Effects of MALAT1 on proliferation and apo- ptosis of human non-small cell lung cancer A549 cells in vitro and tumor xenograft growth in vivo by modulating autophagy

Abstract

OBJECTIVE:

To explore the ability of MALAT1 to influence non-small cell lung cancer (NSCLC) A549 cells in vitro and tumor xenograft growth in vivo by modulating autophagy.

METHODS:

LncRNA MALAT-1 in normal HBE cells and human NSCLC cells was measured. A549 cells were treated with si-MALAT-1, negative control and si-MALAT-1 $+$ rapamycin. The mRNA levels of MALAT-1, P62 and LC3 was determined by the qRT-PCR and the protein levels of autophagy-related proteins by the western blotting. The CCK8 assay was performed for cell proliferation, the scratch test for cell migration, the Transwell assay for cell invasion, and the flow cytometry for cell cycle and apoptosis. Tumor xenograft in nude mice is performed to test tumorigenesis of the transfected A549 cells.

RESULTS:

The expression level of MALAT-1 in A549, SPC-A-1 and NCI-H460 cells was increased compared to HBE cells. And A549 with a high expression level of MALAT-1 were selected for cell transfection. si-MALAT-1 decreased cell proliferation, migration, invasion, and LC3-II/LC3-I ratio, reduced cell cycle progression, and increased cell apoptosis and P62 protein expression. No significant difference was found between A549 cells and A549 cells transfected with si-MALAT-1 $+$ RAPA, A549 cells transfected with NC and A549 cells transfected with si-MALAT-1 $+$ RAPA. Nude mice injected with A549 cells transfected with si-MALAT-1 had smallest tumor on size and weight among other nude mice.

CONCLUSION:

Downregulation of MALAT1 may promote apoptosis and suppress proliferation, migration and invasion of human NSCLC A549 cells by inhibiting autophagy, thereby suppressing the development of NSCLC.

1. Introduction

Lung cancer is one of the leading contributors to new cancer diagnoses (about 1.3 million new cases, representing 12.4% of total new cancer cases) and to cancer-related deaths (about 1.1 million deaths, representing 17.6% of total cancer deaths) [1]. Non-small cell lung cancer (NSCLC) is any type of epithelial lung cancer other than small cell lung carcinoma (SCLC), comprising approximately 85% of all lung cancers [2]. Intriguingly, NSCLC patients are not very sensitive to chemotherapy and/or radiotherapy, so surgery remains the major choice for treating the majority of early-stage NSCLC patients [3]. However, up to 65% of patients are diagnosed with locally advanced or metastatic disease, presenting poor outcomes even following potential interventions [4]. The heterogeneity of clinical presentation of NSCLC is attributable to various molecular mechanisms underlying malignant transformation as well as dissemination of the primary cancer. Autophagy is defined as type II programmed cell death, a highly conserved self-digestion process, and promotes cell survival in response to nutrient starvation and other metabolic stresses [5]. And its dysregulation is proposed to participate in malignant transformation [6, 7]. To develop better diagnostics as well as more effective therapeutic strategies, studies over the past years have focused on an association between molecular changes and autophagy in NSCLC [8, 9].

Accumulating evidence suggests that noncoding RNA (ncRNA) genes are involved in malignant transformation as well as cancer development [10, 11]. Long non-coding RNAs (lncRNAs), longer than 200 nucleotides, play a significant role in regulating and controlling biological processes, including cell proliferation, differentiation, apoptosis and migration [12]. Among 3000 human lncRNAs, less than 1% are functionally characterized [13]. Metastasis associated in lung adenocarcinoma transcript 1 (MALAT1), a highly conserved mRNA-like lncRNA, was initially identified with high expression in advanced or locally metastatic NSCLC [14]. Also, Functional studies show that MALAT1 is overexpressed in a variety of other human cancers, including renal cell carcinoma (RCC), esophageal squamous cell carcinoma, breast cancer, prostate cancer and pancreatic cancer [15, 16, 17, 18, 19]. Thus, fine-regulation of MALAT1 is of great importance to cancer development and progression. However, the molecular mechanism underlying MALAT1 and autophagy in malignant transformation remain unknown or controversial due to dual role of autophagy in malignancy, for instance, Li et al. reported that MALAT1 promoted cell proliferation and migration by activating autophagy in aggressive pancreatic cancer [20]. Here we performed an in vitro study to explore effects of MALAT1 on cell proliferation, migration, invasion and apoptosis by its regulation on autophagy in NSCLC.

2. Materials and methods

2.1. Cell culture

Normal bronchus endothelial cell line HBE cells and NSCLC cell lines (A549 cells, SPC-A-1 cells, NCI-H460 cells) were purchased from Shanghai cell bank of Chinese Academy of Sciences. All cells were routinely cultured in RPMI-1640 culture medium (Hyclone, Logan City, UT, USA) containing 10% fetal calf serum (Gibico, Grand Island, NY, USA) in a constant-temperature incubator at 37${}^{\circ }$C with 5% CO${}_{2.}$The culture medium was changed once every two days. Cell passage was conducted at a ratio of 1:3 when cell reached 90% confluence. Expression of MALAT-1 was detected using quantitative real-time polymerase chain reaction (qRT-PCR).

2.2. Cell transfection

Small interfering plasmid targeting against MALAT-1 (sequence: GCTCCTTGGTGAATTGATA) and negative control (NC) plasmid (sequence: TTCTCCGAA CGTGTCACGT), purchased from Promega Corporation (Madison, Wisconsin, USA) were prepared. Using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) reagents, A549 cells were separately treated with si-MALAT-1, NC and si-MALAT-1 $+$ rapamycin (RAPA) (namely the A549 cells were initially treated by si-MALAT-1 and maintained in RPMI-1640 culture medium supplemented with the inhibitor of autophagy, 250 mmol/L RAPA). A549 cells transfected with FAM-labeled si-MALAT-1 (25, 50 and 100 nmol/L) were incubated for 24, 48 and 72 h in a dark room, and then washed using pre-cold phosphate buffer saline (PBS). The ratio of green fluorescent A549 cells in total A549 cells was calculated. The experiment was conducted for three times to obtain averages.

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

Cells in each group were collected and isolated for total RNA extraction using a Trizol total RNA isolation kit. Then 5 $\mu$l of RNA was obtained and diluted in RNA-free ultrapure water by 20 times. The absorbance in the ultraviolet spectrophotometer with the wave length of 260 nm and 280 nm were recorded to calculate the RNA density and purity. Those with ratio of OD260/OD280 between 1.7$\sim$2.1 indicate for a high purity, which meet the requirement for further experiments. The cDNA template was reversely transcripted in a PCR amplifier and an ABI7500 quantitative PCR instrument was applied for qRT-PCR. The PCR reaction was conducted under following conditions: pre-denature at 95${}^{\circ }$C for 10 min, followed by 50 cycles of 95${}^{\circ }$C for 15 s, 60${}^{\circ }$C for 1 min and 72${}^{\circ }$C for 40 s. The primers used are listed in Table 1. PCR was conducted using $\beta$-actin as the internal control and analyzed using 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\mathrm{Ct}}$ method. The experiment was conducted for three times to obtain averages.

Table 1.

Primer sequences of MALAT-1, LC3, for P62 and $\beta$-actin for qRT-PCR

Gene	Primer sequences
MALAT-1	5’-GAATTGCGTCATTTAAAGCCTAGTT-3’
	5’-GTTTCATCCTACCACTCCCAATTAAT-3’
LC3	5’-CTTCGCCGACCGCTGTAA-3’
	5’-CTGGGAGGCGTAGACCATAT-3’
P62	5’-AGTCCAGAATTCCTGCCTGA-3’
	5’-TTCATTCAACTTCACATGAA-3’
$\beta$-actin	5-ACAGTCAGCCGCATCTTCTT-3’
	5-GACAAGCTTCCCGTTCTCAG-3’

Note: qRT-PCR, quantitative real-time polymerase chain reaction.

2.4. Western blotting

Total cells on A549 cells among groups were extracted 48 hour after transfection., which was then electrophoresed in 10% sodium dodecyl sulfate-polya- crylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene difluoride membranes (PVDF) (Millipore, Billerica, MA, USA). Then cells were blocked for one hour at room temperature in 5% not-fat milk powder, followed by washing PBS. Primary rabbit anti human antibodies for LC3 (1:1000), P62 (1:2000) and GAPDH (1:5000) were added for incubation at 4${}^{\circ }$C at a shaking table for overnight (all primary antibody purchased from Abcam, Cambridge, MA, USA). Then phosphate buffer saline with Tween20 (PBST) was used for washing. Secondary goats anti rabbit antibodies (1:10000, Abcam, Cambridge, MA, USA) were added for incubation at room temperature for one hour. After that, chemical luminescence reagent was used for color development and the grey value of the protein expression was analyzed by Image software. GAPDH was considered as internal reference. The protein expression of target protein was the ratio of the grey value of the target protein and GAPDH.

2.5. CCK-8 assay

A549 cells at 48 hours after cell transfection were inoculated in a 96-well plate and were measured at certain time points (0 hour, 24 hours, 48 hours and 72 hours) for cell numbers. The detailed experiment procedures were as follows. Firstly, the culture medium was replaced with 100 ul fresh culture medium containing 10 ul of CCK-8 reagent (Beyotime Biotechnology, Shanghai). Then the plate was placed in a CO${}_2$ incubator for 2 hours and a microplate reader (Bio-Rad, Hercules, CA, USA) was used to measure the OD value at a wavelength of 450 nm. Cell proliferation was calculated based on formula: cell proliferation rate (%) $=$ (experimental OD value $-$ control OD value)/control OD value $\times$ 100%. Six repeated wells were set for experiment.

2.6. Flow cytometry

Propidium iodide (PI) and flow cytometry were applied for detection on cell cycle. A549 cells at 48 hours after cell transfection were inoculated in five culture disks (10 cm) with the density of 1 $\times$ 10${}^6$ cells in an incubator containing 5% CO${}_2$ at 37${}^{\circ }$C. After 24 hours, cells were washed in pre-cold PBS twice and fixed in 75% frozen ethanol at $-$20${}^{\circ }$C for one hour. Washed in pre-cold PBS, cells were re-suspended in 200–500 uL pre-cold PBS. Then 20 uL of Rnase A solution was added and water bathed at 37${}^{\circ }$C for 30 min. After cells were infiltrated by 400 filter screen, 400 uL of PI was added and mixed at 4${}^{\circ }$C for 30 min–1 hour without light exposure. The final sample was filtered with yellow nylon filter screen. Flow cytometry (BD, Franklin Lakes, NJ, USA) was used for cell cycle measurement. During the cell collection and washing, the mechanical cell injury shall be avoided as much as possible, to prevent DNA loss.

Annexin V/PI double staining was applied for detection on cell apoptosis. A549 cells at 48 hours after cell transfection were inoculated in five culture disks (10 cm) with the density of 1 $\times$ 10${}^6$ cells in an incubator containing 5% CO${}_2$ at 37${}^{\circ }$C. After 24 hours, cells were made into single cell suspension and added with 500 uL of Annexin V Binding Buffer, 5 uL of Annexin V-FITC and 5 uL of PI for mixture at room temperature for 10 min without light exposure. Cell apoptosis was measured using flow cytometry (BD Pharmingen, Franklin Lakes, NJ, USA) after cells were infiltrated using a yellow nylon filter screen.

2.7. Scratch test

After cell transfection for 48 hours, cells were inoculated in a 6-well plate with the density of 5 $\times$ 10${}^5$. Once the cell adherence reached 80%, then 1 ml of sterile pipettor spearhead was used to scratch a vertical line in the cell plate, which was followed by PBS washing. Culture medium was added then and incubated in a 5% CO${}_2$ incubator at 37${}^{\circ }$C for 24 hours and fixation in 75% alcohol at 4${}^{\circ }$C for 30 min. HE staining was performed. Neutral resins were used for sealing. The scratch width under three slides was randomly chosen under a light microscope. Cell migration distance was assessed using Image-Pro Plus Analysis software (Media Cybemetics, Silver Spring, MD, USA) to obtain the average value.

2.8. Transwell assay

A total of Matrigel gel which was dissolved at 4${}^{\circ }$C was added into the pre-cold Transwell chamber in the cell incubator for one hour. After 48 hours of cell transfection, cell density of the serum free culture medium was adjusted into 1 $\times$ 10${}^5$/100 $\mu$l. Cell suspension (100 $\mu$l) was added into the upper Transwell chamber (Becton Dickinson, San Jose, CA, USA) in the 24-well plate, while the lower Transwell chamber was added with 500 $\mu$l of culture medium containing 10% FBS in a 5% CO${}_2$ incubator at 37${}^{\circ }$C for 48 hours. Then the chamber was taken out and was scrapped the cells on the cotton swabs, which was then fixed using 4% paraformaldehyde for 15 min. Washed the chamber in PBS. Crystal violet was used for staining for 10 min. After chambers were washed in PBS, a high-power microscope (Olympus, Japan) was used to calculate the penetrated cell numbers on the upper, down, right, right and middle fields.

2.9. Nude mice bearing xenograft of A549 cells [21]

Forty nude mice were randomly grouped into blank, si-MALAT-1, NC and si-MALAT-1 $+$ RAPA groups. Nude mice in the blank group were normal, nude mice in the si-MALAT-1 group were injected with A549 cells transfected with si-MALAT-1, nude mice in the NC group were injected with A549 cells transfected with NC, and nude mice in the si-MALAT-1 $+$ RAPA group were injected with A549 cells transfected with si-MALAT-1 $+$ RAPA. Cell suspension was prepared using 5 $\times$ 10${}^7$ cells and 1 ml PBS (0.01 mol/L, pH7.2). Each nude mouse was given 0.2 ml cell suspension containing 1 $\times$ 10${}^7$ cells via tail-vein injection once a day for 21 days. With the injection terminated, nude mice were observed for 10 days. Nude mice were anesthetized and sacrificed by cervical dislocation. Tumors were cleaned and weighed. And the length of the longest diameter and the length of diameter perpendicular to it were measured. Animal protocols in this study were approved by the Animal Care and Use Committee at our hospital.

2.10. Statistical analysis

SPSS version 20.0 software (SPSS Inc., Chicago, IL, USA) was applied for statistical analysis. Measurement data were expressed as mean $\pm$ standard deviation and unpaired Student $t$-test was used for mean comparison between two groups. Analysis of multi-group was performed by one-way analysis of variance (ANOVA) test. $P$ values 0.05 were accepted as significant. Enumeration data were expressed as rate or percentage and chi-square test was used for the comparison between two groups.

3. Results

3.1. The expression levels of MALAT-1 in NSCLC A549, SPC-A-1 and NCI-H460 cells

Results of qRT-PCR was presented in Fig. 1, which indicated that compared with that in the HBE cells, the expression levels of MALAT-1 in NSCLC A549, SPC-A-1 and NCI-H460 cells were significantly elevated (all 0.05). A549 cells had a high expression level of MALAT-1 ( 0.05), thus were selected for further studies.

Figure 1.

The expression levels of MALAT-1 in HBE, A549, SPC-A-1 and NCI-H460 cells. Note: *, compared with HBE cells, 0.05; #, compared with A549 cells, 0.05.

3.2. Identification of transfection efficiency

As indicated on flow cytometry (Fig. 2), silence efficiency of MALAT-1 in A549 cells transfected with si-MALAT-1 in 24 h, 48 h and 72 h reached 12.47 $\pm$ 0.32%, 65.84 $\pm$ 1.67% and 49.36 $\pm$ 0.88% respectively. The expression level of MALAT-1 in A549 cells transfected with si-MALAT-1 was lower than that in A549 cells and A549 cells transfected with NC. The best time to silence MALAT-1 was 48 hours.

Figure 2.

Silence efficiency of MALAT-1 in A549 cells transfected with si-MALAT-1 in 24 h, 48 h and 72 h. Note: A, silence efficiency of MALAT-1 at different time points; B, the expression levels of MALAT-1 in untransfected A549 cells, A549 cells transfected with NC, and those transfected with si-MALAT-1 at 48 h after transfection; *, compared with the blank group, 0.05.

3.3. Downregulated MALAT-1 inhibited A549 cell proliferation by inhibiting autophagy

CCK-8 assay for cell proliferation found no significant difference on proliferation rate at 24 h after transfection among groups (all $P>$ 0.05). The cell proliferation rates at 48 and 72 h after transfection in si-MALAT-1 group were significantly lower than those in the blank group. However, cell proliferation rates at 0, 24, 48 and 72 h did not differ significantly among blank, NC and si-MALAT-1 $+$ RAPA groups (all $P>$ 0.05) (Fig. 3). The results indicated that silencing MALAT-1 inhibited A549 cell proliferation by inhibiting autophagy.

Figure 3.

Cell proliferation among blank, NC, si-MALAT-1 and si-MALAT-1 $+$ RAPA groups detected by CCK-8. Note: *, compared with the blank group, 0.05.

3.4. Downregulated MALAT-1 suppressed cell cycle progression in A549 cells by inhibiting autophagy

Cells in the G0/G1 phase accounted for 56.70 $\pm$ 2.36% in the si-MALAT-1 group, which was significant higher than those in the blank group (47.22 $\pm$ 1.59), NC group (48.35 $\pm$ 1.31) and si-MALAT-1 $+$ RAPA group (47.68 $\pm$ 1.47) (all 0.05). Cells in the S phase cells in the blank group accounted for 18.00 $\pm$ 1.49%, which was significantly higher than those in the si-MALAT-1 group ( 0.05). However, cell cycle progression did not differ significantly among blank, NC and si-MALAT-1 $+$ RAPA groups (all $P>$ 0.05) (Fig. 4). These results suggest that silencing MALAT-1 resulted in more cells arrested in the G0/G1 phase, thereby suppressing A549 cell transition from G1 to S phase by inhibiting autophagy

Figure 4.

Cell cycle among blank, NC, si-MALAT-1 and si-MALAT-1 $+$ RAPA groups detected by flow cytometry.

3.5. Downregulated MALAT-1 promoted A549 cell apoptosis by inhibiting autophagy

Annexin V/P flow cytometry found that, 48 h after transfection, the apoptotic rate in the blank group (6.87 $\pm$ 0.22) was significantly lower than those in the si-MALAT-1 group (15.56 $\pm$ 0.88) ( 0.05). However, cell apoptotic rates did not differ significantly among blank group, NC group (6.80 $\pm$ 0.12) and si-MALAT-1 $+$ RAPA group (7.03 $\pm$ 0.24) (all $P>$ 0.05) (Fig. 5). The data suggested that silencing MALAT-1 promoted A549 cell apoptosis by inhibiting autophagy.

Figure 5.

Cell apoptosis among blank, NC, si-MALAT-1 and si-MALAT-1 $+$ RAPA groups detected by flow cytometry.

3.6. Downregulated MALAT-1 inhibited A549 cell migration and invasion by inhibiting autophagy

As indicted by scratch test, the migration distance in the si-MALAT-1 group (43.56 $\pm$ 6.35) was reduced compared with blank group (72.12 $\pm$ 7.13). However, cell migration distance did not differ significantly among blank group, NC group (73.36 $\pm$ 7.32) and si-MALAT-1 $+$ RAPA group (70.15 $\pm$ 6.38) (all $P>$ 0.05) (Fig. 6A and B). Results by Transwell assay showed that cells penetrating chambers did not differ significantly among blank group (25.15 $\pm$ 2.82), NC group (25.60 $\pm$ 2.77) and si-MALAT-1 $+$ RAPA group (24.78 $\pm$ 2.63) (all $P>$ 0.05). Cells penetrating chambers in the si-MALAT-1 group (6.65 $\pm$ 1.69) were lower than those in the blank group ( 0.05) (Fig. 6C and D). The results implied that silencing MALAT-1 inhibited A549 cell migration and invasion by inhibiting autophagy.

Figure 6.

Cell migration and invasion among blank, NC, si-MALAT-1 and si-MALAT-1 $+$ RAPA groups detected by scratch test and transwell assay. Note: A, cell migration images; B, statistics of cell migration distance; C, cell invasion images; D, the numbers of Matrigel-permeating cells; *, compared with blank group, 0.05.

3.7. The expression of autophagy-related proteins in transfected A549 cells

Results of qRT-PCR (Fig. 7A) indicated that compared with the blank group, the si-MALAT-1 group had increased mRNA expression of P62 and decreased mRNA expression of LC3 (both 0.05). However, P62 and LC3 mRNA expression did not differ remarkably among blank, NC and si-MALAT-1 $+$ RAPA groups (all $P>$ 0.05). Results of western blotting (Fig. 7B and C) showed that compared with the blank group, si-MALAT-1 group had reduced ratio of LC3-II/LC3-I and increased protein expression of P62 (both 0.05). However, the ratio of LC3-II/LC3-I and the protein expression of P62 did not change evidently among blank, NC and si-MALAT-1 $+$ RAPA groups (all $P>$ 0.05). Together, our results showed that silencing MALAT-1 inhibited A549 cell autophagy.

Figure 7.

The expression levels of autophagy-related proteins among blank, NC, si-MALAT-1 and si-MALAT-1 $+$ RAPA groups detected by qRT-PCR and western blotting. Note: A, P62 and LC3 mRNA expressions; B, gray values of P62, LC3-I and LC3-II protein bands; C, the expression levels of P62, LC3-I and LC3-II protein; *, compared with blank group, 0.05.

3.8. Downregulated MALAT-1 suppressed A549 cell tumorigenic ability in vivo

A549 cell tumorigenic ability did not differ significantly in nude mice among blank, NC and si-MALAT-1 $+$ RAPA groups (all $P>$ 0.05). Nude mice in the si-MALAT-1 group had smaller tumors on size and weight than those in the blank group (both 0.05). The results suggested that silencing MALAT-1 suppressed A549 cell tumorigenic ability in vivo by inhibiting autophagy (Fig. 8, Table 2).

Figure 8.

Tumor sizes of nude mice among blank, NC, si-MALAT-1 and si-MALAT-1 $+$ RAPA groups.

Table 2.

Tumor size and weight in nude mice injected with transfected A549 cells

	Blank	NC	si-MALAT-1	si-MALAT-1 $+$ RAPA
Tumor size (mm${}^3)$	823 $\pm$ 382	855 $\pm$ 413	538 $\pm$ 118*	831 $\pm$ 421
Tumor weight (g)	0.62 $\pm$ 0.21	0.67 $\pm$ 0.18	0.31 $\pm$ 0.09*	0.70 $\pm$ 0.17

Note: NC, negative control; RAPA, rapamycin; *, 0.05 compared to the blank group.

4. Discussion

By comparing human NSCLC A549, SPC-A-1 and NCI-H460 cells in terms of MALAT1 expression, the study was conducted in vitro to investigate effects of MALAT1 on cell proliferation, migration, invasion and apoptosis by its regulation on autophagy in NSCLC. Subsequently, A549 cells with the lowest expression of MALAT1 were chosen for cell transfection. Consequently, our study confirmed that downregulation of MALAT1 inhibited autophagy in NSCLC cells and downregulation of MALAT1 suppressed the advanced progression of the tumor.

Importantly, our main finding indicated that, the si-MALAT-1 group had decreased cell proliferation, migration and invasion, but increased cell apoptosis compared with the blank and NC groups, suggesting that downregulation of MALAT-1 promoted apoptosis and suppressed proliferation, migration and invasion of A549 cells. Despite its mRNA-like characteristics, MALAT-1 is localized to nuclear speckles in the eukaryotic cell, the area involved in the aggregation, modification and (or) storage, processing, and has been implicated in the occurrence and development of tumors [22]. Also, it may be proposed that siRNA-mediated MALAT-1 impaired cell motility of cancer cells in vitro and exerted significant influences on the expression of many motility-related genes including CCT4, HMMR, CTHRC1, or ROD1 in lung cancer. And knockdown of any one of these genes remarkably inhibited cell invasion and migration [23]. Similarly, Lai et al. reported that inhibition of MALAT1 can significantly reduce cell viability, motility, invasion, and strengthen the sensitivity to apoptosis in human hepatocellular carcinoma HepG2 cells, predicting tumor recurrence following liver transplantation [24]. As for the effect of si-MALAT1 on cell cycle progression, our results indicated, in the si-MALAT-1 group, A549 cells arrested in the G0/G1 stage but decreased in S stage in comparison with the blank and NC groups. Tripathi et al. detected MALAT1 expression in osteosarcoma U20S cells during cell cycle and found low expression of MALAT1 in G0 and G1 and high expression in G1/S and M stages. And knockdown of MALAT1 resulted in cell cycle arrest by up-regulating cell cycle-related genes, such as p53, p21, p27 and Cyclin A2 [25]. On the other hand, Ning et al. revealed that MALAT1 notably facilitate the migration, invasion and tumorigenesis in vivo, indicating its critical role in the bone metastasis of NSCLC [26]. In our study, tumor xenograft in nude mice showed that silencing MALAT-1 suppressed A549 cell tumorigenic ability in vivo by inhibiting autophagy. Autophagy is beneficial to normal cells and tumor cells for their adaptive ability to metabolic stress, promoting cancer cell survival [27]. Cancer cells were characterized by more active metabolism as well as higher demand for nutrition and energy than normal cells, but the tumor microenvironment is often unsatisfactory [28]. In the process of cancer formation, autophagy increased in different stress, such as nutrient starvation [29], unfolded protein response during hypoxia [30], and toxicity of chemotherapy drug [31], and can protect tumor cells from various unfavorable conditions, so as to improve the viability of cancer cells and promote cancer development and progression [32]. Consistent with our study, Karpathiou et al. demonstrated that excessive autophagy, as suggested by the intense presence of “stone-like” structures (SLSs), was strongly linked with a poor outcome in NSCLC [33].

Additionally, our study also found that P62 mRNA and protein expressions were increased while LC3 mRNA expression and LC3-II/LC3-I ratio were reduced compared with the blank and NC groups, which implied that inhibition of MALAT1 might inhibit autophagy in A549 cells. Autophagy-related proteins in-clude p62, NBR1 and LC3. Sequestosome 1 (SQSTM1/p62), a multifunctional protein, participates in signal transduction, protein degradation and as well as transformation, which is degraded by autophagy [34]. Also, the conversion of LC3I to LC3II can reflect autophagic activity [35]. Nonetheless, the mechanism underlying MALAT1 modulating autophagy remains unknown. Whether MALAT1 functions on autophagy by targeting autophagy-related proteins or autophagy-related signaling pathway is required for further study and verification.

In conclusion, the present study provides evidence that down-regulation of MALAT1 may promote apoptosis and inhibit cell proliferation, migration and invasion by inhibiting apoptosis in human NSCLC A549 cells. Credibly, our study highlights the new roles of MALAT1 on protumorigenic functioning and anticancer therapy in NSCLC. At the same time, it is usual to observe cellular behavior discrepancies among various cell lines following same treatment even they is obtained from same type of malignancy, which will be significantly addressed in the further study.

Conflict of interest

None.