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Abstract

Circular RNAs (circRNAs) have been revealed to involve in the chemoresistance of various cancers, including non-small cell lung cancer (NSCLC). Here, we further investigate the role of circRNA_100565 in NSCLC cisplatin (DDP) resistance. The expression of circRNA_100565 and microRNA (miR)-337-3p, and ADAM metallopeptidase domain 28 (ADAM28) mRNA was detected using quantitative real-time polymerase chain reaction. Cell viability and apoptosis were measured by cell counting kit-8 assay and flow cytometry, respectively. Western blot was used to detect the level of ADAM28 and autophagy-related protein. The interaction between miR-337-3p and circRNA_100565 or ADAM28 was confirmed by dual-luciferase reporter assay or pull-down assay. In vivo experiments were conducted via the murine xenograft model. We found CircRNA_100565 was up-regulated in NSCLC DDP-resistant tissues and cell lines, and its high expression was associated with shorter overall survival of NSCLC patients. CircRNA_100565 deletion mitigated DDP resistance, reflected by the suppression of proliferation and autophagy, the reduction of IC50 value, as well as enhancement of apoptosis in DDP-resistant NSCLC cells. MiR-377-3p was confirmed to directly bind to circRNA_100565 or ADAM28 3’-UTR. Moreover, circRNA_100565 indirectly regulated ADAM28 expression by sponging miR-377-3p in NSCLC cells. Additionally, circRNA_100565 deletion-induced sensitivity of NSCLC resistant cells to DDP could be remarkably attenuated by miR-377-3p inhibition or ADAM28 re-expression. Meanwhile, circRNA_100565 knockdown contributed to the anti-tumor effects of DDP on NSCLC in vivo.

CONCLUSION:

CircRNA_100565 was an independent prognostic factor for NSCLC patient survival, and enhanced the resistance of NSCLC cells to cisplatin by regulating cell proliferation, apoptosis and autophagy via miR-337-3p/ADAM28 axis, shedding light on the development of a novel therapeutic strategy to boost the effectiveness of NSCLC chemotherapy.

Keywords

circRNA_100565 miR-337-3p ADAM28 NSCLC cisplatin resistance

1. Introduction

Lung cancer is counted as the leading cause of cancer-induced mortality worldwide. Non-small cell lung cancer (NSCLC) is one of the main sub-types of lung cancer, represents approximately 85% of all lung cancer cases, and has a high incidence and low five-year survival rate of approximately 17.1% [1, 2]. Currently, platinum-based chemotherapy is a well-recognized standard adjunctive treatment strategy in patients with advanced NSCLC, among these, cisplatin (DDP)-based chemotherapy is one of the most widely used therapeutic strategies in NSCLC [3]. Unfortunately, obvious resistance to DDP has been emerged in a large proportion of NSCLC patients receiving chemotherapy, and limits the clinical application of DDP in NSCLC treatment [4]. Thus, extensive efforts on developing low-toxic treatment strategies are necessary to manage the survival of NSCLC patients.

Circular RNAs (circRNAs) are a class of highly stable RNA molecules with the covalently closed-loop structures, which result in resistance to regular mechanisms of linear RNAs decay [5]. Currently, growing investigations have indicated that circRNAs act as critical functional modulators of physiological or pathological cellular processes implicated in cell cycle, growth, differentiation, apoptosis, and metastasis in tumors [6, 7]. Besides that, many circRNAs have also been exhibited to involve in the chemoresistance of various cancers by regulating cellular apoptosis and autophagy. For example, circRNA AKT3 inhibited gastric cancer apoptosis to promote DDP resistance by up-regulating PIK3R1 through miR-198 [8]. Overexpressed circ-Cdr1as sensitized ovarian cancer to DDP by mediating the DDP-induced cell apoptosis via the regulation of miR-1270/SCAI axis [9]. Circ_0035483 enhanced the resistance of renal cancer cells to gemcitabine by regulating autophagy through sponging miR-335 [10]. CircPAN3 facilitated doxorubicin resistance in acute myeloid leukemia by influencing autophagy and apoptosis [11]. In the previous research of our team, it was demonstrated that circ_100565 promoted NACLC cell proliferation, migration and invasion via increasing HMGA2 expression through absorbing miR-506-3p, indicating the oncogenic role of circ_100565 in NSCLC [12]. However, despite some advanced findings, the expression and possible carcinogenic involvement of circ_100565 in NSCLC chemoresistance remain largely unclear.

MiRNAs are small non-protein coding RNAs that modulate gene expression at post-transcriptional level [13]. MiRNAs have been reported to participate in many pathological processes of cancers by involving in cell inflammation, metabolism, metastasis, autophagy, apoptosis and drug resistance [14, 15, 16]. MiR-337-3p is a well-characterized tumor suppressor in many cancers, such as hepatocellular carcinoma, clear cell renal cell carcinoma, and cervical cancer, and have been found to inhibit tumor progression by regulating tumor cell proliferation, apoptosis, and metastasis [17, 18, 19]. Besides that, Du et al. revealed that miR-337-3p sensitized NSCLC cells to paclitaxel by regulating STAT3 and RAP1A expression, indicating the role of miR-337-3p in chemoresistance. ADAM metallopeptidase domain 28 (ADAM28) is a member of the ADAM family, which are found to implicate in a variety of biological events, such as cell-cell and cell-matrix interactions [20]. ADAM28 was found to be elevated in NSCLC, and was associated with cell proliferation, lymph node metastasis and tumor size [21]. However, the role of ADAM28 in chemoresistance of NSCLC remains unclear.

In this study, we attempted to investigate the expression pattern and effects of circRNA_100565 in DDP-resistant NSCLC, and evaluated the regulatory effects of circRNA_100565, miR-337-3p and ADAM28 in NSCLC with sDDP-resistance.

2. Materials and methods

2.1 Patients and specimens

Tumor tissues and matched normal tissues were obtained from 50 NSCLC patients who underwent surgical resection at The First Affiliated Hospital of Hainan Medical University and immediately stored in liquid nitrogen until used. All patients were diagnosed by histopathological examination and only received DDP-based neo-adjuvant chemotherapy before surgery. The patients were divided into two groups depending on the sensitivity to DDP: DDP-sensitive group ( $n=$ 22, tumor remission after 6 cycles of chemotherapy) and DDP-resistant group ( $n=$ 28, tumor stabilization or progression after 6 cycles of chemotherapy). This study was permitted by the Ethics Committee of The First Affiliated Hospital of Hainan Medical University and all subjects had signed the written informed consents.

2.2 Cell culture and transfection

NSCLC cell lines (A549 and H1299) were purchased from Shanghai Academy of Life Science (Shanghai, China). The stable DDP-resistant lines A549/DDP and H1299/DDP were established by exposing stepwise increasing concentrations of DDP (Sigma, St. Louis, MO, USA) over 6 months. Parental cells were grown in the Dulbecco’s modifed Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) harboring with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco) with 5% CO ${}_{2}$ at 37 ${}^{\circ}$ C, and DDP-resistant lines were cultured in the same media supplemented with DDP (18 $\mu$ M for A549/DDP cells and 16 $\mu$ M for H1299/DDP cells) to retain their drug-resistant phenotype.

The miR-337-3p mimic (miR-337-3p), miR-337-3p inhibitor (anti-miR-337-3p), and their corresponding negative control (miR-NC and anti-NC) were purchased from RIBOBIO (Guangzhou, China). The short hairpin RNA (shRNA) targeting circRNA_100565 covalent closed junction (sh-circRNA_100565), shRNA scramble control (sh-NC), empty vector (vector), pcDNA-ADAM28 overexpression vector (ADAM28) were synthesized by Genepharma (Shanghai, China). The transfection of oligonucleotides was performed using Lipofectamine ${}^{\text{TM}}$ 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA).

2.3 Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the standard procedure, and then was interacted with Rnase R (Epicentre, Madison, WI, USA), followed by incubation with RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA, USA). Subsequently, complementary DNA (cDNA) was synthesized using a High Capacity cDNA Reverse Transcription Kit (Qiagen), and then quantitative PCR was conducted using SYBR Green methods on the ABI7500 system. Glyceraldehyde 3-phosphate dehydrogenase (GADPH) and U6 small nuclear B noncoding RNA (U6) were used as internal references to normalize the relative transcription expression using 2 ${}^{-\text{Ct}}$ method. The specific primer sequences were presented as follows: circRNA_100565: F 5’-GCTCACACCAATCCCCTG-3’, R 5’-ACAGATTCGCTATGATCA-3’; miR-337-3p: F 5’-TGCTGCTCCTATATGATGCCTTTCT-3’, R 5’-CCTGCTCCTATATGATGCCTTTCTT-3’; p62: F 5’-CTCCCTAAAGAGGGCCACGA-3’, R 5’-AAAGAC GGACATCCACCTCG-3’; Beclin 1: F 5’-CCGAGGG ATGGAAGGGTCTA-3’, R 5’-GCTGTTGGCACTTT CTGTGG-3’; ADAM28: F 5’-GATGACACACTCATT CCCTG-3’, R 5’-CACACAGCATGTCCTTTGCA-3’; GADPH: F 5’-GATATTGTTGCCATCAATGAC-3’, R 5’-TTGATTTTGGAGGGATCTCG-3’; U6: F5’-CTCGCTTCGGCAGCACA-3’, R 5’-ACGCTTCAC GAATTTGCGT-3’.

2.4 Cell viability assay

The DDP-sensitivity of cells was analyzed by cell counting kit- 8 (CCK-8) assay. Transfected DDP-resistant cells (5000 per well) were seeded into 96-well plates overnight and then were incubated with different concentrations of DDP (1, 10, 20, 40, 80, 160 $\mu$ M) for 48 h. After that, 10 $\mu$ L CCK-8 solution was added into per well and interacted for 2 h. Subsequently, the absorbance at 450 nm was determined by a microplate reader in the indicated time. Besides that, the half maximal inhibitory concentration (IC50 value) of drugs was assessed according to the relative survival curve.

2.5 Cell apoptosis assay

Transfected DDP-resistant cells were incubated with DDP for 48 h in six-well plates. Subsequently, cells were resuspended with binding buffer, followed by staining with 10 $\mu$ L fluorescein isothiocyanate (FITC) annexin V and propidium iodide (PI) (BD Biosciences, San Jose, CA, USA). Finally, the proportion of apoptotic cells was analyzed by FlowJo software.

Figure 1.

CircRNA_100565 expression in DDP-resistant NSCLC tissues and cells and its correlation with overall survival. (A) The expression of circRNA_100565 was detected using qRT-PCR in NSCLC tumor tissues (DDP-sensitive group ( $n=$ 22) and DDP-resistant group ( $n=$ 28)) and paired normal tissues. (B) The expression of circRNA_100565 was detected using qRT-PCR in DDP-resistant NSCLC cell lines (A549/DDP and H1299/DDP) and corresponding parental NSCLC cell lines (A549 and H1299). (C) Kaplan-Meier OS curves for 50 patients with NSCLC classified according to relative circRNA_100565 expression level. (D, E) The abundances of circRNA_100565 and MLLT10 mRNA were analyzed using qRT-PCR in A549/DDP and H1299/DDP cells after treatment with actinomycin D for 0, 8, 16 and 24 h, respectively. (F) The expressions of circRNA_100565 and MLLT10 mRNA were evaluated in A549/DDP and H1299/DDP cells after RNase R treatment. (G, H) Comparison of the abundance of circRNA_100565 in the nuclear and cytoplasmic using qRT-PCR. * $P<$ 0.05.

2.6 Western blot

Proteins were extracted using RIPA lysis buffer (Beyotime, Beijing, China), and then were quantified by the bicinchoninic acid method according to the standard protocol. After that, the equal amount of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, shifted onto a polyvinylidene fluoride membrane, and blocked with 5% non-milk. Subsequently, the membrane was probed with primary antibodies against light chain 3B (LC3B) (1:3000, ab51520, Abcam, Cambridge, MA, USA), Beclin 1 (1:3000, ab62557, Abcam), p62 (1:10000, ab109012, Abcam), Ki-67 (1:5000, ab92742, Abcam), ADAM28 (1: 5000, ab28292, Abcam), and GAPDH (1:10000, ab181602, Abcam). After interaction with HRP-conjugated secondary antibody (1:1000, ab9482, Abcam), protein bands were detected using the chemiluminescence chromogenic substrate (Beyotime).

2.7 Dual-luciferase reporter assay

The circRNA_100565 and 3’ UTR of ADAM28 containing the wild-type (WT) or mutant (MUT) binding sites of miR-337-3p were cloned into the pmiR-RB-Report (Promega, Shanghai, China), respectively. Subsequently, cells were co-transfected with constructed luciferase reporter vectors and miR-337-3p or miR-NC using Lipofectamine ${}^{\text{TM}}$ 3000 (Invitrogen). Finally, the luciferase activity was determined using a dual luciferase assay kit (Promega).

2.8 Pull-down assay

MiR-337-3p and miR-NC were biotinylated to generate Bio-miR-337-3p and Bio-miR-NC by GenePharma Company (Shanghai, China), and then these biotinylated oligonucleotides were transfected into NSCLC-resistant cells for 48 h. After that, cells were collected and lysed, and the lysate was incubated with streptavidin-coated magnetic beads. After elution, the biotin-coupled RNA complex was pulled down and then analyzed by qRT-PCR.

2.9 Xenograft experiments in vivo

BALB/c athymic mice ( $N=$ 6, five weeks old) were used to establish xenograft models according to the guidelines permitted by the Animal Research Committee of The First Affiliated Hospital of Hainan Medical University. H1299/DDP cells stably transfected with lentivirus-(lenti)-shcircRNA_100565 or lenti-sh-NC were subcutaneously inoculated into the flanks of the nude mice, followed by intravenous injection with DDP (3 mg/kg) every 7 days after 7 days of administration. Subsequently, the tumor sizes were detected every week and the tumor volumes were calculated. After 35 days, all mice were killed and tumor masses were harvested for following weight and molecular analysis.

2.10 Statistical analysis

Data from at least three independent experiments were expressed as the mean $\pm$ standard deviation (SD) and analyzed by GraphPad Prism 7 software (GraphPad Inc., San Diego, CA, USA). The statistical difference between each group was analyzed by Student’s $t$ -test or one-way analysis of variance (ANOVA). The Kaplan-Meier method was employed to estimate probability of survival. The association between markers and survival outcomes was analyzed using the log rank test and Cox model. The correlation analysis was performed using Spearman’s correlation test. $P$ values $<$ 0.05 suggested statistically significant.

3. Results

3.1 CircRNA_100565 expression in DDP-resistant NSCLC tissues and cells and its correlation with overall survival

Table 1
Correlation between circ_100565 expression and the clinical pathological features of 50 NSCLC patients

Characteristic	All cases	circ_100565 expression		$P$ -value
		High( $n=$ 25)	Low ( $n=$ 25)
Gender				0.370
Male	33	18	15
Female	17	7	10
Age (years)				0.152
$<$ 60	21	8	13
$\geqslant$ 60	29	17	12
Smoking status				0.083
Smokers	30	18	12
No-smokers	20	7	13
Differentiation				0.248
Well/Moderate	30	17	13
Poor	20	8	12
Lymph node metastasis				0.018*
No	32	20	12
Yes	18	5	13
TNM Stages				0.024*
I/II	24	16	8
III/IV	26	9	17

* $P<$ 0.05.

The expression of circRNA_100565 in NSCLC tumor tissues which were divided into DDP-sensitive group ( $n=$ 22) and DDP-resistant group ( $n=$ 28), and paired normal tissue samples from 50 patients was first detected. The results showed that circRNA_100565 was significantly up-regulated in NSCLC tumor tissues, especially in DDP-resistant group compared with the normal tissue (Fig. 1A). Similarly, circRNA_100565 was also increased in the DDP-resistant NSCLC cell lines (A549/DDP and H1299/DDP) compared with the corresponding parental NSCLC cell lines (A549 and H1299) (Fig. 1B). After that, patients with NSCLC were grouped into two groups based on the median level of circRNA_100565, then we found the overall survival (OS) of patients in the high circRNA_100565 group was markedly shorter than that in the low circRNA_100565 group (Fig. 1C). Furthermore, higher circRNA_100565 expression was closely associated with lymph node metastasis and advanced TNM stages (Table 1). Meanwhile, the correlation between OS and pathological factors was investigated using a COX risk proportional regression model. Univariate analysis suggested that the difference in OS caused by lymph node metastasis, TNM stage or circRNA_100565 expression was statistically significant (Table 2); multivariate analysis showed that lymph node metastasis and circRNA_100565 expression both represented independent factors related to the OS of NSCLC patients involved (Table 2). CircRNA_100565 arose from its host gene MLLT10 and consisted of the head-to-tail splicing of exon 16 to 20. To confirm the stability of circRNA_100565, actinomycin D and RNase R exonuclease were adopted, results showed circRNA_100565 could resist actinomycin D and RNase R exonuclease degradation in A549/DDP and H1299/DDP cells, indicating that circRNA_100565 was indeed circular (Fig. 1D–F). Additionally, the location of circRNA_100565 was explored, qRT-PCR analysis showed circRNA_100565 was prominently distributed in the cytoplasmic fraction in A549/DDP and H1299/DDP cells (Fig 1G and H). These data indicated that abnormal expression of circRNA_100565 might be associated with DDP resistance in NSCLC, and its high expression was associated with shorter overall survival of NSCLC patients.

Table 2

Univariate analysis for factors related to overall survival using the COX proportional hazard model

Characteristics		Univariate analysis
		$P$	HR	95%CI
Gender	Male vs female	0.715	0.855	0.568–1.286
Age (years)	$<$ 60 vs $\geqslant$ 60	0.503	1.220	0.681–2.186
Smoking status	Smokers vs No-smokers	0.765	0.795	0.435–1.452
Differentiation	Well/Moderate vs Poor	0.423	1.330	0.689–2.568
Lymph node metastasis	No vs Yes	0.035*	1.939	1.156–3.251
TNM Stages	I/II vs III/IV	0.021*	2.209	1.453–3.357
circ_100565 expression	High vs low	0.011*	2.429	1.321–4.465

* $P<$ 0.05.

Figure 2.

CircRNA_100565 deletion mitigates DDP resistance by regulating resistant cell proliferation, apoptosis and autophagy in NSCLC. CircRNA_100565 was silenced in A549/DDP and H1299/DDP cells using sh-circRNA_100565. (A) The relative expression of circRNA_100565 was measured using qRT-PCR. (B, C) The CCK-8 assay was performed to detect cell proliferation. (D-F) The IC50 value for DDP was assessed by CCK-8 assay. (G) Cell apoptosis was analyzed using flow cytometric analysis. (H-J) The expression of autophagy-associated protein LC3II/I, Beclin-1 and p62 was measured using qRT-PCR and western blot. * $P<$ 0.05.

3.2 CircRNA_100565 deletion mitigates DDP resistance by regulating resistant cell proliferation, apoptosis and autophagy in NSCLC

To explore the biological effects of circRNA_100565 on DDP resistance of NSCLC, circRNA_100565 was silenced in DDP-resistant NSCLC cells using sh-circRNA_100565. As expected, the level of circRNA_100565 was greatly down-regulated in A549/ DDP and H1299/DDP cells (Fig. 2A). Subsequently, CCK-8 assay indicated that circRNA_100565 silence inhibited the proliferation of A549/DDP and H1299/DDP cells (Fig. 2B and C). Furthermore, we analyzed the IC50 of A549/DDP and H1299/DDP cells to DDP, which was more than 2-fold lower in circRNA_100565-decreased cells than that of sh-NC transfected-A549/DDP and H1299/DDP cells (35.8 vs. 15.2 in A549/DDP cells, 25.8 vs. 11.9 in H1299/DDP cells), suggesting that circRNA_100565 deletion led A549/DDP and H1299/DDP cells sensitive to DDP (Fig. 2D–F). Immediately, flow cytometry analysis showed the apoptotic A549/DDP and H1299/DDP cells, reflected by the lower and upper right quadrants, were increased by circRNA_100565 silencing (Fig. 2G). Besides that, we also found knockdown of circRNA_100565 down-regulated the ratio of LC3II/I and the expression of Beclin-1, while up-regulated p62 expression in A549/DDP and H1299/DDP cells, suggesting circRNA_100565 knockdown inhibited resistant cell autophagy in NSCLC (Fig. 2H–J). Taken together, knockdown of circRNA_100565 sensitized NSCLC resistant cell to DDP by inducing cell apoptosis, and inhibiting cell proliferation and autophagy.

Figure 3.

CircRNA_100565 is a sponge of miR-337-3p and negatively regulates its expression. (A) The putative binding site between circRNA_100565 and miR-337-3p was presented. (B-D) The interaction between circRNA_100565 and miR-337-3p was confirmed by the dual-luciferase reporter assay and pull-down assay in A549/DDP and H1299/DDP cells, respectively. (E) The expression of miR-337-3p in A549/DDP and H1299/DDP cells transfected with sh-NC or sh-circRNA_100565 was detected by qRT-PCR. (F, G) The expression of miR-337-3p in DDP-resistant NSCLC tissues and cell lines was measured by qRT-PCR. (H) The correlation between circRNA_100565 and miR-337-3p was analyzed using Spearman’s correlation test. * $P<$ 0.05.

3.3 CircRNA_100565 is a sponge of miR-337-3p and negatively regulates its expression

To detect the potential pathways in which circRNA_100565 involved in NSCLC DDP resistance, we used the Circinteractome database to search the potential miRNA targets of circRNA_100565, and miR-337-3p had putative binding sites of circRNA_ 100565 (Fig. 3A). Then the dual-luciferase reporter assay was performed and results showed overexpressed miR-337-3p reduced the luciferase activity of the circRNA_100565-WT reporter vector but not circRNA_100565-MUT reporter vector in A549/DDP and H1299/DDP cells (Fig. 3B and C). Moreover, RNA pull-down assay displayed that circRNA_100565 enrichment in Bio-miR-337-3p-probe group was markedly higher than the negative control group (Fig. 3D). These results indicated the direct interaction between circRNA_100565 and miR-337-3p. After that, we found miR-337-3p was increased by the knockdown of circRNA_100565 in A549/DDP and H1299/DDP cells (Fig. 3E). Additionally, miR-337-3p was down-regulated in DDP-resistant NSCLC tissues and cells compared with the controls (Fig. 3F and G), and also was negatively correlated with circRNA_100565 expression in tissues (Fig. 3H). In all, we confirmed that circRNA_100565 directly interacted with miR-337-3p and negatively regulated its expression in NSCLC cells.

Table 3
Multivariate analysis for factors related to overall survival using the COX proportional hazard model

Characteristics		Univariate analysis
		$P$	HR	95%CI
Lymph node metastasis	No vs Yes	0.046*	1.705	1.234–2.356
TNM Stages	I/II vs III/IV	0.204	0.631	0.278–1.432
circ_100565 expression	High vs low	0.029*	2.101	1.089–4.053

* $P<$ 0.05.

Figure 4.

Knockdown of circRNA_100565 sensitizes NSCLC resistant cells to DDP by sponging miR-337-3p. A549/DDP and H1299/DDP cells were transfected with sh-NC, sh-circRNA_100565, sh-circRNA_100565 $+$ anti-NC, or sh-circRNA_100565 $+$ anti-miR-337-3p. (A) qRT-PCR analysis of miR-337-3p expression was conducted. (B, C) Cell proliferation was analyzed using CCK-8 assay. (D) The IC50 value for DDP was assessed by CCK-8 assay. (E) Flow cytometric analysis was applied to detect cell apoptosis. (F-I) Western blot was used to examine the levels of autophagy-associated protein LC3II/I, Beclin-1 and p62. * $P<$ 0.05.

Figure 5.

ADAM28 is a target of miR-337-3p and circRNA_100565 regulates ADAM28 by sponging miR-337-3p. (A) The putative binding site between ADAM28 and miR-337-3p was listed. (B, C) The interaction between ADAM28 and miR-337-3p was confirmed by the dual-luciferase reporter assay in A549/DDP and H1299/DDP cells. (D) The expression of ADAM28 in NSCLC tissues was measured using qRT-PCR and western blot. (E, G) The correlation between miR-337-3p and circRNA_100565 or ADAM28 was analyzed using Spearman’s correlation test. (F) ADAM28 expression in A549/DDP and H1299/DDP cells transfected with miR-NC or miR-337-3p was detected using qRT-PCR. (H, I) ADAM28 protein expression was determined using western blot in A549/DDP and H1299/DDP cells transfected with sh-NC, sh-circRNA_100565, sh-circRNA_100565 $+$ anti-NC, or sh-circRNA_100565 $+$ anti-miR-337-3p. * $P<$ 0.05.

3.4 Knockdown of circRNA_100565 sensitizes NSCLC resistant cells to DDP by sponging miR-337-3p

Based on the relationship between circRNA_100565 and miR-337-3p, we further gained insight into whether miR-337-3p involved in circRNA_100565 deletion-induced inhibition of DDP resistance in NSCLC. A549/DDP and H1299/DDP cells were transfected with sh-NC, sh-circRNA_100565, sh-circRNA_100565 $+$ anti-NC, or sh-circRNA_100565 $+$ anti-miR-337-3p. Then qRT-PCR analysis showed the expression of miR-337-3p was significantly promoted by circRNA_100565 deletion, but was decreased by following miR-337-3p inhibition (Fig. 4A). After that, rescue assay was performed and results exhibited that the introduction of miR-337-3p inhibitor greatly attenuated circRNA_100565 deletion-induced suppression of cell proliferation (Fig. 4B and C), drug sensibility to DDP (Fig. 4D), and promotion of apoptosis (Fig. 4E) in A549/DDP and H1299/DDP cells. Furthermore, western blot showed that miR-337-3p inhibition reversed circRNA_100565 silencing-mediated decrease of LC3II/I ratio and Beclin-1 level, as well as the increase of p62 expression in A549/DDP and H1299/DDP cells (Fig. 4F–I). Therefore, we illustrated that circRNA_100565 deletion weakened DDP resistance by sponging miR-337-3p in NSCLC cells.

Figure 6.

Knockdown of circRNA_100565 promotes the sensitivity of resistant cells to DDP in NSCLC by regulating ADAM28 inhibition. A549/DDP and H1299/DDP cells were transfected with sh-NC, sh-circRNA_100565, sh-circRNA_100565 $+$ vector, or sh-circRNA_100565 $+$ ADAM28. (A) The expression of ADAM28 protein was evaluated by western blot. (B, C) Cell proliferation was examined using CCK-8 assay. (D) The IC50 value for DDP was calculated by CCK-8 assay. (E) Flow cytometric analysis was used to detect cell apoptosis. (F-H) Western blot was used to examine autophagy-associated protein LC3II/I, Beclin-1 and p62. * $P<$ 0.05.

3.5 ADAM28 is a target of miR-337-3p and circRNA_100565 regulates ADAM28 by sponging miR-337-3p

The target genes of miR-337-3p were further explored by using Targetscan online database, results showed that ADAM28 had putative binding sites in miR-337-3p (Fig. 5A). Then the declined luciferase activity in A549/DDP and H1299/DDP cells co-transfected with ADAM28-WT and miR-337-3p confirmed the interaction between miR-337-3p and ADAM28 (Fig. 5B and C). ADAM28 expression was found to be elevated in DDP-resistant NSCLC tissues (Fig. 5D), and was negatively correlated with miR-337-3p expression (Fig. 5E); importantly, ADAM28 expression in A549/DDP and H1299/DDP cells was reduced by miR-337-3p mimics (Fig. 5F). All these results suggested that miR-337-3p targeted ADAM28 and negatively regulated its expression in NSCLC cells. In addition, we also observed a positive correlation between ADAM28 and circRNA_100565 expression in NSCLC (Fig. 5G); synchronously, it was found that ADAM28 expression was inhibited by circRNA_100565 deletion, but was up-regulated by following miR-337-3p inhibition (Fig. 5H and I). Altogether, we confirmed that circRNA_100565 could indirectly regulate ADAM28 expression by serving as a sponge of miR-337-3p in NSCLC cells.

Figure 7.

CircRNA_100565 deletion contributes to DDP-mediated repression on NSCLC tumor growth in vivo. (A) Tumor volumes were calculated every 7 days. (B) Mice were killed on day 35 days, and then tumor masses were excised and weighed. (C, D) The expression of circRNA_100565 and miR-337-3p were determined using qRT-PCR. (E) Western blot was used to detect the level of ADAM28 and autophagy-associated protein. (F) The expression levels of Ki-67, ADAM28 and LC3-II in tumors were analyzed using ICH. * $P<$ 0.05.

3.6 Knockdown of circRNA_100565 promotes the sensitivity of resistant cells to DDP in NSCLC by regulating ADAM28 inhibition

Due to the regulatory network of circRNA_100565/ miR-337-3p/ADAM28, we wanted to know whether ADAM28 participated in the regulation of circRNA_ 100565 deletion on DDP resistance in NSCLC cells. Firstly, A549/DDP and H1299/DDP cells were transfected with sh-NC, sh-circRNA_100565, sh-circRNA_ 100565 $+$ vector, or sh-circRNA_100565 $+$ ADAM28. After transfection, we found ADAM28 protein expression was reduced by circRNA_100565 deletion, which was rescued by ADAM28 re-expression (Fig. 6A), suggesting the successful transfection. After that, rescue assay was performed and results indicated ADAM28 overexpression partially reversed circRNA_100565 knockdown-mediated suppression on proliferation (Fig. 6B and C), drug sensibility to DDP (Fig. 6D), promotion on apoptosis (Fig. 6E), as well as repression on autophagy (Fig. 6F–H) of A549/DDP and H1299/DDP cells. Altogether, knockdown of circRNA_100565 enhanced the sensitivity of resistant cells to DDP in NSCLC by regulating ADAM28 inhibition.

3.7 CircRNA_100565 deletion contributes to DDP-mediated repression on NSCLC tumor growth in vivo

We further investigated the effects of circRNA_ 100565 on DDP-mediated tumor growth in vivo. As shown in Fig. 7A and B, circRNA_100565 silencing reinforced DDP-induced repression on the tumor growth, reflected by the decreased tumor volume and lowered tumor weight in sh-circRNA_100565-transfected H1299/DDP cells group. Moreover, molecular analyses displayed circRNA_100565 deletion reduced the level of circRNA_100565 and ADAM28, but promoted the level of miR-337-3p in excised tumor masses from sh-circRNA_100565-transfected group (Fig. 7C–E). Meanwhile, western blot also showed circRNA_100565 deletion decreased the LC3II/I ratio and Beclin-1 level, while increased p62 expression in tumor masses from the sh-circRNA_100565-transfected group (Fig. 7E). Besides that, the ICH assay indicated circRNA_100565 knockdown decreased the level of Ki-67, ADAM28 and LC3-II in tumors (Fig. 7F). Collectively, all these results implicated circRNA_100565 deletion reinforced DDP-mediated repression on NSCLC tumor growth in vivo through suppressing autophagy by regulating ADAM28 and miR-337-3p.

4. Discussion

Although recent chemotherapeutics have markedly improved the outcome of patients with advanced NSCLC, invariably, almost all NSCLC patients become chemoresistance accompanied by distant metastasis in the end recurrence [4, 22]. Thus, clarifications of the mechanism of chemoresistance in NSCLC and identifications of new therapeutic markers might provide novel strategies to improve the prognosis of NSCLC. In this study, we found circRNA_100565 was up-regulated in NSCLC tissues, especially in DDP-resistant tissues, its high expression was associated with shorter overall survival in patients; what’s more, univariate and multivariate analysis revealed that circRNA_100565 expression represented a significant independent prognostic marker in NSCLC. Furthermore, circRNA_100565 was also elevated in DDP-resistant NSCLC cell lines, then knockdown of circRNA_100565 mitigated DDP resistance in NSCLC by inducing apoptosis and inhibiting proliferation and autophagy in cells. Besides that, the murine xenograft model analysis also showed that circRNA_100565 knockdown contributed to the anti-tumor effects of DDP on NSCLC in vivo.

At present, stimulation of cell death and suppression of cell survival are the key principles of cancer therapy; most anticancer therapies, such as chemo-, radio- and immunotherapy primarily function by inducing cell death including apoptosis and autophagy in cancer cells [23, 24]. Currently, growing evidence has indicated that circRNAs are involved in cell viability, apoptosis and autophagy in NSCLC. For example, circ-HIPK3 induced cell proliferation and repressed cell apoptosis in NSCLC by interacting with miR-149 to promote tumor progression [25]. Circ-HIPK3 facilitated NSCLC development by impairing cell autophagy and induced cell proliferation and metastasis through miR-124-3p-STAT3-PRKAA/AMPK $\alpha$ signaling in NSCLC [26]. Besides that, some circRNAs have also been identified to regulate chemotherapy resistance. For example, circRNA hsa_circ_0004015 enhanced the resistance of NSCLC cells to gefitinib by regulating miR-1183/PDPK1 axis [27]. Circ_0076305 promoted DDP resistance of NSCLC via positively regulating STAT3 by binding to miR-296-5p [28]. Therefore, it is imperative to disclose the role of circRNA_100565 in DDP resistance of NSCLC cells.

It has been reported that circRNAs function as miRNA sponges to regulate gene expression [29]. Here, we further investigated the underlying mechanism of circRNA_100565 on the DDP-resistance of NSCLC. We found miR-377-3p directly bound to circRNA_100565 or ADAM28 3’-UTR. Moreover, circRNA_100565 could indirectly regulate ADAM28 expression by sponging miR-377-3p in NSCLC cells. Additionally, circRNA_100565 deletion-induced sensitivity of NSCLC resistant cells to DDP could be remarkably attenuated by miR-377-3p inhibition or ADAM28 re-expression.

Previous studies have clarified that miR-337-3p was a crucial biomarker in many cancers progression. For example, miR-337-3p inhibition inhibited CoCl ${}_{2}$ -mediated apoptosis and cytotoxicity, and ameliorated oxidative stress and MMP by activating JAK2/STAT3 pathway in PC12 cells [30]. MiR-337-3p acted as a target of lncRNA CRNDE to involve in CRNDE/miR-337-3p/SIX1 axis-mediated regulation on hepatocellular carcinoma cell proliferation and migration [31]. ADAM28 has been revealed to be elevated in human malignant tumors and plays important effects on tumor cell proliferation and progression, including NSCLC [32, 33]. Besides that, Yan et al. revealed ADAM28 might act as a serum biomarker to monitor therapeutic efficacy of treatment in NSCLC [21]. In this study, we found miR-337-3p down-regulation or ADAM28 overexpression increased the ratio of LC3II/I and the expression of Beclin-1, while decreased p62 expression in circRNA_100565-down-regulated A549/DDP and H1299/DDP cells, thus promoting cell autophagy. Therefore, the identification of the circRNA_100565/miR-337-3p/ADAM28 is of great clinical significance in DDP resistance in NSCLC.

In conclusion, these findings demonstrated that circRNA_100565 might serve as a significant independent prognostic marker in NSCLC; besides, circRNA_100565 sensitized NSCLC cells to DDP by regulating cell proliferation, autophagy and apoptosis via miR-337-3p/ADAM28 axis, providing a novel insight into the development of the therapeutic strategy to boost the effectiveness of NSCLC chemotherapy.

Footnotes

Conflict of interest

The authors declare that they have no competing interests.

Funding

This study was supported by Natural Science Foundation of Hainan Province (Grant No. 818MS147).

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CircRNA_100565 contributes to cisplatin resistance of NSCLC cells by regulating proliferation,apoptosis and autophagy via miR-337-3p/ADAM28 axis

Abstract

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Patients and specimens

2.2 Cell culture and transfection

2.3 Quantitative real-time polymerase chain reaction (qRT-PCR)

2.4 Cell viability assay

2.5 Cell apoptosis assay

2.7 Dual-luciferase reporter assay

2.8 Pull-down assay

2.9 Xenograft experiments in vivo

2.10 Statistical analysis

3. Results

3.1 CircRNA_100565 expression in DDP-resistant NSCLC tissues and cells and its correlation with overall survival

Table 1 Correlation between circ_100565 expression and the clinical pathological features of 50 NSCLC patients

Table 3 Multivariate analysis for factors related to overall survival using the COX proportional hazard model

3.7 CircRNA_100565 deletion contributes to DDP-mediated repression on NSCLC tumor growth in vivo

4. Discussion

Footnotes

Conflict of interest

Funding

References

Table 1
Correlation between circ_100565 expression and the clinical pathological features of 50 NSCLC patients

Table 3
Multivariate analysis for factors related to overall survival using the COX proportional hazard model