NFIB-Mediated lncRNA PVT1 Aggravates Laryngeal Squamous Cell Carcinoma Progression via the miR-1301-3p/MBNL1 Axis

Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignant tumors of head and neck cancers. In the past decades, although the therapy strategies of LSCC have made considerable improvement, the terrible outcomes of LSCC still bring an enormous burden to the world health care system. Novel therapeutic targets for LSCC are urgently needed. lncRNAs exert important roles in various biological progressions, including LSCC. Here, we aimed to investigate the function of lncRNA PVT1 in LSCC progression and its underlying molecular mechanisms. By conducting multiple experiments, our results showed that lncRNA PVT1 was upregulated in LSCC cell lines and regulated LSCC cell proliferation, apoptosis, and its cell susceptibility to natural killer (NK) cells. Moreover, it was found that lncRNA PVT1 promotes MBNL1 expression to regulate LSCC cellular progression through sponging miR-1301-3p. Our study might provide novel targets for LSCC basic research or clinical management.


Introduction
Laryngeal squamous cell carcinoma (LSCC), as one of the most common cancer types of head and neck malignancies [1], has become an enormous burden to the world health care system. Although significant improvements in LSCC clinical intervention, including surgery, chemotherapy, and radiotherapy, have been made in the past decades, the outcomes of LSCC patients remain poor [2]. Understanding the mechanisms underlying the development of LSCC is urgently needed for developing novel therapeutic targets.
Recently, accumulating evidence indicated that lncRNAs play an important role in the initiation or progression of LSCC [11][12][13]. Cao et al. elucidated that lncRNA IGKJ2-MALLP2 suppresses LSCC through interacting with miR-1911-3p to regulate p21 expression [14]. Xu et al. revealed that lncRNA HULC regulates LSCC development through modulating PTPRO [15]. Li et al. demonstrated the function of lncRNA SNHG20/miR-140 in the progression of LSCC [16]. All those research suggested that lncRNAs have a promising prospect to be novel LSCC therapeutic targets.
In the current study, we aimed to investigate the biological function of lncRNA PVT1 in LSCC development. A previous study has reported that lncRNA PVT1 was upregulated in LSCC tissues and promoted LSCC progression. However, its underlying mechanisms demand further study [17]. Here, we found that the expression level of lncRNA PVT1 in LSCC cell lines was upregulated. Next, by conducting a serial in vitro experiment, our results showed that lncRNA PVT1 regulates LSCC proliferation, apoptosis, and NK cell-mediated cytotoxicity towards LSCC cells. Through investigating the underlying molecular mechanism, a novel lncRNA PVT1/miR-1301-3p/MBNL1 axis was revealed. Furthermore, we elucidated that lncRNA PVT1 expression in LSCC cells could be regulated by NFIB. Our research might provide a new insight for LSCC therapeutic target research.
2.2. qRT-PCR. Total RNAs from LSCC tissue samples and cell lines were isolated using the TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. The Super-Script IV First-Strand Synthesis System (Invitrogen, CA) was used to conduct the reverse transcription process of miRNA and lncRNA following the manufacturer's instruction. With GAPDH as the internal control, the qualification of RNAs was conducted using the 2 -△△Ct method. All primers used in the current study are as follows (Table 1).

Western
Blotting. Proteins were isolated from cells using the RIPA protein extraction reagent (Beyotime, China) and added with a protease inhibitor cocktail (Roche, USA) in accordance with protocols. Collected proteins were separated by SDS-PAGE gels and electrically transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Subsequently, membranes were incubated with primary antibodies overnight at 4°C. Next, membranes were washed by TBST three times and then subjected to second antibodies for 2 h at room temperature. A Fusion FX5 image analysis system (Vilber Lourmat, France) was used to visualize the results. Antibodies used in the current study are as follows: MBNL1 (sc-47740, 1 : 1000, Santa Cruz Biotechnology) and actin (ab8226, 1 μg/mL, Abcam). Table 1 Gene Sequence Journal of Immunology Research   2.5. Cell Colony Formation Assay. Collectively, cells were seeded in a 35 mm dish at a density of 1000 cells per well after transfection. Next, cells were cultured in the dishes for 2 weeks. After that, cells were washed by PBS and added with 4% paraformaldehyde for 30 min. Next, 0.1% crystal violet solution was applied to stain cells at room temperature for 15 min. Results were visualized by the image capturing.
2.6. Apoptosis Analysis. Cell apoptosis analysis was conducted using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) cell apoptosis kit (Invitrogen) following the manufacturer's instruction. Briefly, pretransfected cells were collected from each group and washed by PBS three times. Next, cells were supplied with 5 μL Annexin V/FITC and 5 μL PI for 10 min at 25°C. Then, results were analyzed using FACScan flow cytometry (BD Biosciences, USA). Experiments were conducted three times, and results were recorded. The apoptotic rate of indicated cells was calculated using the sum value of the right upper results plus the right lower results.

lncRNA Pull-Down
Assay. An in vitro transcription kit from Thermo Fisher Scientific (K0441) was used to acquire the full-length sense or antisense of PVT1 following the manufacturer's instruction. Subsequently, RNA Pull-Down assay was performed using a Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific) according to the protocol. Biotinylated probes were synthesized and obtained from GeneChem (Shanghai, China) Company. Experiments were conducted according to published research [18]. Experiments were conducted in triplicate.

Chromatin Immunoprecipitation (ChIP).
A ChIP kit (CST, USA) was applied to conduct ChIP assays in the current study according to the instructions provided by the manufacturer. In summary, formaldehyde was subjected to cells for crosslinking. Subsequently, cells were sonicated to a length between 200 and 1000 bp. Next, the anti-IgG (CST, #3900S, 2.5 mg/mL) antibody and anti-NFIB (Abcam, ab186738, 1 : 30) antibody were used to perform immunoprecipitation. Results were analyzed by the qRT-PCR assay.

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Si-PVT1#1 Si-PVT1#2 Si-NC (f) Figure 2: lncRNA PVT1 regulates the natural killer cell-mediated cytotoxicity to LSCC cells. (a, b) NK cell cytotoxicity to LSCC cells was evaluated using calcein release assay. (c, d) Perforin polarization assay was conducted to measure the perforin-containing NK cells, which were against lncRNA PVT1 knockdown LSCC cells. (e, f) Conjugation assay was applied to measure the conjugate formation between NK cells and lncRNA PVT1 knockdown LSCC cells. All assays were conducted three times. * P < 0:05, * * P < 0:01.  2.13. Statistical Analysis. All statistical analyses in the current study were calculated using SPSS (23.0V, USA) or Prism 9 software (7.0V, USA). All experiments were performed at least three times. Statistical differences between the two groups were analyzed using Student's t-test. The comparison among three or more groups was analyzed using the one-way ANOVA method. All results were presented as the mean ± standard deviation (SD). * P < 0:05 was considered statistically significant.

The Functional Role of lncRNA PVT1 in LSCC
Progression. To investigate whether lncRNA PVT1 participates in LSCC progression, firstly, we examined the expression level of PVT1 in LSCC cell lines. Results of qRT-PCR showed that PVT1 expression in FD-LSC-1 and TU-177 was significantly upregulated compared with that in normal cell line HOK (Figure 1(a)). Thereafter, PVT1 knockdown cell models were generated as indicated to elucidate the bio-logical function of PVT1 in LSCC cellular progression (Figure 1(b)). Next, results of CCK-8 (Figure 1(c)) and colony formation assays (Figure 1(d)) showed that downregulated PVT1 suppressed LSCC cell proliferation levels. Cell apoptosis detection results showed that PVT1 knockdown increased LSCC cell apoptotic rates (Figure 1(e)). Based on those results, we conclude that lncRNA PVT1 participates in LSCC progression.

lncRNA PVT1 Regulates the Natural Killer Cell-Mediated
Cytotoxicity to LSCC Cells. Previous studies have demonstrated that both the linear and circular forms of PVT1 play an important role in the progression of the immune response [19]. However, little is known about the function of lncRNA PVT1 in the immune system. It has been well documented that the NK cell, as an innate antitumor immune response character, exerts essential roles in various tumorigeneses [20][21][22]. To determine whether lncRNA PVT1 plays its role in LSCC progression via influencing the susceptibility of NK cells, calcein release assay (Figures 2(a) and 2(b)), perforin polarization assay (Figures 2(c) and 2(d)), and conjugation assay (Figures 2(e) and 2(f)) using lncRNA PVT1 knockdown LSCC cells were conducted. Interestingly, we found that lncRNA knockdown repressed the NK cell-mediated cytotoxicity towards LSCC cells. From those results, we conclude that lncRNA PVT1 plays its biological function in the interaction of LSCC cells and NK cells.

miR-1301-3p
Regulates LSCC Proliferation and Impacts the Susceptibility of LSCC Cells to NK Cells. We have demonstrated that miR-1301-3p is a downstream target of lncRNA PVT1 in LSCC cells. However, the biological functions of miR-1301-3p in LSCC cells have not been studied. Therefore, we conducted loss-of-function experiments using miR-1301-3p downregulated LSCC cells (Figure 4(a)).
Results of dual-luciferase reporter gene assay showed that NFIB overexpression markedly increased the fluorescence intensity when cotransfected with PVT1-WT, not PVT1-MUT, suggesting that NFIB was directly binding with the PVT1 promoter in LSCC cells (Figures 7(g) and 7(h)). From the above results, we conclude that lncRNA PVT1 expression in LSCC cells could be transcriptionally mediated by NFIB.

Discussion
LSCC ranks as the second malignancy tumor of head and neck cancers. Due to its tendency of local invasion, metastasis, and chemotherapy resistance, its morbidity and mortality are constantly growing [25,26]. It has been reported that the incidence of LSCC in China is near four times higher than that in the USA. However, with the development of clinical treatment strategies in recent years, the LSCC patients' survival rate is still low [27][28][29]. With the highthroughput technology innovation in the past two decades, the dysregulation and dysfunction of RNAs, especially lncRNAs, have got wildly scientific interest and are considered to be one auspicious direction for disease management. However, the underlying mechanisms of lncRNAs in LSCC development are still not fully understood.   . (a, b) Relative expression level of lncRNA PVT1 in knockdown LSCC cell models pretransfected with indicated siRNAs was measured by qRT-PCR. (c) Relative lncRNA PVT1 expression in NFIB overexpression LSCC cells was detected by qRT-PCR. (d) ChIP assay using anti-IgG and anti-NFIB was performed to evaluate the molecular relationship between the PVT1 promoter and NFIB in LSCC cells; results were analyzed using the qRT-PCR assay. (e) The digraph binding sequence of NFIB was obtained from the JASPAR dataset. (f) The predicted binding region of the PVT1 promoter was obtained from the JASPAR dataset. (g, h) The molecular relationship between the PVT1 promoter and NFIB was assessed by luciferase reporter gene assay. All assays were conducted three times. * * * P < 0:001.
In the current study, we aimed to demonstrate the function of lncRNA PVT1 and the molecular mechanisms underlying LSCC development. lncRNA PVT1 has been studied in multiple cancer types, including gastric cancer [30,31], ovarian cancer [32], and pancreatic cancer [18]. And the molecular functions of lncRNA PVT1 in those cancers have been intensely investigated, which emphasized the important role of lncRNA PVT1 in various tumorigeneses. Published research from Zheng et al. has revealed that lncRNA PVT1 is upregulated in LSCC tumor tissues and promoted LSCC cellular progression via sponging miR-519d-3p [17]. However, our group decided to further explore the biological and mechanical role of lncRNA PVT1 in LSCC progression. Firstly, we confirmed that the expression level of lncRNA PVT1 in LSCC cell lines was upregulated. Next, we found that downregulated lncRNA PVT1 inhibited LSCC cell proliferation and promoted LSCC cell apoptotic rates. Moreover, dysregulation of lncRNA PVT1 is capable of influencing LSCC cell susceptibility to NK cells.
The NK cell is one of the most important members of the innate lymphoid cell family [33,34]. Its essential role in cancer progression has been elucidated by multiple research [21,35,36]. Interestingly, our results showed that lncRNA PVT1 downregulation in LSCC cells increased its susceptibility to NK cells, making LSCC cells less venerable to NK cell-mediated cytotoxicity. However, its molecular mechanisms demand further study.
Our results have elucidated the downstream mechanisms of lncRNA PVT1 in LSCC progression. We also noticed that lncRNA PVT1 expression in lung cancer cells could be regulated by a transcription factor YY1 [37]. The important role of the transcription factor in lncRNA regulation is emerging [23,24,38]. To better understand the function of lncRNA PVT1, we investigated the upstream factor of lncRNA PVT1. Our results found that NFIB could regulate lncRNA PVT1 expression in LSCC cells through directly binding to the promoter region of PVT1. Our results renewed the molecular pattern of lncRNA PVT1 in LSCC.
Collectively, our study has partially demonstrated that lncRNA PVT1 regulates LSCC cell viability and impacts the susceptibility of LSCC cells to NK cells. Furthermore, a novel NFIB/lncPVT1/miR-1301-3p/MBNL1 axis has been partially elucidated in LSCC progression. Our study might provide a new insight for LSCC basic research and novel therapeutic targets for LSCC clinical intervention.

Data Availability
The data performed to support the findings of this study are included within the article.