E2F1-induced long non-coding RNA MCF2L-AS1 modulates Cyclin D1 mRNA stability through ELAVL1 to induce Gefitinib resistance in non-small cell lung cancer

Purpose: Gefitinib is a widely used therapeutic drug for non-small cell lung cancer (NSCLC), and its acquired resistance has become one of the barriers to the successful use of the drugs to treat NSCLC patients. Long non-coding RNA (lncRNA) has an essential role in developing cancer drug resistance. Hence, this study aimed to investigate the effect and modulatory mechanisms of lncRNA MCF2L-AS1 in Gefitinib resistance in NSCLC. Methods: IBEAS-2B and A549 cells and human NSCLC tissues were used. A549/GR cell line was constructed by continuous exposure to Gefitinib. Cell viability, apoptosis, migration, colony formation, and protein expression studies were done in transfected cells. Interactions of MCF2L-AS1, ELAVL1, and Cyclin D1 (CCND1 was also investigated. Results: In patients with Gefitinib-resistant NSCLC, MCF2L-AS1 and CCND1 were both up-regulated. Knock-down of MCF2L-AS1 reduced Gefitinib-resistant NSCLC cell progression, indicating that inhibition of MCF2L-AS1 restrained Gefitinib-resistant NSCLC. Mechanically, MC-F2L-AS1 enhanced CCND1 mRNA stability via combining with ELAVL1, thereby elevating the resistance of NSCLC cells to Gefitinib. Moreover, E2F1 could transcriptionally up-regulate MCF2L-AS1. Conclusion: The results manifest that lncRNA MCF2L-AS1, as an oncogene of NSCLC, controls CCDN1 via ELAVL1 to drive the growth of NSCLC cells and Gefitinib resistance.


INTRODUCTION
Lung cancer (LC) is a malignant tumor with the highest morbidity and mortality worldwide and has extensive drug resistance (Wang et al., 2020).Non-small cell lung cancer (NSCLC) is the major subtype of LC, taking up about 85-90% of all LC cases.Research advances have the diagnosis and treatment approaches to NSCLC.The currently used therapeutic methods include surgery, radiotherapy, chemotherapy, and targeted drug therapy.
However, the prognosis of NSLC remains poor, with low survival rates.
Epidermal growth factor receptor (EGFR) is an oncogenic receptor tyrosine kinase that is crucial in establishing and spreading NSCLC.Gefitinib is a first-generation EGFR-tyrosine kinase inhibitor (TKI) that reversibly binds with the ATP cleft at the EGFR kinase domain, thus blocking EGFR auto-phosphorylation, hence effective in NSCLC (Kwon et al., 2015).However, increased resistance to Gefitinib has been recently reported among NSCLC patients, resulting in treatment failure.Consequently, understanding the mechanisms of Gefitinib resistance to NSCLC is necessary to develop an effective therapy.
Several reports have confirmed the role of long noncoding RNA (lncRNA) in cancer drug resistance.According to the previous investigations, LINC01116 induces gefitinib resistance in NSCLC via controlling IFI44 while lncRNA UCA1 controls FOSL2 through competition with microRNA (miR)-143 to elevate Gefitinib resistance in NSCLC (Chen et al., 2020).Further, lncRNA H19 has also been shown to contribute to the resistance of Gefitinib to LC (Lei et al., 2018).A study reported that lncRNA MCF2L-AS1 drives colorectal cancer (CRC) progression through the miR-874-3p/FOXM1 axis (Zhang et al., 2021).LncRNA MCF2L-AS1 up-regulates CCND1 through sponging miR-874-3p, thereby elevating the invasiveness of CRC (Huang et al., 2021).LncR-NA MCF2L-AS1 is enhanced in LC tissues and cells and drives NSCLC stem cell-like characteristics via down-regulating miR-873-5p (Ajani et al., 2015).In the meantime, cancer stem cells (CSCs) have the ability of self-renewal and multi-lineage differentiation, which are linked with the occurrence, progression, metastasis, recurrence, and resistance of tumors to drugs (Li et al., 2021).Nevertheless, no studies have reported the role of lncRNA MC-F2L-AS1 in the resistance of Gefitinib to NSCLC.
LncRNA, being a latent modulator of various cellular processes, has several roles, including modulation of RNA binding proteins (RBPs), motivation of mRNA transcription, and post-transcriptional controlling through sponging miRNAs (Sana et al., 2012).For instance, NHG12 binds with the RNA binding protein HuR, stabilizes YWHAZ, and accelerates the proliferation of gastric cancer (Zhang et al., 2021).LncRNA TSLNC8 drives the growth and metastasis of pancreatic cancer through HUR-mediated CTNNB1 mRNA stability (Chai et al., 2021).Meanwhile, HuR (ELAVL1) has been reported as the common RNA binding protein of lncRNA MCF2L-AS1 and CCND1.The previous investigation confirmed an increased ELAVL1 in NSCLC tissues (Ni et al., 2020).Cyclin D1 (CCND1) is an essential molecule in the cell cycle transition from the G1 phase to the S phase, and it is elevated in numerous tumors.Linc00703 has been confirmed to inhibit NSCLC progression via repressing CCND1 (Sun et al., 2020), while LncRNA PCNA-AS1 enhances NSCLC cells proliferation via up-regulating CCND1 (Wu et al., 2020).The current study hypothesized that lncRNA MCF2L-AS1 might accelerate the stability of CCND1 mRNA via combining with ELAVL1 to elevate Gefitinib resistance in NSCLC.

Ethical statement and clinical samples
The research approval was done by the Ethics Committee of the Second People's Hospital of Kunshan.Written consent was obtained from all the study participants.The NSCLC human samples were collected from 2016 to 2019.The samples were divided into the control group (not receiving Gefitinib treatment, n=10) and the Gefitinib resistant group (relapse after Gefitinib treatment, n=15, NSCLC patient tissues).The tissues were immediately frozen in liquid nitrogen and stored at -80°C for further experiments.

Cell culture and treatment
Normal human lung epithelial cells BEAS-2B cells and lung adenocarcinoma A549 cells were purchased from ATCC (Rockville, MD, USA).The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin, 100 μg/mL streptomycin.The medium was changed daily, and cells were harvested at the logarithmic growth phase for further experiments.

Gefitinib-resistant cell line establishment
Gefitinib-resistant cell line (A549/GR) was established as reported elsewhere (Rho et al., 2009).Briefly, the parental A549 cells were exposed to 5 µM Gefitinib (Santa Cruz Biotechnology, CA, USA) for 48 h in DMEM supplemented with 10% FBS.The cells were then washed and incubated in a drug-free medium until the surviving cells' population reached 80% confluence.Next, the surviving cells were continuously exposed to increasing Gefitinib dosages which were sequentially increased to 5, 10, 15, 20, 25, and 30 M (after every 2 months).The constructed resistant cell line was maintained by culturing in DMEM containing 30 M Gefitinib.

Cell counting kit (CCK)-8 assay
Cells in the logarithmic phase were trypsinized for 2 min at 37°C to a single cell suspension.The cells were resuspended in 1 mL of complete media and counted.Next, a 100 µl suspension containing 4×10 3 cells was plated per well in a 96-well culture plate.The cells were cultured for 24, 48, and 72 h after which 10 μL CCK-8 solution from the CCK-8 assay kit (Dojindo, Tokyo, Japan) was added and incubated for 4 h, following the manufacturer's instructions.The absorbance was then determined in a microplate reader at an absorption wavelength of 450 nm.Each assay was done in triplicates.

Colony formation assay
Approximately 300 cells per well were introduced in a 6-well plate in triplicates.The cells were then grown for 9 days, and colonies were fixed in methanol and stained in crystal violet (0.1%) at room temperature for 1 h.Later, the plates were completely washed by submerging them in a water bath for 1 hour.Finally, the cell colonies were counted, and the percentage rate of colony formation was determined as colony number per 300 cells ×100, as described elsewhere (Rice et al., 2019).The assay was done in triplicates.

Transwell
Determination of cell migration was done using a transwell assay.Summarily, a 6.5 mm transwell chamber containing 8 μm pores (Corning Costar Corp., USA) was used.The ability of cells to migrate was investigated by re-suspension of the stably transfected cells (2×10 4 cells) in 200 μl of RMPI-1640 medium (serum-free) and eventually plating them in the top chamber.Later, 500 μl of the media supplemented with 10% fetal bovine serum was introduced into the lower well chamber.Cells were then incubated under chemotactic conditions for 24 h at 37°C.Cells were later stained in 1% crystal violet for 30 min, and cells on the upper surface of the membrane were eliminated using cotton swabs.Cells at the bottom of the membrane were then counted and imaged microscopically (Olympus Corp. Tokyo, Japan) in four random fields.

Flow cytometry test of apoptosis
The cells from a logarithmic phase were detached using 0.25% trypsin (without ethylene diamine tetraacetic acid) (PYG0107, Boster, Wuhan, China), washed in cold PBS, and collected in flow cytometry tubes.The cells were then centrifuged, and the supernatant was discarded.The Annexin-V-fluorescein isothiocyanate (FITC) Cell Apoptosis Detection Kit (K201-100, Biovision, USA) was applied to assess apoptosis following the manufacturer's instruction.The Annexin-V-FITC, propidium iodide (PI), and HEPES buffer solutions were appropriately prepared, and 1×10 cells were resuspended per 100 µL staining solution, incubated and 1 mL HEPES buffer was finally added.The apoptosis rate was finally detected using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA), and data were analyzed by CellQuest Pro (BD Biosciences, Franklin Lakes, NJ, USA).The experiment was repeated three times and data were averaged.

RNA stability analysis
In analyzing the half-life of CCND1 mRNA, HEK293T single clonal cell lines were treated using The effect of lncRNA MCF2L-AS1 on Gefitinib resistance in NSCLC 10 μg/mL actinomycin D (Sigma) for transcription inhibition and were harvested at 0, 2, 4, 6, and 8 h.RT-qPCR was done to determine the decay in RNA abundance.Next, linear regression was then done between log values of the expression levels.Time points and the half-life were finally calculated.

Biotin RNA-pull down assay
The pull-down assay was done as described elsewhere (Barnes et al., 2016).The biotinylated biotinlabeled MCF2L-AS1 and ELAVL1 (Bio-MCF2L-AS1/ ELAVL1-AS/sense) (GeneCreate, Wuhan, China).(50 nM).Moreover, mutant plasmids (50 nM) were added to lysis buffer (500 µl, 0.5 M NaCl, 20 mM Tris-HCl, and 1 mM EDTA, pH 7.5) and incubated at 25°C with streptavidin-coated magnetic beads for 3 h.The cell pellets were then resuspended using the lysis buffer (1 ml/100 mg of cell pellet), and cell lysates were then incubated using the probe-coated beads.Lastly, the RNA mixture was finally eluted, and extraction was done for the northern blot using magnetic support, separating the beads from cell lysate.The supernatant was removed, and beads were washed using wash buffer (900 µl, 0.5% SDS, 2X SSC).Digoxin-labeled probes were prepared using the DIG Northern Starter kit (Roche Diagnostics GmbH) described earlier.Total RNA samples were resolved on 15% polyacrylamide-urea gels or 2% agarose and transferred to Hybond-N+ membranes (Amersham; Cytiva).The membranes were then dried, cross-linked by UV radiation, and hybridized using digoxin-labeled miR-140-5p probes at 42°C or 65°C overnight; the digoxin-labeled U6 probes were utilized as controls.The blot was visualized on a ChemiDoc XRS system (Bio-Rad Laboratories, Inc.).

RNA immunoprecipitation (RIP)
Approximately 1.7×10 6 cells were plated in a 10 cm dish and cultured for 48 h.Cells were then washed twice using chilled PBS, collected using a cell scraper into ice-cold PBS, and centrifuged at 500×g for 5 min at 4°C.The pellets were resuspended using RIP lysis buffer (2 mL, 50 mm Tris/HCl, 300 mm NaCl, pH 7.4, 30 mm EDTA and 0.5% Triton X-100) supplement containing a cocktail of protease inhibitor and 200 U/ml RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher) and incubated for 10 min on ice.The lysate was passed through a small needle severally and centrifuged for 15 min at 20 000×g and 4°C.Later, a supernatant (80 μL) was saved as input (10%) for the extraction of RNA, and another supernatant (80 μL) was set aside for analysis of protein.Dynabeads Protein G (50 μL) (Thermo Fisher) was washed twice using RIP lysis buffer (0.7 mL) and resuspended with RIP lysis buffer (150 μL).Next, rabbit anti-FMRP polyclonal antibody (10 μg) (Abcam, ab17722) or control normal rabbit IgG (10 μg) (Cell Signaling Technology) was added to the resuspended magnetic beads.The mixture was continuously centrifuged for 30 min at room temperature.The beadsantibody complex was washed four times using RIP lysis buffer (0.7 mL), mixed with cleared cell lysate (0.8 mL), and continuously centrifuged at 4°C for 3 h.The tubes were briefly centrifuged and placed on a magnetic separator.The supernatant (80 μL) was set aside as flow-through (10%) for protein analysis; beads were collected and washed (five times) at 4°C with RIP lysis buffer (0.7 mL) and resuspended with RIP lysis buffer (0.5 mL).Later, some mixture (50 μL) was saved as IP (10%) for analysis while 90% of the beads were transferred to a new tube, collected and resuspended using proteinase K digestion solution (150 μL), 15 μL of SDS (10%) and proteinase K (10 μL) (20 mg/ml; Thermo Scientific)].In parallel, nuclease-free water (25 μL), 10% SDS (15 μL), and proteinase K (10 μL) were added to the input sample thawed on ice.Digestion of Proteinase K was done for 30 min at 55°C with shaking at 1200 rpm.The tubes were then briefly centrifuged and placed on a magnetic separator.The supernatant was transferred into a Phase Lock Gel Heavy tube, and NT2 buffer (250 μL) was added, followed by phenol:chloroform: isoamyl alcohol (400 μL) (Sigma-Aldrich).Tubes vortexing was done for 10 s and rotated for 10 min at 15000×g and 4°C.The aqueous phase (400 μL) was transferred into a new tube.Recovering RNA from the aqueous phase was done by ethanol precipitation (overnight) with Pellet Paint Co-Precipitant (Millipore) according to the manufacturer's instructions.Purified input and immune-precipitated RNA were used in RT-qPCR.Additionally, input, IP, and flow-through samples were analyzed using SDS-PAGE and western blotting.

Chromatin immunoprecipitation (ChIP)
ChIP assays were done by Magna ChIP Kit (Millipore, Bedford, MA, USA) according to the manufacturer's instructions.The cells were treated with formaldehyde to generate DNA-protein cross-links.Cells were then lysed by sonication to generate 200-300 bp chromatin fragments.Finally, the cell lysates were immune-precipitated with an E2F1 specific antibody (Millipore) or IgG as a control for immunoprecipitation.After recovering the precipitated chromatin DNA, RT-qPCR analysis was performed.

The luciferase activity assay
The JASPAR (https://jaspa r.ger0eg.net/)online database was used to predict the latent transcription factor E2F1 binding motif in the promoter region of MCF2L-AS1.The various sequence of fragments was synthesized and then inserted into the pGL3-basic vector (Promega, Madison, WI, USA).Luciferase reporter experiment was done by seeding HEK293T cells (3.5×10 4 per well) into 48 well plates and transfected with the various plasmids (100 ng/well) using Lipofectamine 3000 transfection reagent (Invitrogen) as per the manufacturer's guidelines.Cells were then harvested 48 h post-transfection.Quantification of relative luciferase units was finally done using Dual-Luciferase ® Reporter Assays (Promega, Fitchburg, WI, USA) following the manufacturer's guidelines on a GLOMAX 20/20 luminometer (Promega, Fitchburg, WI, USA).

Immunohistochemistry
The excised tumor tissues were fixed using PFA, embedding in paraffin, and the paraffin sections were routinely marked.After dewaxing and dehydration of the sections, extraction of the antigens was done by heating in a citric acid buffer solution, and the samples were incubated with the primary and secondary antibodies of Ki67 (1:1000, ab16667, Abcam).Finally, the samples were sealed with neutral glue.The prepared sections were Observed and photographed under a microscope (Nikon, Tokyo, Japan).

TdT-mediated dUTP-biotin nick end-labeling (TUNEL) staining
After dewaxing of paraffin sections with xylene and hydration with gradient ethanol, the samples were treated with proteinase K.Then, after incubation of the sections in 3% H 2 O 2 and 0.1% TritonX-100 and biotinylation, the sections were stained with Diaminobenzidine and hematoxylin following the TUNEL kit instructions (Roche, USA).After dehydration with gradient ethanol, the sections were treated with xylene and sealed with neutral resin.The samples were finally observed and photographed under a microscope (Nikon).

Data analysis
Statistical analysis of data was done using SPSS 19.0 (IBM, Chicago, IL, USA).The data were presented as mean ± standard deviation.Each experiment was repeated at least three times.Comparison of the differences between the two groups was made via Student's t-test while the difference in multiple groups was determined using done one-way analysis of variance.Estimation of the correlation between the two genes was investigated by Pearson correlation analysis.P<0.05 was considered statistically significant.

LncRNA MCF2L-AS1 is up-regulated in Gefitinib-resistant NSCLC patients
To understand the association of lncRNA MCF2L-AS1 and gefitinib-resistance in NSCLC, RT-qPCR was The results confirmed an elevated lncRNA MCF2L-AS1 mRNA expression in the Gefitinib-resistant compared to the Gefitinib sensitive tissues.Further RT-qPCR results also confirmed significantly increased CCND1 mRNA expression in the Gefitinib-resistant than in the Gefitinib-sensitive NSCLC tissues (Fig. 1A).Investigation of ELAVL1 expression through western blot confirmed significantly increased ELAVL1 protein in the Gefitinib resistant tissues compared to the sensitive group (Fig. 1B).Finally, the Pearson correlation analysis determined the correlation between CCND1 and lncRNA MCF2L-AS1.The results confirmed a positive correlation between lncRNA MCF2L-AS1 and CCND1 (Fig. 1C) (P<0.01).These results confirmed that lncRNA MCF2L-AS1 was up-regulated in Gefitinib-resistant NSCLC patients.

Knockdown of lncRNA MCF2L-AS1 inhibits Gefitinib resistance in NSCLC
In studying the effect of lncRNA MCF2L-AS1 on gefitinib resistance in LC, A549 cells were treated with increasing concentrations of Gefitinib to achieve clones of NSCLC Gefitinib-resistant cells (A549/GR).The cell viability determination confirmed that the IC50 value of Gefitinib-resistant NSCLC cells (A549/GR) was significantly increased than in the parent cells (A549) (Fig. 2A) (P<0.01).Next, RT-qPCR was used to assess the lncR-NA MCF2L-AS1 expression in BEAS-2B, A549, A549/ GR, and the cells transfected with sh-MCF2L-AS1-1, sh-MCF2L-AS1-2, or sh-NC.The results confirmed that lncRNA MCF2L-AS1 mRNA expression was significantly increased in A549/GR cells compared to parental and BEAS-2B cells (Fig. 2B) (P<0.01).Next, a CCK-8 assay was carried out to determine the cell viability after the knockdown of MCF2L-AS1-1.The results confirmed a significantly increased cell growth in A549/GR compared to A549 cells.However, the cell viability was significantly reduced following the knockdown of MCF2L-AS1-1 in the sh-MCF2L-AS1-1 and sh-MCF2L-AS1-2-transfected cells (Fig. 2C).The colony formation assay also confirmed increased colonies in the A549/GR cells compared to the parental 549 cells.However, the colony numbers were significantly reduced in the sh-MCF2L-AS1-1 and sh-MCF2L-AS1-2 compared to the sh-NC groups (Fig. 2D).Migration was also studied by transwell assay.The results confirmed significantly increased migration in the A549/GR cells compared to the parental A549 cells.However, the migration was significantly reduced in the sh-MCF2L-AS1-1 and sh-MCF2L-AS1-2 compared to the sh-NC group (Fig. 2E).Investigation of apoptosis through flow cytometry confirmed significantly reduced apoptosis in the A549/GR than the parental cells.After the knockdown, apoptosis was significantly increased in the sh-MCF2L-AS1-1 and sh-MCF2L-AS1-2 compared to the sh-NC cells (Fig. 2F) (P<0.01).Finally, a western blot was used to study the expressions of various apoptosis markers.The observations confirmed significantly increased expressions of the proliferation marker protein Ki67 and the anti-apoptotic protein Bcl-2 in the Gefitinib-resistant cells, while the pro-apoptosis proteins (Bax and cleaved caspase 3) were significantly reduced.These results were, however, reversed after knocking down MCF2L-AS1 (Fig. 2F) (P<0.01).According to these observations, knockdown of lncRNA MC-F2L-AS1 down-regulated Gefitinib resistance in NSCLC.

LncRNA MCF2L-AS1 enhances CCND1 mRNA stability through combining with ELAVL1
To investigate the mechanisms and association of ELAVL1 and CCND1, RT-qPCR was used to determine ELAVL1 and CCND1 expression in BEAS-2B, A549, and A549/GR cells.The observations confirmed significantly increased ELAVL1 and CCND1 mRNA expressions in A549 and A549/GR cells compared to the BEAS-2B cells (Fig. 3A).Assessment of the association of ELAVL1 with MCF2L-AS1 confirmed an interaction between lncRNA MCF2L-AS1 and ELAVL1 in Gefitinib-resistant LC cells (Fig. 3B).There was also a significantly increased enrichment of MCF2L-AS1 and CCND1 in the ELAVL1 than the control IgG (Fig. 3C) (P<0.01).Western blot was then used to determine the level of ELAVL1 in the A549/GR cells transfected with sh-ELAVL1, sh-NC, sh-MCF2L-AS1, and sh-NC.The results confirmed significantly reduced ELAVL1 expression in sh-ELAVL1 transfected cells compared to sh-NC cells.However, ELAVL1 expression was significantly increased in the sh-MCF2L-AS1 compared to its sh-NC counterpart (Fig. 3D) (P<0.01).Meanwhile, after ELAVL1 silencing, MCF2L-AS1 mRNA expression was not significantly changed, while CCND1 was greatly reduced compared to the sh-NC (Fig. 3E) (P<0.01).Finally, the cells were treated with actinomycin D (transcription inhibitor) to observe the impacts of MCF2L-AS1 and ELAVL1 on the stability of CCND1 mRNA.The results confirmed that knockdown of MCF2L-AS and ELAVL1 independently significantly reduced the half-life of CCND1 mRNA than the sh-NC (Fig. 3F) (P<0.01).In summary, the results clarified that lncRNA MCF2L-AS1 motivated CCND1 mRNA stability in LC Gefitinibresistant cells via combining with ELAVL1.

E2F1 transcription modulates lncRNA MCF2L-AS1
To further study the upstream modulatory mechanism of lncRNA MCF2L-AS1, the JASPAR bioinformatics tool was used to predict a possible binding.The results confirmed that the transcription factor E2F1 and MCF2L-AS1 promoter region had latent binding sites (Fig. 5A).The dual-luciferase assay confirmed significantly increased luciferase activity in the MCF2L-AS1-WT groups transfected with the transcription factor specificity protein 1 (SP1) (oe-SP1) compared to the oe-NC-transfected group.However, there was no significant change in the MCF2L-AS1-MUT group (Fig. 5B).The ChIP analysis also confirmed that the transcription factor E2F1 interacted with the MCF2L-AS1 promoter (Fig. 5C) (P<0.01).Finally, RT-qPCR was used to determine MCF2L-AS1 expression after the elevation of E2F1.The results confirmed a significantly reduced MCF2L-AS1 expression in the oe-SP1 compared to the oe-NC group (Fig. 5D) (P<0.01).These observations confirmed that E2F1 transcriptionally up-regulated the MCF2L-AS1 expression.

DISCUSSION
Gefitinib is a chemotherapeutic drug that disrupts EGFR-mediated signaling in the targeted cells.Gefitinib has been regarded as the standard first-line therapy for advanced NSCLC patients with EGFR-activating mutations.In over 50% of the cases of Gefitinib treatment failure, T790M mutation in EGFR is always implicated (Wang et al., 2020).Recent findings have proven that lncRNAs play crucial functions in malignant tumors pathogenesis by controlling genes associated with cell proliferation, apoptosis, metastasis, and radio-or chemoresistance (Lin et al., 2018).Nevertheless, the mechanisms involved in lncRNA-initiated Gefitinib resistance to therapy are yet to be clarified.
In the current investigation, we constructed a Gefitinib-resistant cell line referred to as A549/GR to assess the lncRNA MCF2L-AS1 and CCND1-mediated mechanisms of resistance to Gefitinib.This cell line has been associated with resistance to Gefitinib (Ma et al., 2017).We discovered that CCND1 is increased in Gefitinibresistant NSCLC patients and is positively linked with lncRNA MCF2L-AS1.Simultaneously, both ELAVL1 and CCND1 were enhanced in Gefitinib-resistant cells.It was confirmed that lncRNA MCF2L-AS1 contributes to CCND1 mRNA stability via combining with ELAVL1.Meanwhile, it was also verified that E2F1 transcriptionally up-regulates lncRNA MCF2L-AS1.Mechanically, it was originally reported that E2F1-transcriptionally modulated lncRNA MCF2L-AS1 elevated CCND1 mRNA stability through combining with ELAVL1 to motivate Gefitinib resistance in NSCLC.
The role of lncRNA in the progression of tumors and the establishment of drug resistance has been widely reported.LncRNA MCF2L-AS1 has been reported to be enhanced in LC tissues and cells and drives NSCLC stem cell-like characteristics via inhibiting miR-873-5p.
Additionally, lncRNA MCF2L-AS1 has been associated with colorectal cancer cells' progression and resistance to Oxaliplatin CRC's presence and progression (Cai et al., 2021).Nevertheless, there are currently no reports on the character and mechanism of lncRNA MCF2L-AS1 in NSCLC and Gefitinib resistance.The present research originally reports that lncRNA MCF2L-AS1 is up-regulated in gefitinib-resistant NSCLC patients and gefitinib-resistant cells and drives Gefitinib resistance in NSCLC in vivo and in vitro.
Previous studies have elucidated that lncRNA control downstream gene expression via combining with RBP to function in various illnesses.For instance, lncRNA OIP5-AS1 contributes to the stability of MEF2C mRNA via combining with HUR to accelerate myogenesis (Yang et al., 2020).Additionally, silencing SCAMP1-TV2 elevates the degradation of INSM1 mRNA via interacting with PUM2, thereby inhibiting the malignant biological behavior of BC cells (Tao et al., 2020).Here, we report an increased ELAVL1 expression in Gefitinib-resistant cells.This finding is in agreement with a previous report that associated NSCLC progression with the binding of HuR (ELAVL1) to CDC6 (Zhang et al., 2020).
Cell cycle progression is determined by a well-tuned cascade of signal transduction, whereby D-type cyclins have a crucial role.Human cyclin D proteins encode three D-type cyclins, including cyclin D1 (CCND1), cyclin D2 (CCND2), and cyclin D3 (CCND3) (Zhang et al., 2021).Each of these genes may interact with other proteins, hence activating cyclin-dependent kinase 4/6, which eventually phosphorylates retinoblastoma (Rb) protein and thus induces the transition of the G1/S phase cell cycle.In this work, CCND1 was shown to cooperate with lncRNA MCF2L-AS1 to induce Gefitinib resistance in NSCLC.This finding confirms the importance of CCND1 in NSCLC as was also previously reported where CCND1 was elevated and associated with the promotion of carcinogenesis, progression of the cell cycle, drug resistance, and metastasis in NSCLC (Liu et al., 2020).
In conclusion, this research fundamentally highlights the molecular mechanism of the lncRNA MCF2L-AS1 in inducing the growth and resistance of Gefitinib to NSCLC.The results manifest that lncRNA MCF2L-AS1, as an oncogene of NSCLC, controls CCDN1 via ELAVL1 to drive the growth of NSCLC cells and Gefitinib resistance.The novel findings of this investigation give a basis for targeting lncRNA MCF2L-AS1 and its downstream molecular axis as a possible alternative for developing effective and sensitive drug therapy against NSCLC.

Declarations
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.