A total of 11 patients (age, 48–62 years) with newly diagnosed cervical carcinoma, who received treatment at the First Affiliated Hospital of Harbin Medical University (Harbin, China) between January 2019 and August 2020, were enrolled in the present study. Ethical approval was obtained from the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (approval no. IRB-AF/SC-04/02.0; 11 November 2020); tumor samples were collected from the patients upon written informed consent. Human carcinoma and paracarcinoma tissues (1–5 cm away from carcinoma tissue) were collected for the culture of isolated primary cells.

The experimental protocols involving animals were approved by the Animal Ethical Care Committee of Qiqihar Medical University (Qiqihar, China; approval no. QMU-AECC-2020-43; 8 May 2020) and complied with the Guide for the Use and Care of Laboratory Animals published by the USA National Institutes of Health (NIH Publication No. 85–23, revised 1996) (27). BALB/c nude female mice (n=10; age, 6 weeks; weight, 18–22 g) were provided by the Animal Center of Qiqihar Medical University and were housed in a dedicated room with a 12-h dark/light cycle, controlled temperature (22±1°C) and constant humidity (55±5%); mice had free access to a standard diet and water. After 1 week of acclimatization, 2×10⁶ U14 mouse cervical carcinoma cells (BeNa Culture Collection; Beijing Beina Chunglian Institute of Biotechnology) were intraperitoneally (i.p.) injected in the C57BL/6 mice to prepare the U14 ascites tumor cells; cells were allowed to grow for 7 days, ensuring the abdominal ascites did not exceed 20% body weight (endpoint weight, 20.3–24.2 g). The abdominal ascites were collected as follows: i) Mice were anesthetized with 150 mg/kg tribromoethanol i.p. (cat. no. M2910; Nanjing Aibei Biotechnology Co., Ltd.) until loss of corneal reflex, muscle tightness and response to skin pinch; ii) the abdomen was swabbed with 75% alcohol, a small incision was made in the center of the skin overlying the peritoneal wall and the skin was firmly pulled to expose the peritoneal wall; iii) i) a 25 G needle was inserted into the peritoneal membrane, avoiding inserting needle into intestine or bladder, and 5 ml saline was injected into the peritoneal cavity; iv) the abdomen was massaged for approximately 10–15 sec, then the needle was withdrawn slowly; v) using one hand, the fluid was pushed to one side of the peritoneum and, with the other hand, the needle was inserted into the side of cavity with plenty of fluid and as much of the fluid as possible was withdrawn from the peritoneum; vi) the needle was removed, the contents of the syringe were dispensed into a centrifuge tube on ice, which was then centrifuged at 4°C, 183 × g for 5 min to collect cell pellet; vii) the U14 ascites tumor cells were resuspended and passaged for 10 days. The U14 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fishers Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere containing 5% CO₂. Following collection of ascetic fluid the mice were euthanized with pentobarbital sodium (100 mg/kg; i.p.).

The U14 cervical cells were subsequently divided into two groups and transfected as follows: i) Short hairpin (sh)RNA negative control (sh-Control) and ii) shRNA against lncRNA MALAT1 (sh-MALAT1). The effectiveness of transfection was confirmed as shown in Fig. S1. The tumor cell density was adjusted to 1×10⁷ cells/ml using normal saline. The right subaxillary skin of the mice was disinfected and inoculated with 0.2 ml U14 cervical cancer cell suspension for the establishment of 20 nude mice bearing U14 cervical tumor xenografts (n=10 mice/group). After 4 weeks, the maximum tumor diameter was 14 mm; tumors were excised after euthanasia using 100 mg/kg pentobarbital injection. The tumor volume was calculated as the follows: Tumor volume=(length × width²)/2. The tumor index included tumor diameter (range, 0.7–1.4 cm), volume (range. 0.1–0.726 cm³) and weight (range, 0.12–0.62 g).

Carcinoma and paracarcinoma tissues were isolated from four patients with cervical cancer and prepared for culture according to the following steps: i) Tissue block was placed into a pre-cooled sterile culture medium on ice at room temperature for 5 min; ii) tissue was were washed at room temperature with non-FBS-containing DMEM for 3 times under and cut; iii) tissue was enzymatically dissociated by the addition of 0.05% trypsin-EDTA for overnight at 4°C; iv) rinsed with 0.2% collagenase I for 10 min and collected the cell supernatant at room temperature v) repeated the step iv until none tissue coarse left; v) cell supernatant was filtered with 100 µm mesh filter and centrifuged at 183 × g for 10 min at 4°C, and the supernatant was discarded; viii) the cell pellet was resuspended in fresh culture medium; and ix) cells were incubated overnight at 37°C in a humidified air atmosphere containing 5% CO₂; x) Upon attainment of dense culture (after 2–3 weeks of primary culture), cells were passaged by mechanical dissociation via trituration with a Pasteur pipet, and transferred into separated culture medium containing plasks at room temperature (28). The culture medium used was DMEM with high glucose containing 5% FBS (Gibco; Thermo Fisher Scientific, Inc.), 1% antibiotics and HEPES (10 mM). The viability of primary cervical cancer cells was evaluated by trypan blue assay; the number of live cells was >70%.

The HeLa cervical cancer cell line was obtained from Shanghai Institutes for Biological Sciences and was cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) supplied with 10% FBS and 1% penicillin/streptomycin (100 µg/ml) in 5% CO₂ at 37°C.

Before treatment, cells (1×10⁶ cells/ml) were seeded and serum-starved with FBS-free restriction treatment medium overnight, according to the study by Qiu et al (29). LPS (Beijing Solarbio Science & Technology Co., Ltd.) dissolved in sterile deionized water was used at a final cell culture concentration of 1 µg/ml and cells were incubated for 24 h in 5% CO₂ at 37°C (28).

Isolated primary cervical carcinoma cells from human patients, U14 and HeLa cells were seeded into 6-well plates at a density of 2×10⁶ cells/well. When the confluence reached 80%, the cells were incubated in serum-free medium for 24 h before transfection. Transfection was performed using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Lentiviral particles (Shanghai GenePharma Co., Ltd.) containing the specific shRNAs (100 nM) for sh-Control (5′-GGGUGAACUCACGUCAGAA-3′), sh-MALAT1-1 (5′-CACAGGGAAAGCGAGUGGUUGGU-3′) and sh-MALAT1-2) (5′-GACAGGUAUCUCUUCGUUAUC-3′) were transduced into the cells for 12 h, and fresh medium was then changed for subsequent 48 h culture in 5% CO₂ at 37°C. The interference efficiency was detected by reverse transcription-quantitative PCR (RT-qPCR). Similarly, sirtuin 1 (SIRT1) shRNA (5′-TGCGGGAATCCAAAGGATAATTTTCAAGAGAAATTATCCTTTGGATTCCCGCTTTTTC-3′) and sh-Control (5′-TGCAACAAGATGAAGAGCACCAATTCAAGAGATGGTGCTCTTCATCTTGTTGTTTTTTC-3′) were also purchased from Shanghai GenePharma Co., Ltd. and used to transfect cells as aforementioned.

The MALAT1 overexpression plasmid (pcDNA3.1- MALAT1; NCBI accession: NR_002819.4, nucleotides 3,207-8,411 bp) was constructed as previously described (26,27). The MALAT1-vector plasmid and the empty vector plasmid were purchased from Shanghai GenePharma Co., Ltd. A total of 50 ng/ml plasmid was transfected into cells using Lipofectamine^® 2000 Transfection Reagent (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. miR-124 mimics (50 nM) and negative control (NC; 50 nM) were transfected into cells using X-treme GENE reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C. The sequences of the miR-124 mimics were as follows: Sense 5′-UAAGGCACGCGGUGAAUGCC-3′ and anti-sense 5′-CAUUCACCGCGUGCCUUAUU-3′. The sequences of the negative controls were as follows: Sense 5′-UUCUCCGAACGUGUCACGUTT-3′ and anti-sense 5′-ACGUGACACGUUCGGAGAATT-3′. After 6 h transfection, the medium was replaced with normal growth medium, and total RNA and protein were extracted at 24 h post-transfection.

Total RNA was extracted from cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA synthesis was performed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The levels of lncRNA MALAT1, miR-124, SIRT1, NLRP3, caspase-1 and ASC were determined by the SYBR Green I (Roche Diagnostics) incorporation method using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation for 30 sec at 95°C; followed by 40 cycles of 5 sec at 95°C and 30 sec at 60°C. GAPDH was used as the internal control for lncRNA MALAT1, SIRT1, NLRP3, caspase-1 and ASC; U6 was used as the internal control for miR-124. The primer sequences are shown in Table I. The relative expression levels were calculated using the 2^−ΔΔCq method (30).

Total protein for western blot analysis was extracted and dissolved in RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) with protease inhibitors (Sigma-Aldrich; Merck KGaA). The BCA method (Beyotime Institute of Biotechnology) was used to quantify the protein concentration. Proteins (80 µg/15 µl into each well) were separated by 10% SDS-PAGE and then transferred to a nitrocellulose membrane. The membranes were blocked with 5% skimmed milk for 2 h at room temperature, then incubated with primary antibodies against SIRT1 (1:1,000, Abcam, Catalog No. ab110304), NLRP3 (dilution at 1:1,000, Abcam, Catalog No. ab263899), caspase-1 (dilution at 1:1,000, Abcam, Catalog No. ab207802), cleaved caspase-1 (dilution at 1:1,000, Cell Signaling Technology, Inc. Catalog No. 89332), ASC (dilution at 1:1,000, Abcam, Catalog No. ab283684) and GAPDH (dilution at 1:500, OriGene Technologies, Inc. Catalog No. TA802519) at 4°C for 12 h. The membranes were then incubated with FITC-conjugated goat anti-rabbit (1:10,000, Invitrogen; Thermo Fisher Scientific, Inc. catalog No. A-11008) or goat anti-mouse secondary antibody (dilution at 1:10,000, Invitrogen; Thermo Fisher Scientific, Inc. Catalog No. A-11001) was incubated for 2 h at room temperature in the dark. Odyssey Infrared Imaging System (LI-COR Biosciences) and its analysis software (Odyssey version 3.0) were used to calculate the relative expression level compared with the internal control (GAPDH).

IL-1β and IL-18 in cell culture medium were determined using Human IL-1β (Interleukin 1 Beta) ELISA Kit (Cat. No. E-EL-H0149) and Human IL-18 (Interleukin 18) ELISA Kit (Cat. No. E-EL-H0253), respectively, from Elabscience Biotechnology, Inc., following the manufacturer's instructions.

Hela cells (seeding density of 70% were fixed with 4% paraformaldehyde solution (Beyotime Institute of Biotechnology) for 30 min at room temperature and permeabilized with 0.4% Triton X-100 (Beyotime Institute of Biotechnology; catalog No. P0096-100 ml) for 30 min at room temperature. After 2-h blocking with goat serum (Beyotime Institute of Biotechnology, Catalog No. C0265) at room temperature, cells were then incubated with anti-NLRP3 (1:100; Abcam, Catalog No. ab263899) antibody at 4°C for 24 h followed by incubation with FITC-labeled goat anti-rabbit immunoglobulin G (1:500; Invitrogen; Thermo Fisher Scientific, Inc. Catalog No. A16118) in the dark for 2 h at room temperature. DAPI (1 µg/ml; Beyotime Institute of Biotechnology) was used for nuclear staining; cells were incubated at room temperature in the dark. Conventional fluorescence microscopy (Nikon Corporation) was used for image capturing, and images were analyzed at ×200 magnification. Three complete and non-overlapping views were randomly selected, and the mean optical density under each view were measured as manufacture of Image-pro plus (version 6.0).

Cell death was assessed using a TUNEL assay kit (Beijing Solarbio Science & Technology Co., Ltd.) in accordance with the manufacturer's instructions. Cells were stained with DAPI for nuclear staining, and three complete and non-overlapping views were randomly selected for examination under an Olympus IX51 fluorescence microscope (Olympus Corporation) at ×100 magnification.

Cell lysate (200 ml) was used for caspase activity detection using Caspase-1 Activity Assay kit (cat. no. C1101; Beyotime Institute of Biotechnology), Caspase-4 Activity Assay kit (cat. no. C1121; Beyotime Institute of Biotechnology) and Caspase-5 Colorimetric Analysis kit (cat. no. 55R-1283; AmyJet Scientific Inc.). Briefly, cells were collected with 50 µl of ice-cold lysis buffer for 1 h. Then, caspase activities were detected by spectrophotometer at A405 nm according to the manufacturer's instructions.

Online bioinformatics tools for lncRNAs, including LncACTdb (http://www.bio-bigdata.net/LncACTdb; version 3.0/Sep. 2021), NPInter (http://bigdata.ibp.ac.cn/npinter; version 4.0/Sep. 2019) and LncBase (http://www.microrna.gr/LncBase; version 3.0/2019) were used to predict putative target miRNAs of lncRNA MALAT1. A Venn diagram was used to search for targets withing the intersections among these databases. Bioinformatic tools for miRNAs and its potential mRNA targets were also used, including starBase (https://starbase.sysu.edu.cn/starbase2/index.php; version 2.0/Sep. 2013), TargetScan (http://www.targetscan.org; version 7.2/March 2018) and miRDB (http://mirdb.org/miRDB; version 6.0/June 2019).

For predicting the downstream target mRNAs of selected miRNAs, first a Venn diagram was used to identify possible candidates among the aforementioned databases. Furthermore, a literature search of the PubMed database (PMID: 33040786; PMID: 29844574; PMID: 33061806; PMID: 32232409; PMID: 34369271) with the key words ‘pyroptosis and cervical cancer’ was used to review possible regulators reported in previous studies. Finally, SIRT1 from bioinformatic databases and literature searches was identified as a potential target of miR-124 and selected for further examination in the present study.

The binding of lncRNA MALAT1 to NLRP3 was detected with a Magna RIP™ RNA-Binding Protein Immunoprecipitation kit (EMD Millipore corp., 17–700). Hela cells (1–3×10⁷ cells from manufacture, were washed with pre-cooled PBS and lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology) in an ice bath for 5 min. The supernatant was collected after centrifugation at 20,000 × g for 10 min at 4°C. An aliquot of the cell extract was used as the input. Cell extract was incubated with rabbit antibody against NLRP3 (1:100; Abcam) at room temperature for 30 min for co-precipitation. A rabbit anti-human IgG (cat. no. ab109489; 1:100; Abcam) was used as the negative control with a volume of 1 ml at room temperature for 30 min. Samples were shaken to the bottom and placed on a magnetic seat in an ice bath, and the supernatant was discarded after 1 min. Then, 1 ml NT-2 was added and the beads were shaken vigorously to mix. After 5-time centrifugate (5,000 × g, 15 sec at 4°C) and washing with RIP Wash Buffer, the precipitate was collected and used as the sample. RNA was extracted after digestion by proteinase K at 55°C for subsequent analysis. lncRNA MALAT1 content in immunoprecipitated RNA was quantified by RT-qPCR and present as relative RNA level compared with IgG IP.

After transfection and/or LPS treatment, HeLa cells were incubated for 24 h in a standard CO₂ incubator, and images were obtained with an inverted phase contrast microscope (Olympus Corporation) to observe and record the morphology of the cells.

Primary isolated cervical carcinoma cells from human patients (10⁴ cells/well) and HeLa cells (10⁴ cells/well) were seeded in a 96-well plate. After 24 h transfection and/or LPS treatment, Cell Counting Kit-8 reagent (Nanjing Jiancheng Bioengineering Institute) was added each well (10 µl/well) and incubated for 4 h at 37°C. The optical density value at 450 nm was determined with a Bio-Rad 680 microplate reader (Bio-Rad Laboratories, Inc.).

Trypan blue staining (Beijing Solarbio Science & Technology Co., Ltd.) was used for cell counting. Primary isolated cervical carcinoma cells (and Hela cells (both 1×10⁶ cells/ml) were resuspended and mixed with 0.1% trypan blue at room temperature for 5 min. Cells were counted using a Countless II FL automated cell counter (Invitrogen; Thermo Fisher Scientific, Inc.) and the ratio of living cells vs. dead cells was analyzed.

Data were analyzed using GraphPad Prism 5.0 software (GraphPad Software, Inc.). Data are presented as the mean ± standard error of the mean. All experiments were repeated three times. Statistical comparisons between two groups were performed by Student's t-test (paired t-test for the matched sample data and unpaired t-test for comparisons of two groups). One- or two-way ANOVA followed by Tukey's post hoc test was used for multiple comparisons. Association analyses were assessed by the linear regression index (r²) (31). P<0.05 was considered to indicate a statistically significant difference.

To clarify the function of lncRNA MALAT1 in cervical carcinoma, insight into its biological process from Gene Ontology (GO) analysis was gained by LncACTdb database. The top two GO term enrichment for lncRNA MALAT1 are ‘regulation of cell proliferation’ and ‘regulation of cell cycle’ (Fig. 1A), which may be associated with cervical tumor progression. Thus, the present study aimed to determine the effect of lncRNA MALAT1 on cervical cancer cell viability. Initially, primary cells were isolated from paracarcinoma and carcinoma tissues of patients with cervical cancer and cultures were established to compare the differential expression of lncRNA MALAT1 in vitro. The expression level of lncRNA MALAT1 was significantly increased in cervical carcinoma cells compared with the paracarcinoma cells (Fig. 1B). Additionally, a positive association was found between lncRNA MALAT1 levels and cell viability, as well as with live cell number, whereas the opposite was observed with the number of dead cells (Fig. 1C and D). These results suggested that lncRNA MALAT1 is positively associated with cervical cancer cell viability, and the loss of lncRNA MALAT1 may exert an inhibitory effect on the development of cervical cancer. To demonstrate this hypothesis, lncRNA MALAT1 was knocked down with sh-MALAT1 in isolated primary cervical carcinoma cells and in U14 cervical cancer cells (Fig. S1A and B, respectively). Knockdown of lncRNA MALAT1 induced a marked increase in primary cervical carcinoma cell death in vitro (Fig. 1E) and also led to a decrease in in vivo tumor diameter, volume, and weight (Fig. 1F-H). These results suggested that specific knockdown of lncRNA MALAT1 may function as a suppressor of cervical cancer cells through mechanisms that remain to be identified.

In addition, lncRNA MALAT1 was overexpressed in isolated primary cervical carcinoma cells for in-depth exploration in vitro (Fig. S1C). Increased lncRNA MALAT1 expression promoted cell viability of cervical cancer cells with no significant change in cell deaths (Fig. S2), which suggested the potential of lncRNA MALAT1 overexpression on promoting cervical cancer growth.

Subsequent in vitro experiments were performed to further investigate the aforementioned effect of MALAT1 knockdown on cell death in HeLa cells (Fig. S1D). After transfection with sh-MALAT1, HeLa cervical cancer cells exhibited an increase in the number of TUNEL-positive cells (Fig. 2A and B) and increased levels of IL-18 and IL-1β in the culture medium (Fig. 2C). Thus, it was hypothesized that pyroptosis may have occurred in HeLa cells following knockdown of lncRNA MALAT1 expression. Thus, the present study examined the activity of pyroptosis-related caspases, the expression levels of NLRP3, caspase-1 and ASC, and examined changes in cell morphology following sh-MALAT1 transfection. It was observed that downregulated lncRNA MALAT1 markedly increased caspase-1 activity (Fig. 2D), increased protein (Fig. 2E and F) and mRNA (Fig. 2G) expression levels of NLRP3, cleaved caspase-1 and ASC (Fig. 2E-G), and increased the number of cells exhibiting morphological evidence of membrane rupture (Fig. 2H). These changes may eventually lead cervical cancer cells to undergo pyroptosis.

Taken together, these results indicated that the reduction of lncRNA MALAT1 expression may inhibit cervical cancer cell viability by inducing pyroptosis.

To explore the effects of lncRNA MALAT1 on cervical cancer cells against pyroptosis, lncRNA MALAT1 was overexpressed in LPS-treated HeLa cells; confirmation of successful overexpression vector transfection was first confirmed in untreated HeLa cells (Fig. S1E). LPS treatment is generally used as a pyroptosis stimulus, which induces typical pyroptotic characteristics, including cell morphology (inflammasome-mediated membrane holes and cell swelling), decreased cell viability and increased levels of inflammatory factors (IL-1β and IL-18), as well as increased expression levels of NLRP3, cleaved caspase-1 and ASC upregulation according to previous studies (29,30). In the current study, lncRNA MALAT1 was overexpressed in LPS-treated cells, which led to a noticeable decrease in the number of swollen cells (Fig. 3A), an increase in HeLa cell viability (Fig. 3B) and downregulated levels of IL-1β and IL-18 (Fig. 3C), in addition to decreased protein expression levels of NLRP3, cleaved caspase-1 and ASC (Fig. 3D-F). The results suggested that lncRNA MALAT1 may block the development of pyroptosis in LPS-treated HeLa cells.

Based on these data, it was concluded that lncRNA MALAT1 may affect the progression of cervical cancer, at least in part through the regulation of pyroptosis. However, the mechanism by which lncRNA MALAT1 initiates pyroptosis remains unclear, including whether lncRNA MALAT1 directly interacts with pyroptotic factors, such as NLRP3, or interferes with them at the post-transcriptional level. NLRP3 is generally recognized to initiate pyroptosis (31), thus, it was proposed that lncRNA MALAT1 may directly affect the expression of NLRP3 as a transcriptional factor. Immunofluorescent staining of NLRP3 showed a downregulated expression in MALAT1 overexpressed cell under the condition of LPS treatment (Fig. 3G and H). RIP assays were performed in HeLa cells to verify whether there is a direct interaction between lncRNA MALAT1 and NLRP3. Unexpectedly, no significant difference was identified in the RIP analysis for an lncRNA MALAT1/NLRP3 regulatory axis (Fig. 3I).

To explore the downstream target of lncRNA MALAT1 in regulating the pyroptotic process in cervical cancer cells, bioinformatics tools, Venn diagrams and literature searches using the key words ‘pyroptosis and cervical cancer’ were used. According to the results from three bioinformatics predictive databases (LncACTdb, NPInter and LncBase) and a Venn diagram, which was used to search for the intersection among the results of these databases, five potential targets of lncRNA MALAT1 were identified, including miR-25-3p, miR-92b-3p, miR96-5p, miR-124 and miR-1 (Fig. 4A).

RT-qPCR was then performed to determine miRNA expression levels and to investigate if these molecules serve a role in lncRNA MALAT1-regulated pyroptosis in HeLa cells. The level of miR-124 exhibited the most significant alteration after LPS treatment (Fig. 4B); in addition, knockdown and overexpression of lncRNA MALAT1 resulted in significant increase and decrease of miR-124 expression levels, respectively (Fig. 4C). These data suggested that miR-124 may be a downstream target of lncRNA MALAT1, particularly in pyroptosis.

Apart from lncRNA MALAT1, other putative targets of miR-124 were investigated; three bioinformatics prediction tools (starBase, TargetScan and miRDB) were used alongside a Venn diagram. A total of 82 potential targets of miR-124 were identified with the result of PubMed database search (using the key words ‘pyroptosis and cervical cancer’). A previous study demonstrated the involvement of SIRT1 during pyroptosis, suggesting SIRT1 could therefore be a target for the effective treatment of cervical cancer (32) (Fig. S3A). Therefore, SIRT1 was selected as a potential target of miR-124 (Fig. S3).

To investigate the potential target of lncRNA MALAT1 during pyroptosis, subsequent experiments were divided into two parts: i) Verification the direct interaction between miR-124 and SIRT1, and ii) SIRT1 mediating the function of lncRNA MALAT1 in LPS-treated HeLa cells.

For the first part, the aim was to verify whether miR-124 mediated the effects of lncRNA MALAT1 on pyroptosis. Thus, cells were co-transfected with lncRNA MALAT1 overexpression vector with miR-124 mimics, attempting to disturb the function of lncRNA MALAT1. After the determination of transfection efficiency of miR-124 mimics in non-treated HeLa cells (Fig. S4) and lncRNA MALAT1 co-transfected HeLa cells (Fig. 5A), overexpression of miR-124, compared to its NC group, led to a decrease in the level of lncRNA MALAT1 expression (Fig. 5B). Additionally, the overexpression of miR-124 also concurrently decreased the activity of HeLa cells (Fig. 5C). Our previous findings suggested that lncRNA MALAT1 could ‘protect’ HeLa cells against pyroptotic damage induced by LPS. However, in this context, we discovered that miR-124 counteracted this ability of lncRNA MALAT1, evidenced by a subsequent increase in the expression of inflammatory factors IL-1β and IL-18 (Fig. 5D), promoted death of HeLa cells, and reduced number of viable HeLa cells (Fig. 5E and F). Further examination of pyroptotic factors NLRP3, ASC, and cleaved caspase 1 confirmed that miR-124 inhibited lncRNA MALAT1's ability to protect HeLa cells against pyroptosis induced by LPS stimulation (Fig. 5G-I).

For the second part, experiments were conducted to verify whether SIRT1 underlies the function of lncRNA MALAT1 on pyroptosis. HeLa cells were co-transfected with lncRNA MALAT1 overexpression vector and sh-SIRT1 or its NC (Figs. S5A and 5B). Transfection of sh-SIRT1 functioned as an antagonist against lncRNA MALAT1, abolishing the protective effects of lncRNA MALAT1 in HeLa cells from LPS, including reducing viable HeLa cells (Fig. 6C and D), increasing death number of HeLa cells (Fig. 6E). Transfection of sh-SIRT1 elevated inflammatory factors IL-1β and IL-18 (Fig. 6F) and pyroptotic factors NLRP3, ASC, and cleaved caspase 1 (Fig. 6G-I). Taken together, these results demonstrated the regulatory effect of the lncRNA MALAT1/miR-124/SIRT1 axis on cervical cancer cell pyroptosis.