Background
Lung cancer is the leading cause of cancer death worldwide, and small cell lung cancer (SCLC) accounts for approximately 15 to 20% of all lung cancers [
1]. The standard chemotherapy regimen for SCLC uses topoisomerase inhibitors in combination with cisplatin. SCLC is characterized by the rapid development of resistance to drugs even when there is an initial response [
2]. Acquired chemo-resistance is considered the major drawback of current chemotherapeutic regimens, but the molecular details have not been completely elucidated. Hence, there is an urgent need to identify the underlying mechanisms of chemo-resistance and to explore effective strategies to overcome resistance.
DNA-damaging agents are the most widely used chemotherapeutic drugs [
3]. DNA-damaging agents, such as doxorubicin (Adriamycin, Dox), prevent cell division and lead to cell death by inhibiting the religation of DNA strands in double-strand breaks (DSBs) [
4]. However, cancer cells may acquire chemo-resistance by altering the cell survival signaling pathway and repairing the DNA damage [
5]. The DNA damage response (DDR) is a molecular mechanism that cancer cells have exploited to activate DNA repair pathways and prevent DNA damage-induced cell death [
6].
Among these DNA repair pathways, homologous recombination (HR) is one of the key pathways for the repair of DSBs [
7]. A variety of DDR proteins are involved in the regulation of HR, resulting in cancer drug resistance [
8]. The expression of DNA repair proteins has recently been reported to be regulated by miRNAs (microRNAs). MiRNAs are small, 20–23-nucleotide noncoding RNAs that are known typically to suppress gene expression by binding to complementary sequences in the 3′-untranslated regions (3′ UTR) of target genes [
9]. Recently, the association between miRNA expression and chemo-resistance in cancer was noted [
10]. Some studies demonstrated that changes in the expression levels of miRNAs may be involved in tumor cell resistance to chemotherapy by regulating the efficacy of DNA damage repair processes [
11,
12]. Nevertheless, the molecular mechanisms underlying the role of miRNAs in chemo-resistance are not yet fully understood in SCLC.
In this study, we conducted an array-based analysis of microRNA expression patterns by comparing Dox-resistant H69AR cells and their parental cells. Among 62 differentially expressed miRNAs, miRNA-7-5p (microR-7-5p) exhibited a remarkably decreased level in H69AR cells. MiR-7-5p has previously been reported to be a tumor suppressor in multiple cancer types and inhibits growth and invasion [
13,
14]. However, to date, no available studies have conclusively demonstrated the association between miR-7-5p and DNA repair in SCLC chemo-resistance. Here, we revealed that miR-7-5p-facilitated HR repair contributes to chemo-resistance in SCLC cells by targeting poly ADP-ribose polymerase 1 (PARP1). Our findings provide an understanding of the function of miR-7-5p in the resistance of SCLC to chemotherapy.
Methods
Cell lines and cell culture
A human SCLC cell line (H69) and a Dox-resistant cell line (H69AR) were purchased from the American Type Culture Collection (Manassas, USA). The H446AR cell line is a Dox-resistant SCLC cell line that was derived from H446 in our lab. These cell lines were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Sigma-Aldrich, USA). The H69AR and H446AR cell lines were challenged monthly to maintain resistance to doxorubicin (MedChemExpress, USA). The Dox-resistant cells were maintained in drug-free medium for at least 2 weeks before any experiments.
MiRNA microarray analysis
Total RNA containing miRNA was extracted from H69 and H69AR cells with Trizol reagent (Invitrogen, USA) and purified with the mirVana miRNA Isolation Kit (Ambion, USA) according to the manufacturer’s protocol. MicroRNA profiling was performed using an Agilent miRNA array. Briefly, miRNAs were labeled using the Agilent miRNA labeling reagent. Total RNA (200 ng) was dephosphorylated and ligated with pCp-Cy3, and the labeled RNA was purified and hybridized to miRNA arrays. Images were scanned with the Agilent microarray scanner, gridded, and analyzed using Agilent Feature Extraction Software version 10.10.
MiRNA transfection
The miR-7-5p mimic and inhibitor were synthesized by Sangon Biotech (Shanghai, China). The sequences of the synthetic oligonucleotides were as follows: miR-7-5p mimic (miR-mimic) sense 5′-UGGAAGACUAGUGAUUUUGUUGUU-3′, antisense 5′-CAACAAAAUCACUAGUCUUCCAUU-3′; miR-7-5p mimic NC (NC-mimic) sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAATT-3′; miR-7-5p inhibitor (miR-inhibitor) 5′-AACAACAAAAUCACUAGUCUUCCA-3′; miR-7-5p inhibitor NC (NC-inhibitor) 5′-CAGUACUUUUGUGUAGUACAA-3′. These synthetic oligonucleotides were transiently transfected into SCLC cells by Lipofectamine 3000 (Thermo Fisher Scientific, USA) and Opti-MEM (Invitrogen, USA) according to the manufacturer’s protocol.
MiRNA isolation and quantitative real-time PCR
Total RNA was extracted from cells and reverse-transcribed into cDNA using the miRNA First Strand cDNA Synthesis Kit (Sangon Biotech, China) according to the manufacturer’s protocol. Quantitative real-time PCR (qRT-PCR) was performed using a SYBR® Prime Script™ RT-PCR Kit (Invitrogen, USA). The qRT-PCR primers were synthesized by Sangon Biotech. The forward primer for miR-7-5p was (5′-GCGCTGGAAGACTAGTGATTTTGTTGTT-3′) and the reverse primer for U6 was (5′-CTCGCTTCGGCAGCACA-3′). The universal reverse primer was (5′-AACGCTTCACGAATTTGCGT-3′). The relative expression of miRNA was calculated by normalization against that of the U6 small nuclear RNA.
Western blot analysis
The total protein from SCLC cells was extracted using RIPA lysis buffer. The protein lysates were separated by 10% SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore, USA). The membranes were incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies. The signals were detected using an ECL system (Thermo Fisher Scientific, USA). The intensity of the protein fragments was quantified with Image Lab software (Version 5.2.1 build 11, Bio-Rad). Anti-PARP1 antibody (Cell Signaling Technology, USA), anti-BRCA1 antibody (Santa Cruz, USA), anti-Rad51 antibody (Abcam, USA), anti-GAPDH and anti-β-tubulin (Cell Signaling Technology, USA) were used as the primary antibodies for the detection of specific proteins.
Cell counting kit-8 (CCK-8) assay
SCLC cells were plated in 96-well plates at 5 × 103 cells per well and treated with doxorubicin for 24 h. The absorbance at 450 nm was measured after incubation with 10 μl CCK-8 reagent (Dojindo, Japan) for 4 h. The obtained values were used to calculate the IC50 using untreated cells as a control. The assay was conducted in three replicate wells for each sample, and three parallel experiments were performed.
Dual-luciferase reporter assay
To determine the target sites of miR-7-5p on the 3′ UTR of PARP1 mRNA, HEK293 cells were seeded in 24-well plates and cotransfected with wild-type or mutated (one point mutation) PARP1 3′ UTR reporter plasmids for 24 h using Lipofectamine 3000. Then, the cells were transfected with miR-7-5p mimic or inhibitor for another 24 h. The cells were harvested for luciferase activity assays that were performed using the Dual-Luciferase Reporter Assay System (Promega, Germany).
HR reporter assay
The HR repair assay was carried out using the DR-GFP reporter system developed by Maria Jasin. Cells were seeded in 24-well plates and cotransfected with pDRGFP (Addgene plasmid 26,475) and pCBASceI (Addgene plasmid 26,477) by Lipofectamine 3000. The percentage of GFP-positive cells determined by flow cytometry provided a quantitative measure of HR efficiency.
Immunofluorescent staining
H446R cells were transfected with miR-7-5p mimic or control miRNA for 24 h, and then cells were treated with 25 μg/ml doxorubicin for another 24 h. The cells were fixed and stained with mouse anti-BRCA1 (1:100 dilution; Santa Cruz, USA) and rabbit anti-Rad51 (1:66.67 dilution; Santa Cruz, USA). Goat anti-mouse IgG-Alexa Fluor® 594 or chicken anti-rabbit IgG-Alexa Fluor® 488 (1:400 dilution; Invitrogen, USA) was used as the secondary antibody. Photos were taken with a fluorescence microscope (Life Technologies, USA) at a magnification of 200 × .
Cells were seeded into 60 mm dishes at 200 cells per dish and treated with ABT-888, Dox or a combination of these for 24 h, and then medium was replaced with fresh medium. The cells were stained using Giemsa stain after 14 d of incubation, and colonies containing > 50 cells were counted. The mean value ± SD for three independent experiments was determined.
Statistical analysis
All of these analyses were carried out using GraphPad Prism from GraphPad Software (San Diego, USA). The data are presented as the mean ± SD from at least 3 separate experiments. Unless otherwise noted, the differences between the groups were analyzed using Student’s two-tailed t test when only 2 groups were compared or were assessed by one-way analysis of variance (ANOVA) when more than 2 groups were compared. P < 0.05 was considered statistically significant.
Discussion
Advanced SCLC is an aggressive disease associated with major morbidity and mortality [
22]. Chemo-resistance is a major obstacle for SCLC treatment. Therefore, there is a great need to clarify the mechanisms of chemo-resistance underlying the clinical behavior of SCLC. Recently, an increasing number of studies has demonstrated that the ectopic expression of miRNAs may be involved in the acquisition of tumor cell resistance to chemotherapy by altering the efficacy of DNA damage repair [
11,
12]. Let-7 was reported to regulate BRCA1 and Rad51 expression and subsequently enhance DNA repair, which results in cisplatin resistance [
23]. However, the molecular mechanisms of miRNA involved in DNA damage repair and chemo-resistance in SCLC cells are still unclear. In this study, we provided data indicating the importance of miRNA expression in the acquisition of SCLC cell resistance to doxorubicin. This was evidenced by the pronounced alteration in the expression of miRNA genes in Dox-resistant cells compared with parental cells. MiRNA profiling revealed that miR-7-5p was dramatically downregulated in Dox-resistant SCLC cells, which was validated by qRT-PCR. MiR-7-5p has been reported to be a tumor suppressor due to its ability to suppress cell growth and induce cell apoptosis [
13,
24‐
26]. Liu and colleagues demonstrated that miR-7-5p mediated SCLC chemo-resistance by repressing the expression of ABCC1 gene, which is a typical ABC transporter [
27]. Guo and colleagues reported that the expression of inwardly rectifying potassium channel Kir2.1 (KCNJ2) was regulated by miR-7-5p that modulated multiple drug resistance in SCLC [
28]. However, the functional role of miR-7-5p in regulating DNA repair during chemo-resistance has not yet been elucidated. Our results demonstrated that miR-7-5p was involved in the chemo-resistance of SCLC cells against doxorubicin via DNA repair pathway.
To explore the potential mechanisms underlying the involvement of miR-7-5p in Dox-resistance in SCLC cells, we utilized bioinformatics analysis and dual-luciferase reporter system to predict the genetic targets of miR-7-5p. The results showed that miR-7-5p directly targeted PARP1 and suppressed the expression of PARP1 in Dox-resistant SCLC cells. PARP1 is a nuclear enzyme that plays a critical role in many biological processes, including DNA repair and gene transcription [
29,
30]. PARP1 is activated by DNA damage and catalyzes the polymerization of ADP-ribose units, which results in the rapid recruitment of DNA repair proteins to sites of DNA damage [
31]. There are two major pathways for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). PARP1 is the key determinant of the HR repair pathway, and HR repair is crucial for maintaining genomic integrity and survival in response to chemotherapy [
32]. Our results further showed that HR repair was increased in Dox-resistant SCLC cell lines compared to that in parental cells. Considering the role of PARP1 in HR repair, we can speculate that the downregulation of miR-7-5p promotes PARP1 expression, which in turn enhances HR repair and causes chemo-resistance.
BRCA1 and Rad51 are key factors in HR repair. Previous studies have demonstrated that PARP1 is important for BRCA1 recruitment to DSBs and that PARP1 depletion results in the loss of BRCA1 foci formation [
33,
34]. PARP1 modulates the initial steps of DSB repair that involve the binding of Rad51 to DNA to stimulate strand exchange during HR. It has been reported that PARP1 chemical inhibitors or siRNAs targeted to PARP1 can inhibit HR by suppressing the expression of BRCA1 and Rad51 [
35]. Consistent with this observation, our results demonstrated that miR-7-5p inhibited the expression of BRCA1 and Rad51 that was induced by doxorubicin in Dox-resistant SCLC cells by suppressing PARP1 expression (Fig.
4f). Our findings suggested that PARP1-mediated HR activity promoted cell survival in doxorubicin-resistant SCLC cells and that miR-7-5p could be used as a sensitizer to overcome chemo-resistance.
Conclusion
The current study shows for the first time the correlation between miR-7-5p expression and HR repair in SCLC cells Dox resistance. Further investigation showed that PARP1-mediated HR activity was impaired by miR-7-5p. Considering the indispensable function of PARP1 during HR repair, our findings suggest that miR-7-5p may serve as a potential biomarker of chemo-resistance and may be used as a new therapeutic approach to overcome chemo-resistance.
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