Knockdown of LINC00511 decreased cisplatin resistance in non-small cell lung cancer by elevating miR-625 level to suppress the expression of leucine rich repeat containing eight volume-regulated anion channel subunit E

Abstract

Background

LINC00511 has been reported as a biomarker related to the prognosis of non-small cell lung cancer (NSCLC), but the molecular mechanism and exact functions of LINC00511 in chemoresistance of NSCLC remain to be elucidated.

Methods

RT-qPCR was used to evaluate the mRNA expression of LINC00511, miR-625, and leucine rich repeat containing 8 volume-regulated anion channel subunit E (LRRC8E). Western blotting detected the protein levels of Ki-67, MMP-9, cleaved-caspase-3. The interaction between miR-625 and LINC00511 or LRRC8E was verified by luciferase reporter assays. CCK-8, TUNEL, and Transwell assays were used to evaluate IC₅₀ value, proliferation, migration, and invasion of NSCLC cells.

Results

In our study, it was discovered that the levels of LINC00511 and LRRC8E were increased, while miR-625 expression was decreased in NSCLC tissues, DDP-resistant NSCLC cells, and non-resistant NSCLC cells. LINC00511 depletion significantly curbed cell growth, IC₅₀ value, and metastasis in DDP-resistant NSCLC cells. In addition, the influence of LINC00511 deficiency on the DDP resistance in NSCLC was overturned by suppressing miR-625. Furthermore, LRRC8E overexpression abolished the promotive effect of miR-625 abundance on the DDP sensitivity in DDP-resistant NSCLC cells.

Conclusion

Our results demonstrated that LINC00511 increased DDP resistance in NSCLC by suppressing miR-625 to upregulate LRRC8E.

Keywords

Non-small cell lung carcinoma lncRNA ceRNA chemoresistance

Introduction

Lung cancer continues to be the leading cause of cancer-related mortality worldwide.¹ Non-small cell lung carcinoma (NSCLC) currently accounts for approximately 85% of all lung cancer diagnoses.² Despite great progress in treatments including surgical resection, chemotherapy, targeted therapy, and radiotherapy, the prognosis of NSCLC remains unfavorable.³ Cisplatin (DDP)-based chemotherapy is widely used for NSCLC treatments, but the inevitable development of resistance to DDP limited the efficacy of clinical treatments.⁴ So far, the biological complexity of DDP resistance in NSCLC is still unknown. Therefore, it is important to explore novel strategies to enhance NSCLC sensitivity to DDP.

Long non-coding RNAs (lncRNAs) belong to a class of RNAs that are longer than 200 nucleotides in length without protein-coding ability.⁵ Accumulating studies reported that dysregulated lncRNAs participated in the cellular activities of human cancers, including NSCLC. For example, lncRNA TRERNA1 promoted the malignancy of NSCLC by modulating FOXL1.⁶ LncRNA FENDRR inhibited the development of NSCLC through miR-761/TIMP2 pathway.⁷ LncRNA XIST accelerated NSCLC progression by triggering the miR-335/SOD2/ROS signaling.⁸ More importantly, the regulatory functions of lncRNAs in DDP-resistant NSCLC have been identified in multiple studies. Xu et al.⁹ revealed that DDP resistance of NSCLC was regulated by lncRNA SNHG14 via miR-133a/HOXB13 axis. Wang et al.¹⁰ described that lncRNA SNHG9 promoted the DDP resistance in NSCLC and led to poor prognosis. Ju et al.¹¹ demonstrated that lncRNA BLACAT1 regulated cyclin D1 expression to promote DDP resistance of NSCLC. In 2016, Sun et al.¹² reported that LINC00511 acted as an oncogene in NSCLC by promoting NSCLC cell viability and mobility. Besides, LINC00511 was reported to be associated with the chemoresistance of various human cancers, such as cervical cancer, glioblastoma, and thyroid carcinoma.^13–15 Hence, we hypothesized that LINC00511 might regulate the DDP resistance in NSCLC.

Herein, the levels and biological role of LINC00511, miR-625, and LRRC8E in NSCLC tissues and DDP-resistant cells were explored. In addition, the relationships between miR-625 and LINC00511 or LRRC8E were also studied in the current research.

Materials and methods

Clinical samples

A total of 40 pairs of NSCLC tissues and adjacent non-tumor tissues were obtained from patients at Harbin Medical University Cancer Hospital. The clinical features of NSCLC patients are displayed in Table 1. All participants involved signed the written informed consents, and the research was approved by the Ethics Committee of Harbin Medical University Cancer Hospital.

Table 1.

The correlation between LINC00511 and clinicopathological features of patients with NSCLC.

Parameters		Case	LINC00511		P value^a
			High = 20	Low = 20
Age
	<60 years	21	10	11	0.211
	≥60 years	19	10	9	0.211
Gender
	Male	25	12	13	0.493
	Female	15	8	7	0.493
Tumor size
	<3 cm	25	11	14	0.147
	≥3 cm	15	9	6	0.147
Histologic type
	SCC	22	10	12	0.409
	AD	18	10	8	0.409
TNM stage
	I-II	23	8	15	0.035^b
	III-IV	17	12	5	0.035^b
Lymph node metastasis
	Yes	16	13	3	0.011^b
	No	24	7	17	0.011^b
Distant metastasis
	Yes	17	13	4	0.038^b
	No	23	7	16	0.038^b

^aChi-square test.

^bp < 0.05.

Cell culture

NSCLC cell lines (A549 and H522), DDP-resistant NSCLC cells (A549/DDP, H522/DDP), and normal human lung bronchial epithelial cells (BEAS-2B) were acquired from BeNa Culture Collection (Beijing, China). All cells were cultured with RPMI-1640 medium (Gibco) supplemented with 10% FBS (Gibco), 100 U/mL penicillin (Gibco) and 100 mg/mL streptomycin (Gibco) with 5% CO₂ at 37°C. In addition, the DDP-resistant cells were cultured with 1 μg/mL DDP (Sigma-Aldrich) to maintain the drug resistance phenotype.

Cell transfection

Small interfering RNA (siRNA) targeting LINC00511 (si-LINC00511) and its control (si-NC) were purchased from KeyGEN Biotech (Jiangsu, China). MiR-625 mimics/inhibitor, NC mimics/inhibitor, and LINC00511 and LRRC8E overexpression plasmid (pcDNA3.1/LINC00511 and pcDNA3.1/LRRC8E) were purchased from GenePharma (Shanghai, China). Cell transfection was performed using Lipofectamine 2000 (Invitrogen).

RT-qPCR

Total RNA was extracted using Trizol reagent (Invitrogen) and cDNA was synthesized using the PrimeScript RT reagent kit (Takara). Next, the reagent of the SYBR Premix Ex Taq™ II kit (Takara) was used to implement qPCR on an ABI 7500 Real-Time PCR System (Applied Biosystems). Relative levels of genes were normalized to GAPDH for LINC00511 and LRRC8E or U6 for miR-625.

Western blotting

Total proteins were extracted from the transfected cells using a RIPA buffer (Beyotime), separated by SDS-PAGE membrane (Solarbio), and transferred onto PVDF membranes (Pall Corporation). Subsequently, the membranes were incubated with primary antibodies against Ki-67, MMP-9, cleaved-caspase-3, or GAPDH, and then incubated with secondary antibodies (Sangon) for 2 h. Protein signals were visualized via ECL method.

CCK-8

To evaluate the cell viability, DDP-resistant NSCLC cells (1x10⁴ cells/well) were cultured in 96-well plates for 24 h. After that, the cells were cultured with 10 μl CCK-8 solution for another 2 h. To measure the IC₅₀ values, cells were treated with increasing concentrations of DDP for 48 h. The absorbance was measured using a microplate reader at 490 nm.

TUNEL

One-Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology) was utilized to analyze cell apoptosis. Briefly, the treated cells were then cultured with the TUNEL reaction mixture for 1 h. Then, the TUNEL-stained cells were washed with PBS and counterstained with DAPI. The apoptotic cells were evaluated using a fluorescence microscope.

Transwell

To detect cell invasive ability, Matrigel pre-coated transwell chambers with filters (8 μm pore size, EMD Millipore) were employed, followed by 1×10⁴ transfected cells were plated in the upper chamber filled with serum-free medium. The lower chamber was supplemented with medium containing 10% FBS. After 24 h, the cells invaded to the lower surface were stained by crystal violet after fixation and imaged with a microscope. The migration assay for measurement of migrated cells was conducted as the same except no Matrigel was coated.

Luciferase reporter assay

The mutant (Mut) and wild-type (WT) sequences of LINC00511 and LRRC8E were sub-cloned into pmirGLO vector (Promega). Subsequently, the above vectors were co-transfected with miR-625 mimic or NC mimic into NSCLC cells. The luciferase activities were assessed with a Dual-Luciferase Reporter Assay System (Promega).

Statistical analysis

The data were statistically analyzed by GraphPad Prim 6.0 Software and presented as mean ± SD. The statistical difference was analyzed by Student’s t-test, one-way ANOVA or chi-square test. p < 0.05 was regarded to be statistically significant.

Results

LINC00511 was upregulated in non-small cell lung carcinoma tissues and DDP-resistant cell lines

To investigate the biological role of LINC00511 in the progression of NSCLC, the expression of LINC00511 in NSCLC was explored, and our results indicated that LINC00511 was increased in NSCLC tissues (Figure 1(a)). Moreover, in comparison with NSCLC with early stages (I+II), the level of LINC00511 was remarkably augmented in advanced NSCLC tissues (III+IV) (Figure 1(b)). In the meantime, LINC00511 expression was significantly correlated with TNM stages, lymph node metastasis, and distant metastasis (Table 1). Next, CCK-8 assay indicated that the IC₅₀ values were enhanced in DDP-resistant NSCLC cells compared with that in A549 and H522 cells (Figures 1(c) and (d)). In addition, LINC00511 expression was significantly upregulated in both DDP-resistant and non-resistant NSCLC cells compared to that in BEAS-2B cells (Figure 1(e)).

Figure 1.

Elevated expression of LINC00511 in NSCLC. (a)RT-qPCR showed the relative expression of LINC00511 in NSCLC tissues and para-cancerous tissues. (b) RT-qPCR indicated LINC00511 expression in I-II stage and III-IV stage. (c) and (d) CCK-8 assay showed the IC₅₀ values in DDP-resistant and non-resistant NSCLC cells. (e) RT-qPCR showed the relative expression of LINC00511 in BEAS-2B cells, NSCLC cell lines, and DDP-resistant NSCLC cells. *p < 0.05; **p < 0.01.

LINC00511 silencing decreased the resistance of non-small cell lung carcinoma cells to DDP

Given that LINC00511 expression was highly expressed in DDP-resistant NSCLC cells, LINC00511 was silenced to explore the impact on cell activities. First, RT-qPCR confirmed that LINC00511 was downregulated after transfection of si-LINC00511 (Figure 2(a)). Subsequently, CCK-8 assay detected markedly blocked cell proliferation (Figure 2(b)) as well as decreased IC₅₀ value (Figure 2(c)) caused by LINC00511 deficiency. Next, TUNEL assay indicated the promotive effect of LINC00511 silencing on the apoptosis of DDP-resistant NSCLC cells (Figure 2(d)). Meanwhile, transwell assays showed that the transfection of si-LINC00511 hindered the migration and invasion of DDP-resistant NSCLC cells (Figures 2(e) and (f)). Finally, western blot assay identified that LINC00511 depletion caused the decreased expression of Ki-67 and MMP-9 and increased level of C-Caspase-3, demonstrating retarded cell growth and invasion and augmented cell apoptosis (Figure 2(g)).

Figure 2.

LINC00511 knockdown ameliorated DDP resistance in NSCLC cells. (a) Transfection efficiency of si-LINC00511 in DDP-resistant NSCLC cells was confirmed by RT-qPCR. (b) and (c) CK-8 assay showed the proliferation and IC₅₀ values in DDP-resistant NSCLC cells after transfection with si-LINC00511. (d)–(f) The apoptosis, migration and invasion of DDP-resistant NSCLC cells were evaluated by TUNEL and Transwell assays. (g) Western blot detected the levels of Ki-67, MMP-9, and C-Caspase-3 in A549/DDP and H522/DDP cells transfected with si-LINC00511. **p < 0.01.

LINC00511 was a ceRNA of miR-625

To uncover the underlying mechanism of LINC00511, Starbase website was utilized and the potent binding sequence between LINC00511 and miR-625 was predicted (Figure 3(a)). Then, Luciferase reporter assay further validated the prediction as miR-625 mimics significantly reduced LINC00511-WT luciferase activity but barely affected that of LINC00511-MUT (Figure 3(b)). Besides, miR-625 was markedly downregulated in NSCLC tissues and was in a negative correlation with LINC00511 expression (Figures 3(c) and (d)). Moreover, compared with BEAS-2B cells, miR-625 expression was also obviously lower in NSCLC cells, especially in DDP-resistant NSCLC cells (Figure 3(e)). RT-qPCR confirmed the overexpression efficiency of pcDNA3.1/LINC00511 (Figure 3(f)) and demonstrated that LINC00511 abundance and deficiency decreased and increased the expressions of miR-625, respectively (Figures 3(g) and (h)).

Figure 3.

LINC00511 was a sponge for miR-625. (a) Predicted binding sites between LINC00511 and miR-625. (b) Luciferase activities of LINC00511-WT and LINC00511-Mut in DDP-resistant cells. (c) Relative miR-625 level in NSCLC tissues and non-cancerous tissues. (d) The correlation between LINC00511 and miR-625 expressions in NSCLC tissues. (e) RT-qPCR analyzed the level of miR-625 in BEAS-2B cells, NSCLC cell lines, and DDP-resistant NSCLC cells. (f) RT-qPCR evaluated the levels of LINC00511 in DDP-resistant NSCLC cells transfected with pcDNA3.1/LINC00511. (g) and (h) RT-qPCR analyzed the impact of LINC00511 overexpression (g) or knockdown (h) on miR-625 expression in DDP-resistant NSCLC cells. *p < 0.05; **p < 0.01; ***p < 0.001.

LINC00511 increased DDP resistance in non-small cell lung carcinoma cells by suppressing miR-625

The functional role of LINC00511/miR-625 axis in DDP-resistant NSCLC was explored by silencing LINC00511 and miR-625. MiR-625 expression was elevated by the transfection of si-LINC00511, while the inhibition of miR-625 abrogated the elevation (Figure 4(a)). Then, the inhibited cell proliferation and IC₅₀ value caused by LINC00511 knockdown were revived by depleting miR-625 (Figures 4(b) and (c)). Meanwhile, TUNEL assay discovered that cell apoptosis was expedited by LINC00511 depletion but such expedition was annulled by silencing miR-625 (Figure 4(d)). Furthermore, LINC00511 silence repressed the abilities of migration and invasion but the repressive effect was reversed by the participation of miR-625 inhibitor (Figures 4(e) and (f)). Finally, the expression of Ki-67, MMP-9, and C-Caspase-3 was evaluated and the results demonstrated a consistent cell activity with the aforementioned assays (Figure 4(g)).

Figure 4.

LINC00511 knockdown abrogated the augmented DDP-resistant phenotypes of NSCLC cells caused by miR-625 depletion. (a) The relative expression of miR-625 in DDP-resistant NSCLC cells transfected with si-NC, si-LINC00511, or si-LINC00511+miR-625 inhibitor. (b)–(f) The effects of miR-625 and LINC00511 on cell viability, the IC₅₀ value, cell apoptosis, cell migration and cell invasion of DDP-resistant NSCLC cells. (g) Western blot evaluated the levels of Ki-67, MMP-9, and C-Caspase-3 in A549/DDP and H522/DDP cells. *p < 0.05; **p < 0.01.

LRRC8E was a downstream gene of miR-625

Four online tools (microT, miRmap, PITA, and RNA22) were used to predict the target gene of miR-625 and a total of 10 potent candidates (PPP6C, PPP2R1A, SDF2, PAPD5, CPSF7, ZSCAN25, LRRC8E, BMF, SMARCC2, and MARK2) were predicted (Figure 5(a)). Next, TCGA database results indicated that LRRC8E was the only one that was significantly upregulated in both lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) tissues among the aforementioned 10 genes (Figure 5(b)). The potent binding sequences between miR-625 and LRRC8E were illustrated in Figure 5(c), which was further confirmed by a luciferase reporter assay (Figure 5(d)). RT-qPCR results showed higher levels of LRRC8E in NSCLC tissues (Figure 5(e)). Besides, a negative correlation between LRRC8E and miR-625 expression was observed in NSCLC tissues (Figure 5(f)). Subsequently, the mRNA expression of LRRC8E was highly expressed in both DDP-resistant and non-resistant NSCLC cells compared with that in BEAS-2B cells (Figure 5(g)). To further confirm the association, miR-625 was silenced or overexpressed (Figures 5(h) and (i)) and LRRC8E expression was elevated and suppressed in DDP-resistant cells, respectively (Figures 5(j) and (k)).

Figure 5.

miR-625 directly targeted LRRC8E. (a) Bioinformatic analysis of potent candidates targeted by miR-625. (b) TCGA database analysis of LRRC8E expressions in LUSC and LUAD tissues. (c) The complementary binding sites between miR-625 and LRRC8E. (d) The luciferase activities of LRRC8E-WT and LRRC8E-Mut in DDP-resistant NSCLC cells. (e) Relative levels of LRRC8E in NSCLC tissues compared with paired non-tumorous tissues. (f) The correlation between LRRC8E and miR-625 levels was analyzed in NSCLC tissues. (g) Relative levels of LRRC8E in BEAS-2B cells, NSCLC cell lines, and DDP-resistant NSCLC cells. (H and I) MiR-625 expressions in DDP-resistant NSCLC cells after transfection with miR-625 inhibitor or miR-625 mimics. (J and K) RT-qPCR analyzed the mRNA expression of LRRC8E following miR-625 depletion or overexpression. *p < 0.05; **p < 0.01.

miR-625 attenuates non-small cell lung carcinoma cell resistance to DDP by inhibiting LRRC8E

Finally, rescue assays were performed to evaluate the function of the miR-625/LRRC8E axis in DDP-resistant NSCLC. RT-qPCR revealed that miR-625 overexpression greatly reduced LRRC8E levels but the participation of pcDNA3.1/LRRC8E re-elevated LRRC8E expressions in DDP-resistant NSCLC cells (Figure 6(a)). Next, CCK-8 assay detected hindered cell growth and reduced IC₅₀ value following miR-625 overexpression, but LRRC8E abundance reversed these effects (Figures 6(b) and (c)). Moreover, LRRC8E overexpression attenuated the cell apoptosis enhanced by miR-625 mimics (Figure 6(d)). Additionally, LRRC8E overexpression rescued the abilities of migration and invasion retarded by miR-625 upregulation (Figures 6(e) and (f)). Lastly, the expression of Ki-67, MMP-9, and C-Caspase-3 measured by western blotting further supported the results obtained from previous assays (Figure 6(g)).

Figure 6.

miR-625 overexpression overturned the impact of LRRC8E abundance on the behaviors in DDP-resistant NSCLC cells. (a) The mRNA levels of LRRC8E in A549/DDP and H522/DDP cells after transfection with NC mimics, miR-625 mimics, or miR-625 mimics+pcDNA3.1/LRRC8E. (b)–(f) The influence of miR-625 and LRRC8E on cell proliferation, IC₅₀, cell apoptosis, cell migration, and cell invasion of DDP-resistant NSCLC cells. (g) Western blot evaluated the levels of Ki-67, MMP-9, and C-Caspase-3 in A549/DDP and H522/DDP cells.*p < 0.05; **p < 0.01.

Discussion

Intrinsic and acquired resistance to chemotherapies induces the recurrence of tumors and significantly attenuates the efficacy of chemotherapeutic treatment for cancer patients.¹⁶ Chemotherapy based on DDP is the primary modality of treatment for NSCLC in the advanced stage.¹⁷ Despite continuous efforts to improve the chemosensitivity of NSCLC to DDP, the overall survival rate for NSCLC is still unsatisfactory.¹⁸ Hence, it is still essential to explore novel therapeutic targets to potentiate the chemo-sensitivity of NSCLC to DDP. In this study, we elucidated the molecular mechanism and functions of LINC00511 in DDP resistant NSCLC. LINC00511 was upregulated in NSCLC, especially in DDP-resistant cells. Besides, chemosensitivity of NSCLC cells to DDP could be enhanced by silencing LINC00511, indicating LINC00511 as a promising biomarker to potentiate DDP-sensitivity in NSCLC.

Interactions between lncRNAs and microRNAs (miRNA) have been widely reported in human cancers.^19–21 LINC00511 was reported to serve as sponges for miRNAs to regulate the progression of malignant tumors. For example, LINC00511 sponged miR-195 to upregulate EYA1 in hepatocellular carcinoma (HCC), leading to promote malignant behaviors of HCC cells.²² LINC00511 absorbed miR-515-5p to facilitate cell growth and invasion in gastric cancer.²³ LINC00511 potentiated the carcinogenesis and invasiveness of osteosarcoma by suppressing miR-185-3p to positively recover the E2F1 level.²⁴ Through a series of experiments, miR-625 was demonstrated as a target gene of LINC00511. MiR-625 was also reported to participate in the regulation of tumor progressions in many cases. For instance, LINC01123 exacerbated the malignancy of colorectal cancer by modulating the miR-625/LASP1 pathway.²⁵ LncRNA MIR503HG depletion ameliorated the malignant behaviors of NSCLC cells by elevating miR-489-3p and miR-625 levels.²⁶ The current research confirmed that LINC00511 negatively regulated miR-625 expression and the inhibition of miR-625 could increase the attenuated DDP resistance in NSCLC caused by depleting LINC00511.

LRRC8E was discovered to function as a pivotal component of the cell volume-regulated anion channel (VRAC)²⁷ which is the Cl-/anion channel ubiquitously expressed in vertebrates.²⁸ LRRC8E was also demonstrated to be implicated in the development of various malignant tumors. LINC00958 promoted the carcinogenesis of cervical cancer by sponging miR-625 to upregulate LRRC8E.²⁹ SP1-induced upregulation of lncRNA PCAT6 promoted breast cancer progression via the miR-326/LRRC8E pathway.³⁰ Our investigation uncovered that LRRC8E had remarkably abundant expression and was specifically targeted by miR-625. Functional assays revealed that miR-625 suppressed the expression of LRRC8E to augment the sensitivity of NSCLC to DDP.

In conclusion, our results indicated that LINC00511, miR-625, and LRRC8E exerted important functions in regulating the DDP-resistance of NSCLC that LINC00511 positively modulates NSCLC resistance to DDP through absorbing miR-625 to upregulate LRRC8E, implicating LINC00511 might be a therapeutic target to alleviate the DDP resistance in NSCLC.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Shidong Xu

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