Jurkat-E6-1 human leukemia T lymphocytes (purchased from Bnbio, Inc., Beijing, China) were cultured in RPMI 1640 (Invitrogen, Carlsbad, California, United States) medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Biological Industries Ltd., Kibbutz Beit HaEmek, Israel) at 37°C in a humidified atmosphere containing 5% CO₂ LncRNA-ENST00000421645 was cloned into the GV502 plasmid (GeneChem Co., Ltd., Shanghai, China). The lentiviral vector and packaging vector were transfected into the packaging cell line 293T (ATCC) with Turbofect transfection reagent (Invitrogen) in accordance with the instruction manual. The lentivirus-containing medium was thus obtained. Then, T lymphocytes were transduced with the lentiviral vectors, and the stable cells were screened with puromycin (5 μg/mL). The lentiviral lncRNA-ENST00000421645 overexpression vector was transduced into T lymphocytes, and the resulting cells were called OE cells. The empty lentiviral vector was transduced into T lymphocytes, and the resulting cells were called NC cells. Kank1 small interfering RNA (siRNA) was transfected into OE cells, and the resulting cells were called OEsiRNA Kank1 cells. OE cells transfected with non-targeting control siRNA were called OEsiRNA NC cells.

The proliferation of T lymphocyte Jurkat-E6-1 cells, NC cells and OE cells was assessed at 24, 48, and 72 h after transfection. CCK-8 solution (10 μL; Beyotime Biotechnology Co., Ltd.) was added and incubated for 3 h, and the absorbance was measured at 450 nm as described previously (Wang et al., 2020).

Cell samples (slides) were naturally dried and immersed in 4% paraformaldehyde fixative solution for 30 min to improve the permeability of the cells. After soaking in PBS, 1% Triton X-100 was added to the samples and incubated at room temperature for 15 min. After washing, nuclease inactivation enzyme was added, and after another wash, protease K was added for cleanup. Then, 100 μL of Streptavidin-TRITC-labeled working solution was added to each sample and soaked at 37°C in the dark for 1 h. After washing, DAPI was added for re-staining, and samples were observed under a fluorescence microscope after washing.

The T lymphocytes were transduced with lentiviral lncRNA-ENST00000421645 overexpression vector and empty lentiviral vector, respectively. The experiment was repeated three times to obtain three groups of OE cells and NC cells that had been transformed for 24 h. Kank1 small interfering RNA (siRNA) was transfected into OE cells and NC cells. After transfection for 24 h, the OE cells, NC cells, OEsiRNA Kank1 and OEsiRNA NC cells were collected for RT-qPCR experiments. Total RNA was isolated from the cells using Trizol reagent (Life Technologies Corporation, Carlsbad, CA, United States). Total RNA yield and purity were analyzed spectrophotometrically (Denovix, United States) as previously reported (Chong, 2001). Then, First-strand cDNA was synthesized using a cDNA synthesis kit (Takara Biotechnology, Dalian, China) and SYBR Premix Ex Taq™ (Takara Biotechnology, Dalian, China). Two-step real-time fluorescence quantitative PCR for Kank1, 14-3-3 and IFN-γ was used to detected the mRNA level of Kank1, 14-3-3 and IFN-γ. One microliters of each cDNA sample was amplified in a 20 μL reaction mixture containing 10 μL of SYBR premix Ex Taq II (2x) (Takara Biotechnology, Dalian, China), the forward primer and reverse primer at 0.25 μM each, and 8 μL Nuclease-Free Water in a 96-well reaction plate with a LightCycler^® 480 PCR-Time PCR System (Roche Diagnostics Ltd., Swit). The precise conditions for PCRs were as follows: in the pre-denaturation stage, the reaction cycle was repeated for 30 s at 95°C; at the denaturation stage, the reaction time was 5 s at 95°C, in the annealing extension phase, the reaction time was 35 s at 60°C; and the denaturation and annealing extension stage was cycled for 40 times (distilled water and negative extraction control) were included in each assay. Three biological and technical replicates were performed. The specific primers used in real-time RT-PCR analyses were listed in Table 1.

Total RNA was extracted from NC cells, OE cells, OEsiRNA NC cells and OEsiRNA Kank1 cells and was subjected to reverse transcription. The cDNA product of reverse transcription was used as the template in subsequent experiments. 1 μL of cDNA extracted from peripheral blood T cells among 3 clinical samples was used in this study and 4 different T cells from culture were dissolved in 9 μL water, respectively. After the mixing process, 1 μL of mixture was added into 9 μL RNA-free water, followed by the same multiple dilution twice. Standard curves of target and reference genes were established from samples which were diluted to 1 × 10⁵, 1 × 10⁴, 1 × 10³, 1 × 10², and 1 × 10¹. The E-values and corresponding R² values were shown in Table 2. The melting curve of PCR showed that PCR amplification had specificity, with only one specific peak, no primer-dimers and no non-specific amplification products. The results showed that the slope differences of standard curves between lncRNA-ENST00000421645 mRNA and β-actin mRNA, IFN-γ mRNA and GAPDH mRNA, Kank1 mRNA and GAPDH mRNA, 14-3-3 mRNA and GAPDH mRNA were less than 0.1, respectively; therefore, we employed 2-ΔΔCt method (Livak and Schmittgen, 2001) to calculate the relative expression level of the target gene. The C_t value was adjusted automatically and the threshold cycle value difference (ΔC_t) between FAM C_t of the target gene (lncRNA- ENST00000421645, Kank1, 14-3-3, and IFN-γ) and FAM C_t of GAPDH (internal control) was used to normalize the amount of Kank1, 14-3-3 and IFN-γ. As long as the target gene and the internal control have similar amplification efficiencies, C_t values are normalized by using the difference (ΔC_t) between the internal control and target gene. This value is calculated for each sample to be quantified. Finally, the amount of Kank1, 14-3-3 and IFN-γ, normalized to an endogenous reference and relative to a calibrator, is given by:

After transfection for 24 h, the IFN-γ concentration in the OE cells, NC cells, OEsiRNA Kank1 and OEsiRNA NC cells culture supernatant was determined with an Human IFN-gamma ELISABASIC kit (HRP) (Neobioscience Technology Co., Ltd., China) according to the manufacturer’s instructions. IFN-γ ELISA kits were solid-phase sandwich enzyme-linked immunosorbent assays. The kit contains a matched pair of monoclonal capture and detection antibodies, Streptavidin-HRP, ELISA standard and standard reconstitution buffer.

The cells were washed with PBS for three times and then added with cell lysate RIPA (1–5 × 10⁶ cells added with 250 μL lysate). The whole process was carried out on ice. The cells were then lysed by ultrasound, and the power was adjusted to 28% for about 10 s until the cell suspension became clear. After ultrasonic lysis, the cells were placed on ice for 30 min, and then cell lysis solution was collected and centrifuged at 13,200 rpm at 4°C for 30 min. The supernatant was carefully absorbed (be careful not to remove precipitation) into a clean 1.5 mL EP tube. 5 × loading buffer, 0.25 times of supernatant volume, was added and fully mixed, heated at 100^°C for 10 min and stored at −20^°C for later use. The membrane transfer process begins immediately after electrophoresis. Then the membrane was incubated first with anti-14-3-3, anti-kank1, anti-p-ikba, anti-t-ikba (all 1:1,000; Abcam, Cambridge, Ma, United States) and anti-GAPDH (1:1,000, Cell Signaling Technology, Danvers, MA, United States) antibodies and then with horseradish peroxidase-conjugated secondary antibodies. Western Chemiluminescent HRP 149 Substrate (ECL, Millipore, United States) was used to visualize the immunoreactivity bands. The images were quantified using ImageJ software.

ChIP was used to investigate the binding of histone H3K27 to the target gene Kank1 in NC cells and OE cells. After 48 h of culture, the chromatin in the cells was first cross-linked using 1% formaldehyde, and the cells were subjected to fixation followed by lysis and enzymatic digestion. Immunoprecipitation was then conducted. The IgG ChIP antibody (2 μg, Cell Signaling Technology, Danvers, MA, United States) and the anti-H3K27 ChIP antibody (2 μg, Cambridge, Ma, United States) (Liu et al., 2021) were added. The immunoprecipitation reaction was performed for 2 h; samples were incubated on a shaking platform at 4°C overnight. After elution of the immunoprecipitates, the DNA was purified. Enrichment of histone H3K27 and the target gene Kank1 was detected by RT-qPCR. All samples were tested in triplicate, and three multiple holes were made in each experiment.

Streptavidin-conjugated magnetic beads were used to pull down lncRNA-ENST00000421645 according to the manual for the Pierce™ Magnetic RNA-Protein pull-down kit (Thermo Fisher Scientific, United States). Biotinylated RNA and T lymphocyte lysates were mixed and incubated. Streptavidin-agarose beads were added to each binding reaction, and the mixtures were incubated at 4°C for 1 h prior to two elution steps with detergent solution. The eluted proteins were detected by mass spectrometry (MS).

Continuous variables are presented in the form of mean ± standard deviation. Non-normal distribution parameters are described by median ± interquartile range. The expression level of 14-3-3 mRNA level was analyzed by using rank sum test. The multiple comparison of 14-3-3 expression level was conducted using Kruskal-Wallis test. The apoptosis rates of OEsiRNA NC cells and OEsiRNA, and the mRNA level and protein level of these two cell lines were analyzed by using Student’s t-test. One-way ANOVA was employed to compare the mRNA levels that measured from more than two groups, and SNK test was used for conducting multiple comparisons. All statistical analyses were performed in SPSS version 20 and a P-value less than 0.05 was considered significant.

This study was approved by the Ethics Committee of Zhongshan Hospital (NO. 2021-147) and was in accordance with the Helsinki Declaration. Participants signed written informed consent prior to participating in the study.

In order to study on the mechanistic of lncRNA-ENST00000421645 based on our previous study (Liu et al., 2017), the vector GV502 was used to construct the lentiviral lncRNA-ENST00000421645 overexpression vector. T lymphocytes were transduced with lentiviral vectors. After transduction for 24 h, the apoptosis rates of NC cells OE cells were 28.91 ± 3.58% and 44.12 ± 5.24%, respectively. The apoptosis rate of OE cells was significantly increased compared with that of NC cells (P < 0.001, Figure 1A). Caspase3 is closely related to cell apoptosis. To further confirm the effect of lncRNA-ENST00000421645 on T lymphocyte apoptosis, the expression of caspase3 in NC cells and OE cells was analyzed by Western blotting after transduction for 24 h. The relative caspase3 protein level in NC cells was found to be 0.228 ± 0.019. The relative caspase3 protein expression level in OE cells was 0.464 ± 0.024, indicating significant upregulation (P < 0.001, Figure 1B). These results suggested that overexpression of lncRNA-ENST00000421645 can promote apoptosis in T lymphocytes.

The samples of IFN-γ had a similar PCR efficiency to samples containing GAPDH. The relative fold change in IFN-γ mRNA expression in OE cells compared to NC cells was 0.23, a significant decrease (P < 0.001, Figure 1C). In addition, the ELISA results were consistent with the RT-qPCR results. By ELISA, the IFN-γ protein concentration was determined to be 84.47 ± 7.63 pg/mL in NC cells and 59.77 ± 7.65 pg/mL in OE cells. Thus, compared with that in NC cells, the content of IFN-γ in OE cells was significantly reduced (P < 0.001, Figure 1D). These results showed that lncRNA-ENST00000421645 inhibits the secretion of IFN-γ.

According to lncRNA expression profile analysis, the gene adjacent to lncRNA-ENST00000421645 is Kank1. The expression of Kank1 in OE cells was analyzed by Western blotting, and the relative Kank1 protein expression level in NC cells was found to be 0.277 ± 0.021. The relative Kank1 protein expression level in OE cells was 0.773 ± 0.021, indicating significant upregulation (P < 0.001, Figure 2).

Bioinformatics showed that the adjacent gene of lncRNA-ENST00000421645 is Kank1 according to our previous published paper (Liu et al., 2017). To investigate the mechanism by which lncRNA-ENST00000421645 upregulates the expression of the adjacent gene Kank1, a lncRNA-ENST00000421645 pull-down assay was performed with streptavidin-conjugated magnetic beads. The precipitated product was analyzed by mass spectrometry to identify the lncRNA-ENST00000421645 interacting proteins. A total of 793 proteins were identified in the sense group, and 638 proteins were identified in the anti-sense group. Mass spectrometry analysis of the precipitated products revealed 155 differential proteins. The top ten differential proteins were listed in Table 3. The changes in chromatin structure caused by the modification of histones play an important role in the regulation of gene expression and transcription. Among them, histone acetylation is particularly important. The acetylation of the N-terminal lysine residues of histones in eukaryotic cells is related to the transcriptional activation of genes. We analyzed that among the top 10 proteins that bind to lncRNA-ENST00000421645, there is a protein named NAT10 related to acetylation. The acetylase NAT10 ranked ninth, indicating that lncRNA-ENST00000421645 acted on this protein.

We verified that lncRNA-ENST00000421645 may promote the acetylation of histone H3K27 adjacent to the Kank1 promoter through the acetylase NAT10, thus promoting the expression of Kank1. ChIP-PCR was performed on OE cells and NC cells. An anti-H3K27 antibody was used to precipitate the cross-linked DNA-protein complexes. Primers targeting the Kank1 promoter sequence were designed, and the DNA sequence enriched by the anti-H3K27 antibody was detected by real-time PCR. The samples of Kank1 had a similar PCR efficiency to samples containing GAPDH. The enrichment of the Kank1 gene promoter in the non-H3K27-acetylated group of OE cells was significantly increased 3.96-fold compared with that in NC cells (P < 0.001, Figure 3). These results suggest that lncRNA-ENST00000421645 promotes the acetylation of histone H3K27 adjacent to the Kank1 promoter, thereby promoting Kank1 expression. All samples were tested in triplicate.

Kank1 siRNA was used to knock down Kank1 gene expression in OE cells, and RT-qPCR was then used to detect the knockdown efficiency and select the siRNA with the highest efficiency—Kank1-776—for subsequent experiments (P < 0.001, Figure 4A). The relative fold change in the Kank1 mRNA level in OE cells compared with NC cells was 6.52 ± 1.75, a significant increase compared with the level in NC cells (P < 0.001, Figure 4B). The relative fold change in the Kank1 mRNA level in OEsiRNA NC cells compared with OE cells was 6.18 ± 1.27, a difference that was not significant. The relative fold change in the Kank1 mRNA level in OEsiRNA Kank1 cells was 1.89 ± 0.38, a significant reduction compared with that in OEsiRNA NC cells (P < 0.01, Figure 4B). Kank 1 has also been shown to promote 14-3-3 protein expression to inhibit NF-kB activation (Kakinuma et al., 2008). The samples of 14-3-3 had a similar PCR efficiency to samples containing GAPDH. The relative fold change in the 14-3-3 mRNA level in OE cells was 4.88 ± 0.58, a significant increase compared with that in NC cells (P < 0.001, Figure 4C). The relative fold change in the 14-3-3 mRNA level in OEsiRNA-NC cells compared with OE cells was 5.43 ± 0.59, a difference that was not significant. The relative fold change in the 14-3-3 mRNA level in OEsiRNA Kank1 cells was 2.93 ± 0.40, a significant reduction compared to that in OEsiRNA NC cells (P < 0.01, Figure 4C). In addition, the Western blot results were consistent with the RT-qPCR results. The relative Kank1 protein levels in NC cells, OE cells, OEsiRNA NC cells and OEsiRNA Kank1 cells were 0.288 ± 0.013, 0.833 ± 0.037, 0.837 ± 0.017, and 0.471 ± 0.018, respectively. The Kank1 protein level in OEsiRNA Kank1 cells was significantly lower than that in OEsiRNA NC cells (P < 0.001, Figure 4D). The relative 14-3-3 protein levels in NC cells, OE cells, OEsiRNA NC cells and OEsiRNA Kank1 cells were 0.246 ± 0.033, 0.817 ± 0.043, 0.813 ± 0.018, and 0.460 ± 0.037, respectively. The relative 14-3-3 protein expression level was also reduced in OEsiRNA Kank1 cells compared with OEsiRNA NC cells (P < 0.001, Figure 4D).

The phosphorylation level of IKBa can indirectly reflect the activation of the NF-kB signaling pathway. P-IKBa/T-IKBa refers to the ratio of phosphorylated IKBa protein to total IKBa protein content, which can reflect the activation degree of NF-kB pathway. Thus, the phosphorylation level of IKBa was analyzed by Western blotting. The relative p-IKBa/IKBa ratios in NC cells, OE cells, OEsiRNA NC cells and OEsiRNA Kank1 cells were 1.200 ± 0.058, 0.482 ± 0.035, 0.503 ± 0.058, and 0.958 ± 0.038, respectively. Compared with that in NC cells, the relative p-IKBa/IKBa ratio was significantly reduced in OE cells (P < 0.001; Figure 4D). Compared with that in OEsiRNA NC cells, the relative p-IKBa/IKBa ratio was obviously increased in OEsiRNA Kank1 cells (P < 0.001, Figure 4D).

This analysis showed that lncRNA-ENST00000421645 upregulated the expression of Kank1. Kank1 promoted 14-3-3 protein expression and inhibited NF-kB activation.

The effect of the lncRNA-ENST00000421645–Kank1 protein interaction on T lymphocyte function was further investigated. The apoptosis rates of OEsiRNA NC cells and OEsiRNA Kank1 cells were 42.35 ± 4.16% and 21.50 ± 3.81%, respectively. Compared with that of OEsiRNA NC cells, the apoptosis rate of OEsiRNA Kank1 cells was significantly reduced (P < 0.001, Figure 5A). To further confirm the effect of Kank1 on T lymphocyte apoptosis, after transfection for 24 h, the expression of caspase3 in OE siRNA NC cells and OE siRNA Kank1 cells was analyzed by Western blotting. The relative caspase-3 protein expression level was significantly decreased in OE siRNA Kank1 cells compared with OE siRNA NC cells (0.347 ± 0.034 vs. 0.829 ± 0.035, P < 0.001; Figure 5B). The relative fold change in the IFN-γ mRNA level in OE siRNA Kank1 cells compared with OEsiRNA NC cells was 2.88 ± 0.65, a significant increase (P < 0.001, Figure 5C). In addition, the ELISA results were consistent with the RT-qPCR results. The IFN-γ protein levels in OEsiRNA Kank1 and OEsiRNA NC cells were 83.20 ± 4.86 pg/mL and 64.42 ± 5.30 pg/mL, respectively. Compared with that in OE siRNA NC cells, the IFN-γ level in OE siRNA Kank1 cells was obviously increased (P < 0.001, Figure 5D). These results indicated that lncRNA-ENST00000421645 was overexpressed in T lymphocytes of neurosyphilis patients, resulting in promotion of apoptosis and inhibition of IFN-γ secretion through the Kank1 protein. The mechanistic diagram of T lymphocyte regulation by lncRNA-ENST00000421645 was shown in Figure 6.