4.1 Cell lines and culture

PC9 lines were obtained from the Tumor Cell Bank of the Chinese Academy of Medical Science (Shanghai, China). Cells were cultivated in RPMI 1640 medium (Hyclon, USA), which was supplemented with 10% fetal bovine serum (Biological Industries), ampicillin (100 U/mL), and streptomycin (100 mg/mL) at 37 ℃ in humidifified air with 5% CO2. Erlotinib-resistant LUAD cells (PC9ER) was established from parental PC9 cells and was preserved in 0.1 μmol/L Erlotinib (fifinal concentration).

4.2 Cell transfection

SAM double vector lentivirus with MALAT-1 gene overexpression and Crispr/Cas9 gene editing lentivirus with MALAT-1 gene knockout were designed by Shanghai Jikai Company. SAM double vector lentivirus was transfected into PC9 cells to overexpress MALAT-1 in PC9 cells.

4.3 Construction of stable MALAT-1 knockout, overexpression and corresponding control cell lines

Cas9 lentivirus transfected PC9ER cells to silence MALAT-1 in PC9ER cells. Inoculate PC9 and PC9ER cells in logarithmic growth period into 6-well plate, and the inoculation amount is 5 × 104/mL, the amount of virus inoculated is the number of cells inoculated × The complex number of infection/virus titer was 100. After 12–16 h of infection, purinomycin was added to screen for one week, and then the selected cell line was continuously inoculated into the 6-well plate. After 24 h of culture, lentivirus carrying the target fragment was added. After 12 h of culture, the medium was changed to culture for 72 h. PC9 cells overexpressing MALAT-1 were added with neomycin to screen for another week. PC9ER cells silencing MALAT-1 were cultured for one week, and then observed and verified with fluorescence microscope.

4.4 Bioinformatics analysis

Online bioinformatics analysis found two binding sequences between MALAT1 and miR-125 (http://bicresources.jcbose.ac.in/zhumur/lncrbase). Possible targets of miR-125 are predicted using two bioinformatics prediction tools: TargetScan (http://www.targetscan.org/) and RNA22 (http://www.rnaseqblog.com/rna22-version-2-0-mirnamre-predictions/). MALAT-1 gene sequence comes from the University of California, Santa Cruz (UCSC) (http://genome.ucsc.edu/index.html).

4.5 Double luciferase report experiment analysis

Cells in logarithmic growth phase were made into cell suspension, counted, inoculated in 24 well culture plate (the number of cells was about 105, depending on the size of cell morphology), and cultured in 37 ℃, 5% CO2 incubator until the cell fusion degree reached about 60%. Transfection with ROCHE: X-tremegene HP transfection reagent (http://lifescience.roche.com/webapp/wcs/stores/servlet/ProductDisplay?partNumber = 3.5.3.18.1.10). Luciferase detection:

(http://cn.promega.com/resources/protocols/technical-manuals/0/dual-glo-luciferaseassay-system-protocol).

4.6 qRT-PCR analysis

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Use PrimeScript RT kit (Takara, Dalian, China) to reverse transcription according to the manufacturer's instructions. SYBR Prime Script RT-PCR kit (Takara) is used for qRT-PCR according to the manufacturer's scheme. All mRNA expression levels were calculated by using the 2 −ΔΔCt method and were normalized to GAPDH, U6 or β- Action expression. All measurements were performed three times independently.

4.7 Cell counting Kit-8 chemosensitivity assay

The cells were inoculated into 96 well plates, added with the corresponding concentration of erlotinib, labeled and put into the incubator for culture. Set the culture time of 24, 48 and 72 h according to the experimental needs, take out the 96 well plate, suck out the culture medium in the plate, prepare the CCK-8 detection reagent according to the concentration of 9:1, put it into the incubator for 1–2 h, take out the 96 well plate and put it into the microplate analyzer to detect the OD value of cells at 450 nm wavelength, calculate the cell vitality and IC50 through the OD value. All experimental procedures were repeated at least three times.

4.8 Immunofluorescence assay

Cells were seeded on glass coverslips in 6-well plates, fixed in 4% formaldehyde solution (Solarbio, Beijing, China), and permeabilized with 0.5% Triton X-100/PBS (Servicebio, Wuhan, China). Cells were sealed with 5% BSA/PBS (Servicebio, Wuhan, China) for 1 h at room temperature and then were incubated with primary antibodies (E-cadherin,N-cadherin,ZEB1 and Rab25) (Cell Signaling,American) at 4 ℃ overnight, followed by incubation with fluorescent dye-conjugated secondary antibody (Servicebio, Wuhan, China) for 1 h. Finally, the cells were stained with DAPI (Servicebio, Wuhan, China), and images were observed under a confocal microscope(TCS SP8 STED, Leica, Wetzlar, Germany). All experiments were conducted independently three times.

4.9 Flow cytometric analysis

Inoculate the exponential growth period into the culture bottle with or without Erlotinib for 24 h, collect the cell suspension in a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 min. The Binding Buffer solution was used to resuspension the cells for precipitation, and was mixed evenly, and the cell concentration was adjusted. Annexin-V (Becton, Dickinson and Company, USA) was added for apoptosis experiment, PI dye (Becton, Dickinson and Company, USA) was added for cell cycle experiment, cells were collected, fixed in 70% ethanol at 4 ℃ for 16 h, and negative control group was set. The results were detected and analyzed by flow cytometry within 1 h. All experiments were conducted independently three times[53].

4.10 Western blot assay

Protein lysates were separated on 10% SDS-PAGE gels and then transferred onto polyvinylidene difluoride membranes (Roche) via electrophoresis. Protein loading was assessed using mouse anti-GAPDH monoclonal antibody. The membranes were incubated with 10% skim milk in TBST at room temperature for 2 h, washed, and then probed overnight at 4 °C with antibodies against ZEB1, E-cadherin, N-cadherin, Rab25, and GAPDH. Following this, the membranes underwent incubation with a secondary antibody conjugated to horseradish peroxidase for 2 h at room temperature. Protein detection was carried out using an enhanced chemiluminescence system and visualized by exposure to X-ray film. All antibodies were procured from Abcam (Cell Signaling Technology, USA). Each sample was subjected to the experiment three times for validation.

4.11 Xenograft model

All BALB/c male nude mice (4–6 weeks old) were purchased from Beijing HFK Bioscience Co., Ltd (certificate number: SCXK (Jing) 2019–0008). The experimental animals were reared in a specific-pathogen-free (SPF) facility of the Key Laboratory of Basic Pharmacology of Ministry of Education, Zunyi Medical University, at a temperature of 20 ± 2 °C, with lighting from 8:00 in the morning to 8:00 in the evening and free access to food and water. The experimental protocol followed the Chinese Animal Protection and Welfare Guidelines and was approved by the Animal Ethics Committee of Zunyi Medical University (2015-07). Mice were randomly divided into four groups: A group (inject PC9 cells); B group (inject PC9 + Control cells); C group (inject PC9 + MALAT-1 cells); D group (inject PC9ER cells). The subcutaneous xenograft model was established by directly subcutaneously injecting 5 × 106 PC9,PC9 + Control,PC9 + MALAT1,PC9ER cells respectively (n = 10 mice/group) into the right flanks of BALB/c nude mice, which were previously suspended in NS. When the average tumor size reached approximately 100 mm3, Erlotinib (Shanghai Tengzhun Biotechnology Co., Ltd) was given through gavage at a concentration of 50 μg/mL and the control group of each were given commensurable 1% of Tween 80 (Solarbio, China); one dose was provided every day, with fourteen total doses. Mouse weight and tumor volume was measured using calipers every 2 days and was calculated by the following formula: (long diameter) × (short diameter)2/2. After two weeks of treatment, all mice were killed, and tumors were excised, paraffin embedded and formalin fixed or stored at − 80 °C until further real-time PCR and Western blot analysis. H&E staining and Ki-67 immunostaining analysis were conducted.

4.12 Histopathology

A portion of transplanted tumor was cut and fixed with 10% formaldehyde, dehydrated with ethanol gradients, and embedded in paraffin. Tumor tissue was cut into 4-µm slices with a tissue slicer (model: RM2245), and sections were deparaffinized with xylene, hydrated with gradient ethanol, stained with conventional HE, and observed under upright optical microscope (Olympus,Tokyo, Japan).

4.13 Immunohistochemistry assay (IHC)

The tumor tissue sections embedded in paraffin were deparaffinized and rehydrated before immunohistochemistry (IHC) processing. Antigen retrieval was performed using high pressure in a 0.01 M sodium citrate buffer solution. Following incubation with primary and secondary antibodies, the sections were treated with diaminobenzidine and counterstained using hematoxylin (Solarbio, China). Images were captured using a microscope at 200 × magnification (Nikon, E100). Primary antibodies used for IHC included anti-Ki67 (Bioss, USA) and anti-β-catenin (Bioss, USA).

4.14 Statistical analysis

SPSS v.21.0 software (IBM, Armonk, NY, USA) was applied for statistical analyses. Mean ± SD was used to present experimental results. Student’s t test or one-way ANOVA was used to detect the differences among groups. p values < 0.05 were considered statistically significant.