and Science and Technology Planning Project of Shenzhen (JCYJ20180306140801775 to Z.Z.).

Conflict of interest statement. None declared.