Long non-coding RNA MALAT1 facilitates the tumorigenesis, invasion and glycolysis of multiple myeloma via miR-1271-5p/SOX13 axis

Serum samples were collected from MM patients (n = 30) and healthy donors (n = 30) in the First Affiliated Hospital of Zhengzhou University. After centrifugation at 3000×g for 15 min, the supernatant serum was transferred into an RNase-free Eppendorf tube and immediately conserved in − 80 °C ultra-low temperature refrigerator for use. All patients and healthy subjects were completely informed and signed the written informed consents. This research got authorization from the Ethic Committee of the First Affiliated Hospital of Zhengzhou University.

Human normal plasma cell line (nPCs) was acquired from Fenghui Biotechnology Co., Ltd (Changsha, China) and MM cell lines (NCI-H929 and OPM-2) were bought from Xiangf Biosciences Co., Ltd. (Shanghai, China). All cells were cultivated in Roswell Park Memorial Institute-1640 (RPMI-1640; Corning Life Sciences, Corning, NY, USA) added with 10% fetal bovine serum (FBS; Serapro, Naila, Germany), and 1% penicillin–streptomycin mixed solution (Transgen, Beijing, China) in humidified air with 5% CO₂ at 37 °C.

The qRT-PCR assay was administrated applying SYBR Green PCR Kit (Applied Biosystems, Foster City, CA, USA) through the ABI Prism 7500 sequence detection system (Applied Biosystems) as previously reported (Jin et al. 2019). The relative expression levels were analyzed via the 2^−∆∆Ct approach (Livak and Schmittgen 2001) using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for normalizing MALAT1 or SOX13 and U6 as the internal reference of miR-1271-5p. Primers sequences used in this report were as follows: MALAT1 (Forward: 5′-AAAGCAAGGTCTCCCCACAA-3′, Reverse: 5′-GGTCTGTGCTAGATCAAAAGGCA-3′); miR-1271-5p (Forward: 5′-CAGCACTTGGCACCTAGCA-3′, Reverse: 5′-TATGGTTGTTCTCCTCTCTGTCTC-3′); SOX13 (Forward: 5′-CTGGACTTCAACCGAAATTTGA-3′, Reverse: 5′-GTTCCTTCCTAGAAACCTCTCC-3′); GAPDH (Forward: 5′-GCATCCTGGGCTACACTG-3′, Reverse: 5′-TGGTCGTTGAGGGCAAT-3′); U6 (Forward: 5′-GCTTCGGCAGCACATATACTAAAAT-3′, Reverse: 5′-CGCTTCACGAATTTGCGTGTCAT-3′).

Cell transient transfection was conducted using Lipofectamine3000 reagent (Invitrogen, Carlsbad, CA, USA) complying with the manufacturer’s instruction. Small interfering RNA (siRNA) against MALAT1 (si-MALAT1), miR-1271-5p mimic and inhibitor (miR-1271-5p and anti-miR-1271-5p) and respective negative controls (si-NC, miR-NC and anti-miR-NC) were purchased from Ribobio (Guangzhou, China). The sequence of SOX13 was cloned into pcDNA vector (Invitrogen) to construct the overexpression vector pcDNA-SOX13 (SOX13) with pcDNA as the negative control.

Firstly, a total number of 2 × 10³ MM cells were plated into 96-well plates overnight. Following transfection for 24 h, 48 h and 72 h, 20 µL MTT (Sangon Biotech, Shanghai, China) were pipetted into the wells, mixing with cells for another 4 h. Next, the supernatant was removed and cells were incubated with dimethyl sulfoxide (DMSO; Sangon Biotech) with 200 µL per well. Ten minutes later, the optical density (OD) value at 490 nm was determined by a microplate reader, which represented the viability of MM cells. The un-transfected cells were used as the transfection control group.

Transfected MM cells were washed by pre-cooled phosphate buffer solution (PBS; Corning Life Sciences) and centrifugally harvested at 2000 rpm for 10 min. Cell pellets were resuspended in 500 μL Binding buffer, then mixed with respective 5 μL Annexin V-fluorescein isothiocyanate (Annexin V-FITC), and propidium iodide (PI) (BD Biosciences, San Diego, CA, USA) for 20 min in the dark. Eventually, apoptotic cells could be distinguished through a flow cytometer (BD Biosciences) and apoptosis rate was calculated.

At the beginning, the low side of the upper chamber of the transwell chamber (Corning Life Sciences) was firstly enveloped with matrigel (Corning Life Sciences). Then cell suspension in serum-free medium was pipetted into the upper chamber, accompanying with the adding of RIPM-1640 containing 10% FBS into the lower chamber. Subsequently, cells were fixated using methanol (Sangon Biotech) and colored with crystal violet (Sangon Biotech) 48 h later. Ultimately, invaded cells were counted through a microscope after uninvaded cells were wiped off with a wet cotton swab.

Cell supernatant was collected post-transfection for 48 h, then glucose and lactate levels were severally examined via Glucose Uptake Colorimetric Assay Kit and Lactate Colorimetric Assay Kit (Biovision, San Francisco, CA, USA) in line with the producers’ directions. ATP/ADP ratio was measured by ApoSENSOR™ ADP/ATP Ratio Bioluminescence Assay Kit (Biovision). Briefly, 1 × 10⁴ cells were inoculated into the luminometer plate and Nucleotide Releasing Buffer was added. Then the values of wells were read at 1 min (A) and 10 min (B) after adding with 1 μL ATP Monitoring Enzyme. Sample values were recorded (C) following the addition of ADP Converting Enzyme. ATP/ADP ratio was calculated according to the formula: A/(C − B).

After the extraction of proteins from serums and cells via radioimmunoprecipitation assay (RIPA) lysis solution (Beyotime, Shanghai, China), 60 μg quantified proteins were isolated on 10% sodium dodecyl sulfate–polyacrylamide gel for 2 h, followed by the transferring of proteins onto the polyvinylidene fluoride membranes (Beyotime). Then, 5% skim milk (Beyotime) was applied for blocking membranes for 3 h, which were incubated with primary antibodies from Abcam (Cambridge, United Kingdom): anti-Hexokinase 2 (anti-HK2; ab209847, 1:1000), anti-Glucose Transporter 1 (anti-GLUT1; ab115730, 1:1000), anti-SOX13 (ab198921, 1:1000) and anti-GAPDH (ab9485, 1:3000) overnight at 4 °C Following the incubation of secondary antibody (Abcam, ab205718, 1:5000) for 1 h, the immunoreactive signals were assayed through enhanced chemiluminescence reagent (Abcam), and the gray levels were analyzed via the Image J software (NIH, Bethesda, MD, USA).

The bioinformatics analysis was executed by Starbase v2.0. Dual-luciferase reporter assay was used for validating the combination between miR-1271-5p and MALAT1 or SOX13. The sequences of wild-type (WT) MALAT1 and 3′UTR of SOX13 (with the binding sites for miR-1271-5p) were inserted into pmirGLO vector (Promega, Madison, WI, USA) to form WT luciferase reporters WT-MALAT1 and SOX13 3′UTR-WT. After the complementary sites for miR-1271-5p in MALAT1 and SOX13 3′UTR were mutated, the mutant-type (MUT) reporters MUT-MALAT1 and SOX13 3′UTR-MUT were constructed. Afterwards, MM cells were respectively co-transfected with above reporters and miR-1271-5p or miR-NC. Finally, the dual-luciferase reporter assay system (Promega) was employed for the detection of luciferase activities from cell lysates. Firefly luciferase activity was standardized by renilla activity and the ratio of firefly/renilla was considered as the relative luciferase activity.

Short hairpin RNA (shRNA) against MALAT1 (sh-MALAT1) was synthesized by GenePharma (Shanghai, China) to establish stably transfected cells via lentivirus mediation with sh-NC as the negative control. After the purchase of BALB/c nude mice (6-week-old) from Vital River Laboratory Animal Technology (Beijing, China), xenograft tumor model was established through the subcutaneous injection into mice with NCI-H929 cells (5 × 10⁶ cells/mice) stably expressed sh-MALAT1 or sh-NC (seven mice per group). Tumor volume (length × width² × 0.5) was recorded every 4 days post-injection 8 days. After 28 days, tumor tissues were excised from euthanized mice and weighed. And the levels of MALAT1, miR-1271-5p, and SOX13 in tissues were measured. This animal experiment was ratified by the Animal Ethics Committee of the First Affiliated Hospital of Zhengzhou University.

Assays in this study were implemented by three repetitive parallels and data were expressed as mean ± standard deviation (SD). SPSS 19.0 was used for data analysis and statistical processing. Graphing was performed in GraphPad Prism 7. The linear relationship was analyzed via Spearman’s correlation coefficient. The difference comparison was analyzed by Student’s t test or one-way analysis of variance (ANOVA) followed by Tukey’s test. Difference was defined as statistically significant with P < 0.05.

We first assayed the expression of MALAT1 in MM by qRT-PCR. Compared with normal serum samples, MALAT1 level was significantly increased in MM serums (Fig. 1a). Also, MALAT1 was expressed higher in NCI-H929 and OPM-2 cells than that in normal nPCs cells (Fig. 1b). Then we found the inverse down-regulation of miR-1271-5p in MM serums (Fig. 1c) and cells (Fig. 1d) by comparison with normal serums and cells. After the analysis of Spearman’s correlation coefficient, a negative relation (r =  − 0.796, P < 0.001) between MALAT1 and miR-1271-5p was exhibited in MM serums (Fig. 1e). These results indicated MALAT1 was enriched while miR-1271-5p was decreased in MM.

To investigate the potential function of MALAT1 in MM, si-MALAT1 was synthesized to disturb the expression of MALAT1 and the interference was successful in NCI-H929 and OPM-2 cells compared to si-NC and control groups (Fig. 2a). Then we applied further experiments to assess cellular processes of MMC cells. MTT showed that the OD value of si-MALAT1 group was obviously lower than that of si-NC group in NCI-H929 (Fig. 2b) and OPM-2 (Fig. 2c) cells. si-MALAT1 transfection caused the promotion of apoptosis rate (Fig. 2d) but the lessening of invaded cells number (Fig. 2e). Moreover, glycolysis process was evaluated. The glucose consumption (Fig. 2f) and lactate production (Fig. 2g) levels were fewer in NCI-H929 and OPM-2 cells transfected with si-MALAT1 compared to the si-NC group. The ATP/ADP ratio also declined after knockdown of MALAT1 (Fig. 2h). Meanwhile, the glycolysis-associated enzymes were examined by Western blot, in which the protein levels of HK2 and GLUT1 were notably repressed following si-MALAT1 transfection (Fig. 2i, j), verifying the inhibition of cellular glycolysis by MALAT1 knockdown again. In short, MALAT1 down-regulation repressed cell viability, invasion, and glycolysis but promoted apoptosis in MM cells.

LncRNAs combine with miRNAs generally (Peng et al. 2018; Su et al. 2019; Wang et al. 2017). After the bioinformatics analysis by Starbase v2.0, we found MALAT1 contained the combinative sites for miR-1271-5p (Fig. 3a), speculating that miR-1271-5p might be a target of MALAT1. Then, miR-1271-5p transfection strikingly decreased the luciferase activity of WT-MALAT1 group but not in MUT-MALAT1 group in NCI-H929 and OPM-2 cells (Fig. 3b, c), affirming the combination between MALAT1 and miR-1271-5p. After MALAT1 was successfully knocked down and overexpressed (Fig. 3d), miR-1271-5p expression was assayed. The results manifested MALAT1 knockdown heightened the miR-1271-5p expression but MALAT1 overexpression displayed the contrary phenomenon (Fig. 3e). To explore the regulatory relation between MALAT1 and miR-1271-5p in MM, NCI-H929 and OPM-2 cells were transfected with si-MALAT1, si-MALAT1 + anti-miR-1271-5p or matched controls. As shown in Fig. 3f, miR-1271-5p inhibition rescued the rising of miR-1271-5p induced by MALAT1 knockdown. MTT proved that si-MALAT1-induced inhibition of cell viability was ameliorated after miR-1271-5p was down-regulated (Fig. 3g, h) a The stimulative effect on cell apoptosis (Fig. 3i) and inhibitory effect on cell invasion (Fig. 3j) caused by si-MALAT1 were alleviated by miR-1271-5p inhibitor. Also, the down-regulation of miR-1271-5p reverted the suppression of glucose consumption (Fig. 3k), lactate production, (Fig. 3l) and ATP/ADP ratio (Fig. 3m), as well as the restraint of HK2 and GLUT1 protein levels (Fig. 3n, o) in NCI-H929 and OPM-2 cells transfected with si-MALAT1. All in all, MALAT1 targeted miR-1271-5p and miR-1271-5p down-regulation relieved the effects of MALAT1 knockdown on MM cells.

miRNAs usually interact with the 3′UTR of downstream gene (Li et al. 2018; Wei et al. 2017, 2019). Starbase v2.0 revealed that SOX13 had the mutual binding sites for miR-1271-5p (Fig. 4a), implying that SOX13 might be a target candidate of miR-1271-5p. Dual-luciferase reporter assay further validated that the luciferase activity in SOX13 3′UTR-WT group was descended by miR-1271-5p, but there was no evident change in SOX13 3′UTR-MUT group (Fig. 4b, c). Subsequently, we found the up-regulation of SOX13 mRNA and protein levels in MM serums (Fig. 4d, e) and cells (Fig. 4f, g) in comparison to normal serums and cells aThere was a negative association (r = − 0.6404, P < 0.001) between miR-1271-5p and SOX13 in MM serum samples (Fig. 4h). QRT-PCR analysis showed the inhibitory effect of anti-miR-1271-5p and overexpressed effect of miR-1271-5p on miR-1271-5p expression were great (Fig. 4i). miR-1271-5p inhibitor elevated SOX13 protein level but miR-1271-5p overexpression triggered the down-regulation of SOX13 expression (Fig. 4j). Therefore, miR-1271-5p negatively regulated SOX13 level.

The regulatory relation between miR-1271-5p and SOX13 was investigated after transfection with miR-1271-5p, miR-1271-5p + SOX13 or respective controls. Firstly, we observed that the inhibition of SOX13 protein expression induced by miR-1271-5p was abated through SOX13 up-regulation (Fig. 5a). Then MTT assay presented that the intervention of miR-1271-5p evoked the refrainment of cell viability, which was abolished by the overexpression of SOX13 (Fig. 5b, c). Additionally, transfection of SOX13 abrogated the promotion of cell apoptosis (Fig. 5d) and the repression of invasion (Fig. 5e) caused by miR-1271-5p. Also, miR-1271-5p led to the reduction of glucose consumption (Fig. 5f), lactate production (Fig. 5g), ATP/ADP ratio, (Fig. 5h) and the protein levels of HK2 and GLUT1 (Fig. 5i, j), whereas ectopic overexpression of SOX13 reverted these effects. Above results demonstrated that SOX13 overexpression reversed the miR-1271-5p-induced inhibition of cell viability, invasion and glycolysis but the enhancement of apoptosis in MM cells.

We transfected si-MALAT1, si-MALAT1 + anti-miR-1271-5p or relative controls into NCI-H929 and OPM-2 cells for the purpose of studying the relation between MALAT1 and SOX13. As Fig. 6a, b described, transfection of si-MALAT1 resulted in the restraint of SOX13 mRNA and protein levels, which was counteracted following the inhibition of miR-1271-5p expression in NCI-H929 and OPM-2 cells. Therefore, MALAT1 could regulate the level of SOX13 through the indirect interaction with miR-1271-5p.

The xenograft tumor model was established to ascertain the impact of MALAT1 on MM in vivo. Through the measurement and record, we found that tumor volume (Fig. 7a) and weight (Fig. 7b) declined in sh-MALAT1 group contrasted to sh-NC group. Moreover, knockdown of MALAT1 caused the down-regulation of MALAT1 and SOX13, as well as the increase of miR-1271-5p expression in excised tissues (Fig. 7c). After the analysis of Western blot, the SOX13 protein level was lower in sh-MALAT1 group than that in sh-NC group (Fig. 7d). These data manifested that MALAT1 knockdown inhibited MM growth by miR-1271-5p/SOX13 axis in vivo.