CN115227708A - Application of LncRNA IFITM4P targeted small interfering RNA in treatment of oral leukoplakia and/or oral cancer - Google Patents

Abstract

The invention discloses an application of a small interfering RNA targeting LncRNA IFITM4P in treatment of oral leukoplakia and/or oral cancer, wherein the LncRNA IFITM4P nucleotide sequence is shown as Seq ID NO:1 is shown in the specification; the sequence of the small interfering RNA is at least one of the sequence shown in SEQ ID NO.2 and the sequence shown in SEQ ID NO. 3. The invention provides an application of a targeted LncRNA IFITM4P small interfering RNA in oral leukoplakia and/or oral cancer treatment, and also provides an application of a PD-1 monoclonal antibody in preparing a medicine for treating high-expression LncRNA IFITM4P oral leukoplakia and/or oral cancer, provides a new way for treating human oral leukoplakia and/or oral cancer, and provides a new method for developing medicines for treating oral leukoplakia and antitumor medicines.

Description

Application of LncRNA IFITM4P targeted small interfering RNA in treatment of oral leukoplakia and/or oral cancer

Technical Field

The invention relates to the technical field of biomedicine, in particular to application of a small interfering RNA targeting LncRNA IFITM4P in treatment of oral leukoplakia and/or oral cancer.

Background

Oral cancer (OSCC) is a common malignant neoplasm with a poor prognosis, with approximately more than 30 million new cases worldwide per year. The precancerous lesions of the oral cavity refer to some clinical or histological changes of the oral jaw and face and have canceration tendency, and comprise leukoplakia, erythema, lichen planus, discoid lupus erythematosus, fibrosis under mucous membrane, papilloma, chronic ulcer, mucous membrane black spot, pigmented nevus and the like. Among them, oral Leukoplakia (OL) is the most typical one of Oral Potential Malignant Diseases (OPMD).

Currently, surgical resection and radiation therapy are the two most effective methods for treating oral leukoplakia and/or oral cancer, and although the survival rate of patients is greatly improved after surgery, the overall recovery is still not optimistic, and the main cause of death of patients is tumor recurrence and metastasis. Therefore, the development of a novel medicament for treating oral leukoplakia and/or oral cancer has important economic value and social benefit.

Disclosure of Invention

In view of the defects in the prior art, the invention aims to provide the application of the LncRNA IFITM4P targeted small interfering RNA in the treatment of oral leukoplakia and/or oral cancer.

The invention provides in a first aspect the use of a small interfering RNA targeting LncRNA IFITM4P in the treatment of oral leukoplakia and/or oral cancer, said LncRNA IFITM4P nucleotide sequence being as defined in Seq ID NO:1 is shown.

In one embodiment of the invention, the sequence of the small interfering RNA is at least one of the sequence shown in SEQ ID NO.2 and the sequence shown in SEQ ID NO. 3.

In a second aspect of the present invention, there is provided an RNA drug for treatment of oral leukoplakia and/or oral cancer, comprising a small interfering RNA targeting LncRNA IFITM4P, the LncRNA IFITM4P nucleotide sequence being as defined in Seq ID NO:1 is shown.

In one embodiment of the invention, the sequence of the small interfering RNA is at least one of the sequence shown in SEQ ID NO.2 and the sequence shown in SEQ ID NO.3

The third aspect of the present invention provides a method for screening a drug for treating oral leukoplakia and/or an anticancer drug, comprising the steps of:

s1, determining the expression level of LncRNA IFITM4P in the oral tissue cells, wherein the nucleotide sequence of LncRNA IFITM4P is as defined in Seq ID NO:1 is shown in the specification;

s2, contacting the candidate medicine with the cell in the step S1;

s3, determining the expression level of LncRNA IFITM4P in the cells after the step S2;

s4, comparing the expression levels of LncRNA IFITM4P determined in step S1 and step S3, wherein a decreased expression level of LncRNA IFITM4P indicates that the drug candidate has the potential to treat vitiligo and/or to prevent cancer.

In a fourth aspect, the present invention provides the use of PD-1 monoclonal antibody in the preparation of a medicament for treating oral leukoplakia and/or oral cancer with high expression of LncRNA IFITM4P, wherein the LncRNA IFITM4P nucleotide sequence is as defined in Seq ID NO:1 is shown. Compared with the prior art, the embodiment of the invention has the following beneficial effects: the application of the reagent for reducing or inhibiting the expression of LncRNA IFITM4P in preparing the medicine for treating oral leukoplakia and/or oral cancer provided by the embodiment of the invention provides the application of the PD-1 monoclonal antibody in preparing the medicine for treating the oral leukoplakia and/or oral cancer of LncRNA IFITM4P with high expression, provides a new way for treating human oral leukoplakia and oral cancer, and provides a new method for developing the medicines for treating the oral leukoplakia and the antitumor medicines.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a graph of the expression profile of IFITM4P in patient tissue; wherein, fig. 1A is a clinical photograph and a pathological diagnosis photograph of a human normal oral mucosa, oral leukoderma and oral squamous cell carcinoma, fig. 1B is a heat map of a chip detection result, fig. 1C is a list of LncRNA differentially expressing the first ten, fig. 1D is a result of LncRNA IFITM4P qRT-PCR, fig. 1E is a pathological diagnosis photograph and a result of LncRNA IFITM4P in situ hybridization of a human normal oral mucosa, oral leukoderma and oral squamous cell carcinoma, fig. 1F is a result of LncRNA IFITM4P quantification in TCGA database, fig. 1G is a schematic diagram of a mouse tongue leukoplakia/squamous cell carcinoma animal model, fig. 1H is a general photograph, a pathological diagnosis photograph and a result of LncRNA IFITM4P in situ hybridization of a mouse tongue normal mucosa and tongue leukoplakia/squamous cell carcinoma, fig. 1I is a result of LncRNA IFITM 4P-PCR after leukin-1 cell line interference, fig. 2A is a result of lecqrt-PCR-1 cell line;

FIG. 2 is a graph showing that IFITM4P promotes proliferation and colony formation of OL and OSS; wherein, fig. 2B is a CCK8 cell proliferation result, fig. 2C is a CCK8 cell proliferation result, fig. 2D is a photograph of a colony formation culture dish, fig. 2E is a colony formation result, fig. 2F is a qRT-PCR result after RNA infection of HN4 cell line, fig. 2G is a CCK8 cell proliferation result, fig. 2H is a CCK8 cell proliferation result, fig. 2I is a photograph of a colony formation culture dish, fig. 2J is a colony formation result, fig. 2K is a photograph of nude mouse tumorigenesis, and fig. 2L is a volumetric diagram of nude mouse tumorigenesis;

FIG. 3 shows PD-L1 is a new downstream target of IFITM4P in OL and OSCC; wherein, fig. 3A is an RNA sequencing heatmap, fig. 3B is a gene set enrichment analysis map, fig. 3C is a gene set enrichment analysis map, fig. 3D is a wien map, fig. 3E is a qRT-PCR result, fig. 3F is a western blotting result, fig. 3G is a qRT-PCR result, fig. 3H is a western blotting result, fig. 3I is a qRT-PCR result, fig. 3J is a TCGA database PD-L1 quantification result, fig. 3K is a pathological diagnosis photograph, lncRNA IFITM4P in situ hybridization result photograph, PD-L1 immunohistochemistry result photograph and immunofluorescence photograph of a human normal oral mucosa, oral leukoderma and oral squamous cell carcinoma, fig. 3L is a qRT-PCR result correlation analysis, and fig. 3M is a qRT-PCR result correlation analysis;

FIG. 4 shows that LPS/TLR4 activates IFITM4P/PD-L1 signaling pathway to promote signaling immunity; wherein, fig. 4A is a working flow chart of in vitro tumor formation test, fig. 4B is a result chart of in vitro tumor formation, fig. 4C is a volume broken line chart of in vitro tumor formation, fig. 4D is a body weight broken line chart of a mouse, fig. 4E is a qRT-PCR result, fig. 4F is a qRT-PCR result, fig. 4G is a qRT-PCR result, fig. 4H is a luciferase report result, fig. 4I is a working flow chart of improved mouse tongue white spot/squamous carcinoma model, fig. 4J is a qRT-PCR result chart and a western blotting result chart, fig. 4K is a correlation analysis of the qRT-PCR result, fig. 4L is a working flow chart of mouse tongue white spot PD-1 monoclonal antibody treatment, fig. 4M is a visual chart of mouse living body tongue, and fig. 4N is a result chart of before and after mouse curative effect;

FIG. 5 is a graph showing that the interaction of IFITM4P with SASH1 promotes the expression of PD-L1 through the TAK-1-NF-KB signaling pathway; wherein, fig. 5A is a diagram of RNA pull-down and western blotting results, fig. 5B is a diagram of RNA binding protein immunoprecipitation results, fig. 5C is a diagram of a truncation work flow, fig. 5D is a diagram of RNA binding protein immunoprecipitation results, fig. 5E is a diagram of qRT-PCR and western blotting results, fig. 5F is a diagram of co-immunoprecipitation and qRT-PCR results, fig. 5G is a diagram of qRT-PCR and western blotting results, fig. 5H is a diagram of western blotting results, fig. 5I is a diagram of qRT-PCR and western blotting results, fig. 5J is a diagram of qRT-PCR results and western blotting results, and fig. 5K is a diagram of luciferase reporting method results;

FIG. 6 shows that ectopic expression of IFITM4P effectively enhances the binding of KDM5A and Pten promoter, and increases PD-L1 abundance: wherein, FIG. 6A shows the result of fluorescence confocal microscope; FIG. 6B is a drawing showing the results of RNA pull-down and western blotting; FIG. 6C is a graph showing the results of immunoprecipitation of RNA-binding proteins; FIG. 6D is a graph showing chromatin immunoprecipitation results; FIG. 6E is a diagram showing a western blotting result; FIG. 6F is a graph of qRT-PCR results; FIG. 6G is a mechanical diagram;

FIG. 7 is a signal path enrichment map;

FIG. 8A is a diagram showing the results of western blotting;

FIG. 8B is a diagram of CCK8 results;

FIG. 8 is a view showing the results of Cwestern blotting;

FIG. 8D is a CCK8 results chart;

FIG. 8E is a graph of cell viability crystal violet staining;

FIG. 8F is a graph of cell viability crystal violet staining;

FIG. 9A is a graph of qRT-PCR results;

FIG. 9B is a graph of the results of qRT-PCR;

FIG. 10 is a diagram showing the results of western blotting;

FIG. 11 is a diagram showing the result of confocal laser scanning microscopy and the result of qRT-PCR;

FIG. 12 is a graph showing the results of immunoprecipitation of RNA-binding proteins;

FIG. 13 is a graph of qRT-PCR results;

FIG. 14 is a graph showing the results of bioinformatics analysis of protein interactions

FIG. 15A is a graph of qRT-PCR results;

FIG. 15B is a graph of qRT-PCR results;

FIG. 15C is a diagram showing the results of western blotting and qRT-PCR;

FIG. 15D is a graph of qRT-PCR results;

FIG. 16A is a graph of qRT-PCR results;

FIG. 16B is a graph of the qRT-PCR results;

FIG. 16C is a graph of qRT-PCR results;

FIG. 17A is a flow chart of an RNA pull-down experiment;

FIG. 17B is a graph showing the results of immunoprecipitation;

FIG. 18 is a graph of qRT-PCR results;

FIG. 19 is a graph of qRT-PCR results;

FIG. 20 is a graph showing the results of correlation analysis of the TCGA common gene levels.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.

Oral cancer (OSCC) is a common malignant tumor with a poor prognosis, with approximately more than 30 million new cases worldwide per year. The precancerous lesions of the oral cavity refer to some lesions of the oral jaw and face which are changed clinically or histologically and have cancerization tendency, and comprise leukoplakia, erythema, lichen planus, discoid lupus erythematosus, submucosal fibrosis, papilloma, chronic ulcer, mucosal melasma, pigmented nevus and the like. Among them, oral Leukoplakia (OL) is the most typical one of Oral Potential Malignant Diseases (OPMD). The progression from leukoplakia stomatitis (OL) to oral cancer (OSCC) is a typical multistage canceration process. Then, effective markers and their mechanisms that predict oral carcinogenesis remain unknown.

The present study found that LncRNA IFITM4P is highly expressed in OSCC compared to normal tissue, and ectopic expression or knockdown of IFITM4P results in increased or decreased cell proliferation in vitro and in xenograft tumors, respectively. Mechanistically, in the cytoplasm, IFITM4P serves as a scaffold in the cytoplasm to promote binding and phosphorylation of SASH1 to TAK1 (Thr 187), thereby increasing phosphorylation of NF- κ B (Ser 536) and concomitantly inducing expression of PD-L1, resulting in activation of an immunosuppressive program that allows OL cells to evade anticancer immunity in the cytoplasm. In the nucleus, IFITM4P upregulates PD-L1 in OL cells by enhancing KDM5A binding to the Pten promoter to reduce Pten transcription.

The present invention will be described in further detail with reference to specific examples.

The materials and methods used in the examples are as follows:

1. OL/OSCC patients and specimens

Tissue samples for lncRNA chip experiments were obtained from human oral cavity NM (n = 3), to OL (n = 5), to OSCC (n = 4) in stepwise settings (table 1).

TABLE 1 clinicopathological information of patients in LncRNA microarray experiments

Wherein NM is normal oral mucosa, OL is oral leukoplakia, and OSCC is oral cancer.

Tissue samples for QRT-PCR validation are shown in table 2, including NM (n = 23), OL (n = 67), and OSCC (n = 46).

TABLE 2 basic characteristics of patients participating in qRT-PCR validation of IFITM4P and PD-L1 expression

Note that all research procedures involving human participants met ethical standards of the institutional and/or national research committee, and helsinki declaration, a later revision thereof, or similar ethical standards in 1964. Histological examination of all subjects was performed by the applicant according to WHO standards. "all patients diagnosed with primary OL or OSCC were not treated prior to biopsy or surgery.

2. Cell culture and drug

Leuk-1cells were cultured in keratinocyte serum-free medium (cat. No.10744, gibco, waltham, MA, USA) and HN4 cells were cultured in DMEM supplemented with 10% fetal bovine serum. The information on the drugs used is shown in Table 3.

TABLE 3

3. lncRNA microarray analysis, RNA-seq and qRT-PCR validation

Total RNA was extracted from cultured cells and tissue samples using TRIzol reagent according to the manufacturer's protocol (TaKaRa, japan Dalian). For microarray analysis, affymetrix (Santa Clara, calif., USA) GeneChip human transcriptome array 2.0 was used according to the manufacturer's protocol.

The RNA-seq sequencing of the sample adopts an Illumina HiSeq X Ten sequencing system and is detected by Novit corporation (Librarian bioinformatics technology corporation in China).

For expression verification, cDNA was synthesized using PrimeScript RT kit with gDNA Eraser (TaKaRa). The expression level of mRNA was detected by SYBR green (TaKaRa) RT-PCR method according to the manufacturer's instructions. The primers used for qRT-PCR are shown in Table 4.

TABLE 4

4. Immunohistochemistry and RNAscope

PD-L1-IHC staining was as described previously. The OL and OSCC sections were tested for IFITM4P expression using RNAscope red manual detection (advanced cell diagnosis, new wak, CA, USA) according to the manufacturer's recommendations. The probes were IFITM4P, hs-PPIB-1ZZ (positive control, 701041) and dapB (negative control, 701021) (Advanced Cell Diagnostics).

5. Cell viability and colony formation assays

Cell viability by enzyme-linked immunosorbent assay (1X 10) in 96-well plates ⁴ Per hole). The cell proliferation reagent CCK-8 is as described above. Absorbance was measured against a 480nm background using a microplate reader.

High expression of IFITM4P in Leuk-1 and HN4 cells, and IFITM4P knock-out and corresponding control cells (1X 10) ³ ) The cells were placed in 12-well plates for 14 days, and then 50 or more cells were counted under a dissecting microscope

6. Co-immunoprecipitation, RNA pull-down assay and RNA immunoprecipitation assay

Detailed methods of co-immunoprecipitation (co-IP) are described. The IFITM4P binding protein was studied by RNA Pull-Down assay analysis using the Pierc Magnetic RNA-Pull-Down Kit (Thermo Fisher Scientific, waltham, MA, USA) according to the manufacturer's instructions. Biotinylated IFITM4P and antisense sequences were synthesized using a transcription helper T7 high-throughput transcription kit (Thermo Fisher Scientific). The cytoplasmic fraction obtained using the NE-PER protein extraction kit (Thermo Fisher Scientific) was incubated overnight with biotinylated IFITM4P, followed by precipitation with streptavidin magnetic beads. The extracted proteins were eluted from the RNA-protein complex and analyzed by immunoblotting or silver staining. Silver staining was performed using a silver staining kit (Beyotime, china) according to the manufacturer's instructions. Ubiquitination sites were studied by MS (Novogene Bio-informatics Technology, beijing, china).

RIP detection was performed using an EZ-Magna RIP kit (microwell). Leuk-1cells (4X 10) ⁸ ) Lysis was performed with intact RIP lysis buffer. Lysates were immunoprecipitated in RIP buffer using anti-HuR antibody-conjugated magnetic beads (Abcam, cambridge, UK), SASH1 antibody or IgG. qRT-PCR analysis was performed on the precipitated RNA. Mouse IgG and HuR RNA were used as negative and positive control groups, respectively.

7. Chromatin immunoprecipitation test

ChIP detection adopts ChIP detection kit (Cell Signaling Technology [ CST)]Danvers, MA, USA). Briefly, leuk-1cells (5X 10) ⁸ ) Fixed with formaldehyde at a final concentration of 1%, crosslinked and sonicated. KDM5A antibody (10 mg/mL, abcam), igG control antibody (2 mg/mL, abcam) were added to the sonicated lysate overnight at 4 ℃ and then incubated with protein A/G Candida mixture (1:1 ratio, CST) at 4 ℃ with another>For 7 hours. Chromatin was eluted, reverse cross-linked and recovered using the achipassay Kit (CST). Input DNA and immunoprecipitated DNA were analyzed by qPCR using promoter DNA specific primers listed in table 4.

Improvement of mouse tongue leukoplakia/squamous cell carcinoma model

PD-1 Ma Ba therapy 8 week old male C57Bl/6J mice were purchased from Shanghai Lingchang Biotechnology, inc. (China). Carcinogen 4NQO (Sigma, st. Louis, MO, USA) solution was stirred overnight at room temperature in 100. Mu.g/mL double distilled water (ddH 2O) in advance, and after 1 day the drinking water was changed. LPS (Solarbio, beijing, china) was put in advance in a solution at a concentration of 10. Mu.g/ml ddH2O. A total of 20 mice were assigned to 4 cages (I-IV), 5 per cage. The drinking regimen and PD-1 mAb treatment for each cage are shown in FIGS. 4I and 4L.

To examine the effect of IFITM4P on anti-pd-1 treatment, another in vivo mouse model was used. For the immunocompetent mouse model, B16F10 cells (5X 10) ⁵ Individual cells were placed in 100mL of medium) cells expressing IFITM4P or empty vector were injected subcutaneously into C57BL/6 mice. Tumor growth was measured using digital calipers and tumor size was recorded. All animal experiments were performed according to the national research council "guidelines for laboratory animal Care and use" (SCXK [ Shanghai 2007-0005)]) Approved by the institutional animal care and use committee of Shanghai.

Example one

RNA extraction and RT-PCR

(1) RNA extraction procedure

a) When extracting tissue RNA, per 50-100 mg tissue, using 1ml Trizol reagent to decompose the tissue, adding 2-3 iron beads into 1.5ml EP tube added with the tissue, and using a low-temperature freezing automatic grinder to break the tissue; when extracting cell RNA, 800g,5min centrifugal precipitation cell, every 5 ~ 106 cells add 1ml Trizol, use 1ml liquid transfer gun blow to mix, cell lysis.

b) And (3) placing the Trizol lysate of the tissues or the cells at room temperature for 5min to fully lyse the tissues and the cells.

c) In the above EP tube, 0.2ml of chloroform (chloroform) was added to 1ml of Trizol, the cap of the EP tube was closed, the tube was shaken for 15 seconds on a vortex shaker, and after standing at room temperature (about 22 ℃) for 3 minutes, the tube was centrifuged at 12000g,4 ℃ for 15 minutes, to separate the organic phase from the aqueous phase containing RNA.

d) The upper aqueous phase containing RNA was transferred to a fresh EP tube, about 400. Mu.l, while an equal volume of isopropanol was added, and RNA was precipitated by shaking on a vortex shaker for 10 minutes at 15s,12000g,4 ℃.

e) The supernatant was discarded, 1ml of pre-cooled 75% glacial ethanol at-20 ℃ was added to the RNA pellet for washing, vortexed in a vortexer for 10 seconds at 12000g, and centrifuged at 4 ℃ for 10min.

f) The supernatant was discarded and the precipitated RNA was allowed to dry naturally at room temperature for 5-10min.

g) The RNA precipitate was dissolved in high pressure RNase-free distilled water, and the RNA concentration was measured by Nandrop, followed by reverse conversion of mRNA into cDNA.

(2) Reverse conversion of RNA to cDNA

mRNA was inverted into a cDNA system using a two-step procedure, the first system being as follows:

TABLE 5 first step System for mRNA inversion into cDNA System

Reagent	Dosage form
		RNA	2μg
dNTP	1μl
		oligo-dT	1μl
DEPC water	13μl

Putting the mixed system into a PCR instrument, reacting for 5min at 65 ℃, then immediately freezing for 2min, preparing a system of the second step after the first step is finished, and adding the following components on the basis of the system of the first step:

TABLE 6 inversion of mRNA into cDNA System second step System

Reagent	Dosage form
		DTT(0.1M)	2μl
Reverse transcriptase buffer	4μl
		RT reverse transcriptase	1μl

Putting the second step system into a PCR instrument, wherein the PCR system comprises the following steps:

elongation at 42 ℃ for 60min

Inactivating reverse transcriptase at 85 deg.C for 5min

Storing at 4 deg.C

After the PCR reaction is finished, collecting cDNA for subsequent RT-PCR target gene expression detection or target gene amplification, wherein the cycle number and conditions of the amplified target gene are determined by the specific target gene, and a common PCR reaction system is as follows:

(3) Real-time quantitative PCR detection of target gene expression

The real-time quantitative PCR reaction system is as follows:

TABLE 7 real-time quantitative PCR System

Reagent	Dosage form
		cDNA	0.5μl
Upstream primer (10. Mu.M)	0.5μl
		Downstream primer (10. Mu.M)	0.5μl
SYBR Green 2x mix	5μl
		Distilled water	3.5μl

The reaction conditions were set as follows:

1. 95℃ 10min

2. 95℃ 30s

3. 60℃ 30s

4.72 ℃ 30s (2-4 steps for 30 cycles)

Storing at 5.4 deg.C

3 multiple holes are made in each detection sample, expression detection of a target gene is carried out by using an ABI 7500 real-time fluorescence quantitative PCR instrument, the number of cycles required by amplification of the target gene for appointed fluorescence intensity is calculated, and then the expression intensity of the target gene is calculated, and a specific data calculation mode adopts a 2-delta CT method for analysis: Δ Ct = Ct (Target gene) -Ct (GAPDH)

(4) Patient samples were taken for real-time quantitative PCR detection of PD-L1 expression. pcr primers were as follows:

example two

To identify lncRNAs differentially expressed in normal mucosa of the oral cavity (NM), OL and OSCC, samples of NM (n = 3), OL (n = 4) and OSCC (n = 5) from chinese patients were subjected to microarray analysis in this example, as shown in fig. 1A and table 1.

Analysis showed 3109 interactions between lncRNA and mRNA transcripts in NM, OL and OSCC groups (p <0.05, fold difference > 2); focus on 10 differentially expressed lncrnas (5 genes up-regulated, 5 down-regulated) with fold difference >2 ×. × <0.0001 (fig. 1C). IFITM4P is most highly expressed in OSCC/OL and OL/NM groups. To validate these observations, IFITM4P expression was detected by qRT-PCR in samples of normal mucosa of the oral cavity (NM) (n = 23), OL (n = 64) and OSCC (n = 43), respectively, IFITM4P being highest in OCSS and lowest in NM (fig. 1D) compared to OL and NM, and these results were validated by RNA Fluorescence In Situ Hybridization (FISH) staining samples of OL, OSCC and adjacent NM, respectively, of the same patient.

In step sampling of the same patient, IFITM4P was not detected in NM, however IFITM4P gradually increased as OL progressed to early aggressive OSCC (fig. 1E). Furthermore, data from cancer genomic maps (TCGA) indicated that IFITM4P expression in head and neck squamous cell carcinoma (HNSC) tissues (n = 519) was higher than normal tissue (n = 44) (. X.p < 0.001) (fig. 1F). The 4-nitroquinoline-1-oxidation (4 NQQ) induced OL/OSCC model led us to study the development of oral epithelial cancers in vivo. 4 NQO-induced immunocompetent mice (C57 BL/6) develop OL between 14-16 weeks and OSCC between 22-24 weeks.

To evaluate the effect of IFITM4P on 4 NQO-induced oral carcinogenesis in mice, a modified C57/B6J mouse leukoplakia linguistica/Squamous Cell Carcinoma (SCC) model was used in this example (fig. 1G). Visually, the tongue of the PBS group mice did not develop any lesions (fig. 1H), and the 4NQO group developed typical white spots and tumors (fig. 1H). To confirm the results obtained by visual inspection, H & E staining, qRT-PCR staining, IFITM4P-FISH staining were performed on the two groups of tongue lesions, respectively. Histopathological diagnosis also proves that 4NQO group has white spots on the back and local early-onset invasive tongue SCC, FISH staining shows that IFITM4P of 4NQO group is strongly stained, and PBS group mice are not stained (figure 1H); in addition, qRT-PCR results showed an increase in IFITM4P for the 4NQO group compared to the PSB group (fig. 1I).

In conclusion, the expression of lncRNA IFITM4P is increased along with the development of normal mucosa to OL and OSCC, and the IFITM4P is a biomarker in the canceration process of the oral cavity.

EXAMPLE III

To determine the role of IFITM4P in oral carcinogenesis, the present example selected Leuk-1 (OL) and HN4 (OSCC) cells, manipulated the expression of IFITM4P by stable transduction of Leuk-1cells with lentiviral vectors carrying cDNA encoding the full length of IFITM4P (fig. 2A), and analyzed these cells for cell growth and colony formation. The results show that exogenous IFITM4P increased cell growth and colony formation of Leuk-1cells (fig. 2B, 2D and 2E).

To verify the opposite results, the present invention depleted IFITM4P in Leuk-1cells by using IFITM4P specific short hairpin RNA (shRNA) (fig. 2A) and observed a significant reduction in cell growth and colony formation (fig. 2B, 2D and 2E). Similar results were seen in HN4 cells with high IFITM4P expression (FIGS. 2F, 2G, 2I and 2J) and deletion (FIGS. 2F, 2H, 2I and 2J). To investigate the effect of IFITM4P in OSCC, HN4-Vector and HN4-IFITM4P cells were transplanted into BALB/C nude mice in this example. High expression of IFITM4P in cells increased growth of OSCC in vivo compared to the vector group (fig. 2K and 2L). Collectively, these data indicate that IFITM4P promotes proliferation of OL and OSCC cells, and that IFITM4P acts as a novel oncogene in oral canceration processes.

Example four

To determine the downstream target of IFITM4P in regulating Leuk-1cell proliferation, RNA sequencing (RNA-seq) was performed to determine the differentially expressed gene between the highly expressed IFITM4P and the vector (FIG. 3A). Gene ontology analysis of RNA-seq data showed that IFITM4P affects many biological processes, including immune responses, innate immune responses and inflammatory responses (fig. 7). Also, IFITM 4P-regulated Gene signature was revealed by Gene Set Enrichment Analysis (GSEA), showing Enrichment of adhesion molecules (FIGS. 3B and 3C). In this example, the OL group was further compared to the OSCC group by GSEA (fig. 3D) and PD-L1 was found to be significantly enriched in both groups, and to verify the effect of IFITM4P on PD-L1 during oral canceration, leuk-1 and HN4 cells were stably transduced with shRNA targeting IFITM4P or cDNA encoding full-length IFITM4P in this example. IFITM4P was shown to induce PD-L1 in both Leuk-1 (FIGS. 3E and 3F) and HN4 cells (FIGS. 3G and 3H) by qRNA and Western Blot (WB).

To further assess the role of PD-L1 in oral cancer, the present example performed qRT-PCR assays on NM (n = 23), OL (n = 67) and OSCC (n = 37) samples (fig. 3I) in humans, which showed significantly higher expression of PD-L1 (. < 0.05) in OSCC samples compared to OL and NM (fig. 3I). TCGA data showed that PD-L1 expression was higher in HNSC tissue (n = 519) than in normal tissue (n = 44) (. P < 0.05) (fig. 3J).

In addition, this example performed PD-L1 Immunohistochemistry (IHC), PD-L1 Immunofluorescence (IF) and IFITM4P-FISH staining of OL, OSCC and NM samples, respectively, with no detection of PD-L1 and IFITM4P, however, as OL progressed to early invasive OSCC, PD-L1 and IFITM4P staining increased (FIG. 3K), analysis showed that in OL (FIG. 3L,. Times.p)<0.05，r ² = 0.443) and OSCC (fig. 3M, # p)<0.05 R2= 0.623) in a sample, IFITM4P and PD-L1 levels are positively correlated.

In summary, PD-L1 is a new target of IFITM4P in OL and OSCC.

EXAMPLE five

In the example, the research verifies that IFITM4P/PD-L1 induced by an LPS/TLR4 pathway promotes the immune escape in mouse tongue cancer.

To determine the role of PD-L1 down-regulation in IFITM 4P-mediated cell proliferation, either vector (control) or IFITM4P was stably transduced into both shPD-L1 and Negative Control (NC) Leuk-1/HN4 cells in this example (fig. 8A and 8B).

In this example, the effect of PD-L1 on cell growth was examined using a CCK-8 cell counting kit (cell counting kit-8, CCK-8). PD-L1 significantly reduced the growth of IFITM4P Leuk-1/HN4 cells to a level similar to that of the control Leuk-1 and HN4 cells (FIGS. 8C and 8D).

In order to further illustrate that the IFITM4P is functionally involved in regulating the expression of PD-L1, a T cell killing experiment is also carried out in the embodiment, and the result shows that after the IFITM4P is co-cultured with T cells, shiftm 4P-leukemia cells and HN4 cells, the cell death of the IFITM4P knockout group is obviously higher than that of the NC group. In addition, increased levels of PD-L1 in shIFTM4P decreased cell death compared to the NC group (FIGS. 8E and 8F).

To test the effect of IFITM4P on anti-pd-1 treatment, C57BL/6 mice treated with anti-pd-1 mab in this example were inoculated with melanoma (B16F 10) cell overexpression control vector or IFITM4P (fig. 4A). Tumor-bearing mice were treated with either PD-1 monoclonal antibody or immunoglobulin G (IgG) isotype (IgG 2 a). Compared to the vehicle group, the tumor volume of the IFITM4P group was significantly increased (. < 0.05) (fig. 4B, fig. 4C); PD-1 mab treated mice showed a significant reduction in tumor volume compared to the control IgG group (fig. 4B and 4C). Fig. 4B and 4C show that the anti-tumor effect of PD-1 mab treatment was significantly better in the IFITM4P group than in the control group (P < 0.05) (fig. 4B and 4C), but no significant difference in mouse body weight was observed between the IFITM4P group and the vehicle group (fig. 4D).

It has been found that oral inflammation promotes the progression of OSCC through TLR 4. In this example, to investigate the role of TLR4 in the process of oral canceration with immune escape from IFITM4P, the TLR4 ligand, LPS, was used as the stimulus. First, effects of LPS on IFITM4P expression were examined and it was found that LPS effectively induced IFITM4P transcription in a dose-dependent manner in Leuk-1cells (fig. 4E and 9A). Polymyxin B (Polymyxin B, pmB) generally neutralizes contaminated LPS by preventing its binding to TLRs. To investigate whether the activator is LPS, pmB was used in this example to bind and inactivate LPS in the culture medium. It should be noted that induction of IFITM4P by LPS could be effectively abolished by addition of PmB in Leuk-1cells, whereas addition of PmB alone did not affect IFITM4P expression (fig. 9B); the role of TLR4, which specifically recognizes LPS, was further demonstrated in this example. In IFITM4P induction, TLR4 deficiency was found to inhibit LPS-induced IFITM4P expression in Leuk-1cells using TLR 4-specific shRNA (fig. 4F and 10).

Next, in this example, the effect of TAK-242 (resortorvid), a small molecule that inhibits the TLR4 signaling pathway and inhibits inflammatory responses, and LPS induces changes in the mRNA levels of Leuk-1cell IFITM4P, was evaluated. The findings that LPS stimulation resulted in increased IFITM4P mRNA levels compared to the control group, and that this response was abolished after addition of TAK-242 (fig. 4G) were confirmed by a luciferase reporter gene assay that showed a significant increase in IFITM 4P-related luciferase activity after LPS stimulation and a significant decrease in IFITM 4P-related luciferase activity after TAK-242 treatment (fig. 4H).

In this example, to investigate the effect of LPS-mediated IFITM4P on 4 NQO-induced oral canceration in mice, a modified C57/B6J mouse tongue canceration model was used (FIG. 4I). The results show that mice in iii and iv cages have heavy leukoplakia of tongue, leukoplakia of dorsum of tongue and local early invasive squamous cell carcinoma of tongue. PD-L1 and IFITM4P staining of SCC on the tongue of mice in cage III and IV was stronger than that of leukoplakia on the tongue of mice in cage II, while normal tongue mucosa of mice in cage I was not stained (FIG. 4J). WB and qRT-PCR analyses consistently showed similar expression of IFITM4P and PD-L1 as IHC and FISH staining (fig. 4K). Furthermore, to verify the effect of IFITM4P on anti-PD-1 treatment, PD-1 mAb was used in this example to treat early stage white tongue spots in mice treated with 4NQO + LPS or 4NQO alone (FIG. 4L). The result shows that the leukoplakia of the tongue mucosa of the mouse is obviously relieved after the PD-1 monoclonal antibody is treated for 12 days. PD-1 mAb was more effective in the 4NQO + LPS-induced group compared to the 4 NQO-induced group (FIGS. 4M and 4N).

In conclusion, LPS accelerates mouse tongue carcinogenesis, upregulates IFITM4P expression, and induces tumor immunosuppression by upregulating PD-L1. Furthermore, increased expression of IFITM4P increases the sensitivity of PD-1 mab treatment. Therefore, high IFITM4P may be an indicator of PD-1 monoclonal antibody treatment sensitivity during oral canceration.

EXAMPLE six

Biotin-labeled RNA pull-down assay analysis was performed by Mass Spectrometry (MS) in this example to identify proteins that interact with IFITM4P.

To reduce non-specificity of the RNA pull-down experiments/MS results, FISH was used in this example to localize the expression of IFITM4P in Leuk-1 cells. FISH staining showed that IFITM4P is predominantly expressed in the cytoplasm (fig. 11). Protein 1 (SASH 1) containing the functional sites SAM and SH3 is a scaffold protein in the TLR4 signaling pathway, assembling signaling complexes downstream of TLR4, activating early endothelial cell response to receptor activation. SASH1 was found in this example to be a potential interacting protein of IFITM4P. In addition, biotin-labeled IFITM4P pull-down experiments were performed in this example using biotin-hur as a positive control and their interaction with SASH1 was verified by WB (fig. 5A). RNA Immunoprecipitation (RIP) assays showed that SASH1 was significantly enriched in IFITM4P compared to control (fig. 5B and fig. 12).

SASH1 proteins can be divided into SH3 (amino acids 554-615), SAM1 (amino acids 633-697) and SAM2 (amino acids 1177-1241) functional sites (FIG. 5C). To identify functional sites mediating interactions with IFITM4P, in this example, a 554-to 615-Myc-tagged sh1 truncation was first generated and RIP detection was performed using Myc antibodies. The full-length protein, as well as SAM1 and SAM2 functional sites of SASH1, bound to IFITM4P, while SH3 functional sites did not (fig. 5D), suggesting that 554-615 amino acid residues are critical for SASH1 binding. Down-regulation of SASH1 results in the inhibition of the expression of PD-L1 (FIGS. 5E and 13). Furthermore, expression of PD-L1 was also significantly reduced following SASH1 knockdown in cells overexpressing IFITM4P (fig. 5E). Next, the interaction of the SASH 1-related protein and TAK1 (MAP 3K 7) as potential interactors of SASH1 was analyzed in this example (fig. 14).

To determine whether there is a physical interaction between SASH1 and TAK1 and whether it is dependent on IFITM4P, we transiently expressed Sh-IFITM4P and performed co-immunoprecipitation (co-IP) assays using TAK 1. SASH1 was unable to bind TAK1 following IFITM4P knockdown in Leuk-1cells (FIG. 5F). To further understand the mechanism of IFITM 4P-mediated PD-L1 regulation, we examined the mRNA levels of TAK1 and SASH1 after high and down-regulation of IFITM4P, respectively. The results show that IFITM4P had no significant effect on TAK1 and SASH1 (fig. 15A and 15B). Next, we examined the phosphorylation states of TAK1 (Thr 187) and TAK1 (Thr 412) with increased IFITM4P expression by WB (fig. 15C). Phosphorylation at the TAK1 Thr187 site was positively correlated with IFITM4P expression, whereas phosphorylation at the TAK1 Thr412 site was not significantly changed (FIG. 15C). Furthermore, deletion of IFITM4P resulted in decreased phosphorylation of TAK1 (Thr 187), while elevation of IFITM4P resulted in increased phosphorylation of TAK1 (Thr 187) (fig. 5G). TAK1 is involved in the activation of the nuclear factor kB (NF-kB). GSEA showed characteristics of malignancy, including NF-kB signaling pathway, significantly enriched in Leuk-1cells with higher than median risk scores (fig. S1A). Consistent with these findings, it was found in this example that stable expression of IFITM4P increased phosphorylation of NF-kB (Ser 536), while down-regulation of IFITM4P decreased phosphorylation of NF-kB (Ser 536) (FIG. 5G). Furthermore, the deletion of SASH1 resulted in a reduction in the expression of NF-kB, P-NF-kB (Ser 536) and P-tak (Thr 187) in the vectors and IFITM4P (FIGS. 5H and 15D) expressing Leuk-1 cells; NF-kB was also reduced in the shIFITM4P-Leuk-1 cells and vice versa (FIGS. 5G,5H and 16A). BAY 11-7082 is an inhibitor of phosphorylation of IkBa, can stabilize IkBa and specifically block NF-kB signal channels. BAY 11-7082 treatment resulted in reduced expression of PD-L1 in vectors and IFITM4P expressing cells (FIGS. 5I and 16B). Furthermore, knockout of TAK1 resulted in reduced levels of PD-L1 in IFITM4P-leuk-1 cells (FIGS. 5J and 16C).

NF-kB-driven luciferase activity was enhanced in HN4, and LPS stimulated Leuk-1 cells. However, transfection of these cells with shTAK1, shSASH1 or shIFITM4P inhibited LPS-induced NF-kB signaling, indicating that NF-kB activity was completely dependent on the expression of TAK1/SASH1/IFITM4P (FIG. 5K). Taken together, these data indicate that IFITM4P promotes immune evasion of OL by modulating the SASH1-TAK1-NF-kB-PD-L1 axis.

Taken together, cytoplasmic IFITM4P interacts with SH3, a functional site of SASH1, promoting the transcription of PD-L1 through the TAK-1/NF-kB pathway in OL.

EXAMPLE seven

In the fifth example, a FISH assay was performed to detect subcellular localization of IFITM4P under LPS stimulation. Upon LPS stimulation, IFITM4P clearly translocated from the cytoplasm to the nucleus in Leuk-1 (FIG. 6A). In the existing research, the increase of the quantity of histone 3 lysine 4 demethylase KDM5A in a mouse tumor model obviously improves the response of PD-1 antibody treatment. The interaction of IFITM4P with KDM5A was confirmed by RNA pull-down experiments and MS analysis of biotin labelling in the present invention (fig. 17A and 17B). In addition, the invention carries out the IFITM4P pull-down experiment of biotin labeling, takes biotin-HuR as a positive control, and verifies the interaction with KDM5A through WB. The results indicate that the interaction of IFITM4P and KDM5A is dependent on LPS stimulation (fig. 6B). In addition, RIP assays showed that KDM5A can be significantly enriched for IFITM4P compared to LPS control (fig. 6C). KDM5A increases the abundance of PD-L1 in tumor cells by inhibiting PTEN expression pathway and inducing PI3K-AKT-S6K signal pathway, and directly interacts with PTEN promoter (transcription initiation site is near 3 kb) to inhibit Pten transcription. We performed a chromatin immunoprecipitation (ChIP) assay to determine whether IFITM4P modulates KDM5A binding to the Pten promoter. The results show that KDM5A preferentially binds to P1 (2, 929-2, 819) and P2 (2, 751-2, 533). Deletion of IFITM4P alone significantly reduced binding of KDM5A to Pten, while stable knock-out IFITM4MP-WT (wild-type) transfection plasmid IFITM4MP-WT reduced inhibition of KDM5A binding to Pten (fig. 6D). qPCR and WB were also used in the invention to demonstrate that overexpression of KDM5A reduced PTEN abundance at the transcriptional and protein level, and that knock-down of IFITM4P could reduce this inhibition (fig. 6E and 18). Furthermore, qRT-PCR showed a significant decrease in PD-L1 expression after KDM5A and NF-kB gene knockdown, while PD-L1 expression in Leuk-1 and HN4 cells was increased after KDM5A or NF-kB gene knockdown (fig. 6E) (fig. 19). To investigate the clinical relevance of IFITM4P to PTEN, TAK1, SASH1 and NF-kB, 519 cases of TCGA were analyzed for HNSC expression in the present invention. Among them, IFITM4P is negatively correlated with PTEN expression (P =0.001, r =0.14, n =519; fig. 20), but there is no correlation between other genes. Taken together, the above data indicate that LPS partially induces IFITM4P into the nucleus, enhancing KDM5A binding to the Pten promoter, decreasing Pten transcription, thereby up-regulating PD-L1 in OL.

In conclusion, the LPS part induces IFITM4P to enter the nucleus, enhances the combination of KDM5A and the Pten promoter, and reduces the Pten transcription.

Example eight

In this example, the sequence of shRNA was designed by bioinformatics,

Sh1-GCCCAAACCTTCTTCATTCCT

Sh2-TGTCCACCATGATCCATATCT。

wherein, seq ID NO: 2. seq ID NO: the nucleotide sequence of the shRNA is also shown in FIG. 3.

In this example, the carriers for knocking down IFITM4P all use PLKO carriers, and PLKO, PMD2G, and PSPAX carriers for packaging viruses, and the specific steps are as follows:

(1) The full 293T cells in the 6cm dish were passaged to a new 6cm dish by trypsinization at a rate of 1:3 for passage.

(2) Plasmid transfection was initiated when the cell density reached 80% -90%. Viral packaging plasmids including PMD2G and PSPAX2 were transfected into 293T cells together with the PCDH (over-expressed)/PLKO (knock-down) plasmid containing the gene of interest, and the specific transfection procedure was as above, 3 plasmid amounts of PCDH/PLKO transfected in 6cm dishes PMD2G: PSPAX2=2 μ G: 1ug.

(3) Complete medium (4 ml) was replaced once more after 24 h.

(4) After 48h, the upper medium was gently collected and the cells were supplemented with fresh complete medium (4 ml).

(5) After 72h of transfection, the supernatant medium was collected and filtered separately from the virus supernatant collected for 48h with a 0.22 μ M filter to obtain virus supernatants, which were then used for the next cell infection or stored in a refrigerator at-80 ℃.

(6) When the density of Leuk-1cells or HN4 cells reaches 60%, adding collected virus supernatant for 48h or 72h for infection, mixing and diluting the virus supernatant and a corresponding cell complete culture medium (the dilution ratio is: virus/complete culture medium = 1:1) when the virus is infected, discarding the culture medium of cells to be infected, replacing the diluted virus, and adding Polybrene (used for dilution of 1000X) of 10 mu g/ml to increase the virus infection efficiency.

(7) And supplementing a proper amount of culture medium to continue culturing after 24 hours of infection.

(8) After 48h of infection, the virus supernatant was discarded, complete medium containing 0.5-2 μ g/ml puromycin (different cells need to be searched for appropriate screening concentrations) was added, infected cells were screened, cells with stable overexpression/knockdown were selected, and gene overexpression/knockdown was verified using RT-PCR or WB.

IFITM4P was depleted in Leuk-1cells in this example by using IFITM4P specific short hairpin RNA (shRNA) (fig. 2A) and a significant reduction in cell growth and colony formation was observed (fig. 2B, 2D and 2E).

Overexpression/knockdown of IFITM4P may promote or inhibit proliferation and clonogenic of OL cells.

In this example, two cell lines, OL cell line Leuk-1 and OSCC cell line HN4, were selected for the experiment. First, we constructed stable transformants using a lentivirus overexpressing IFITM4P in Leuk-1cells (see fig. 18), fig. 18 cells were further investigated for proliferation and colony forming ability by stably overexpressing IFITM4P in Leuk-1cells by lentivirus transfection, or by knocking out IFITM4P by specific shRNA, establishing Leuk-1cells stably overexpressing or knocking down IFITM4P by puromycin screening.

According to the above embodiments, the present invention discloses an LncRNA IFITM4P, which is activated by LPS/TLR4 during oral canceration and up-regulates PD-L1 by dual mechanisms.

From the above examples, it can be seen that IFITM4P enhances KDM5A binding to Pten promoter in the nucleus and reduces Pten transcription, thereby up-regulating PD-L1 in OL cells. KDM5A may have multiple mechanisms that facilitate PD-L1 abundance. KDM5A induces PI3K-AKT-S6K signal transduction by inhibiting PTEN expression, and increases the abundance of PD-L1 in tumor cells. In the invention, LPS can induce IFITM4P part to enter the nucleus, and enhance the combination of KDM5A and Pten promoter to reduce Pten transcription, thereby up-regulating the PD-L1 of OL cells.

SASH1 is a large protein with a predicted molecular mass of 137kDa, and belongs to SAM and SH3 adaptor family proteins. It consists of an SH3 functional site (SLY 1) expressed in lymphocytes and a hematopoietic adaptor (HACS 1, also called SLY 2) containing both SH3 and SAM functional site 1. caspase-3 activation and cleavage of SASH1 has been shown to mediate an NF-kB dependent apoptotic response. These findings reveal that SASH1 may be an aptamer to a variety of signaling pathways. In the present invention, it was found that the IFITM4P/SASH1 complex leads to NF-kB P65 activation in OL and OSCC cells by binding TAK1 complex as a scaffold molecule. NF-kB has been shown to be a key positive regulator of PD-L1 expression in a variety of cancers. This process is largely deregulated in cancer. The up-regulation of PD-L1 in cancer cells is controlled by downstream signals of NF-kB, including oncogenes and stress-induced pathways as well as inflammatory cytokines. TLR4 is proved to have the function of protecting the tumor from immune attack in HNSC/OSCC. In the invention, TLR4 ligand LPS35 promotes the canceration of white spots of mouse tongue by increasing levels of IFITM4P and PD-L1. The above examples of the invention also show that the LPS/TLR4 pathway significantly induces IFITM4P during oral canceration.

Taken together, IFITM4P is gradually induced from OL to OSCC cells via the LPS/TLR4 pathway, and high expression of IFITM4P leads to increased OSCC cell proliferation and enhanced immune escape via induction of PD-L1 expression. Mechanistically, IFITM4P induces PD-L1 via a dual pathway.

In cytoplasm, IFITM4P is used as a scaffold to promote SASH1 aggregation binding and phosphorylation of TAK1 (Thr 187), further increase NF-kB phosphorylation (Ser 536) and induce PD-L1 transcription; in the nucleus, IFITM4P down-regulates PD-L1 in OL cells by decreasing Pten transcription through enhanced KDM5A binding to Pten promoter. In addition, the IFITM4P high-expression tumor mice show obvious treatment sensitivity to the PD-1 monoclonal antibody treatment. In conclusion, IFITM4P can be used as a new therapeutic target for blocking canceration of the oral cavity, and PD-1 monoclonal antibody can be used as an effective reagent for treating OSCC with high expression of IFITM4P.

The drawings that accompany the detailed description can be further detailed and explained as follows: typical macroscopic and microscopic findings (H & E staining) for NM, OL and OSCC (100 x) in fig. 1A; in fig. 1B hierarchical clustering microarray analysis on NM (n = 3), OL (n = 4) and OSCC (n = 5) samples from chinese patients are based on differentially expressed RNA transcripts (p <0.05, fold change > 2) in microarray data, each column representing one sample, each row representing one transcript, the expression level of each gene in a single sample being described according to color scale; FIG. 1C shows that regulated lncRNAs were identified in OL/NM and OSCC/OL and 10 fold changes >2 were listed and p <0.001 differentially expressed lncRNAs; in fig. 1D, qRT-PCR analysis showed highest IFITM4P expression in OSCC samples and lowest expression in NM samples, { P } <0.001, compared to OL and NM samples; in fig. 1E, IFITM4P staining became stronger during OSCC development in OL (200 x) in stepwise samples from the same patient, IFITM4P staining was negative at adjacent NM; in fig. 1F, TCGA data showed that IFITM4P expression was higher in HNSC tissue (n = 519) than in normal tissue (n = 44); P <0.001; FIG. 1G is a time-representative view of the tongue white spot/squamous cell carcinoma model; the 4NQO group in FIG. 1H exhibited typical white spots and squamous cell carcinoma. Histopathological diagnosis also confirmed white spots on the back of the tongue and local early invasive squamous cell carcinoma of the tongue in the 4NQO group, FISH showed strong IFITM4P staining in the 4NQO group, while no staining was found in the PBS group (n = 6); in fig. 1I, the qRT-PCR results showed an increase in IFITM4P in the 4NQO group compared to the PBS group, (n = 6), { P <0.001.

In FIG. 2A, stable overexpression of IFITM4P and knock-out by viral transduction using specific shRNA in white Leuk-1 cells; establishing stable cells after screening puromycin; in FIG. 2B, CCK-8 analysis showed that overexpression of IFITM4P significantly promoted cell proliferation; in FIG. 2C, CCK-8 analysis showed that IFITM4P gene knock-out significantly inhibited cell proliferation of cell line Leuk-1; FIG. 2D represents a culture dish, and the quantitative analysis in FIG. 2E of overexpression of surface IFITM4P significantly increased cell colony formation, while knock-out inhibited cell colony formation in Leuk-1; fig. 2F shows stable overexpression and knock-out of IFITM4P using specific shRNA by viral transduction in HN4 cells; establishing stable cells after screening puromycin; in fig. 2G, CCK-8 analysis showed that overexpression of IFITM4P significantly promoted proliferation of HN4 cells; in fig. 2H, CCK-8 analysis showed that the knockout of IFITM4P significantly inhibited cell proliferation in HN 4; figure 2I represents a petri dish, and the results, quantified in figure 2J, show that overexpression of IFITM4P significantly increased cell colony formation in HN4, while knockdown inhibited cell colony formation; in fig. 2K, IFITM4P promoted HN4 cell growth in vivo (n = 6); in figure 2L, overexpression of IFITM4P in HN4 significantly increased tumor volume. Wherein the data of panels a, B, C, F, G and H are shown as mean ± SD × p <0.05 of three independent experiments. Data for panels E, J and L are shown as mean ± SD × p <0.05 of six independent experiments.

FIG. 3A, heat map of the genes associated with frontal cell adhesion (analyzed by GSEA) showing the strongest upregulation in Leuk-1cells in the IFITM4P group; FIG. 3B, enrichment plots of the resulting vectors compared to IFITM 4P-expressing Leuk-1cells by GSEA analysis of the aligned gene expression data (left, up-regulation [ red ]; right, down-regulation [ blue ]). The concentration fraction is shown as green lines (concentration fraction = 0.52;. P < 0.001); FIG. 3C, comparison of OL and OSCC enrichment plots generated by GSEA analysis of aligned gene expression data (left, up-regulated [ red ]; right, down-regulated [ blue ]), concentration scores are shown as green lines (concentration score = 0.49;. P < 0.001); FIG. 3D, venn diagram shows the overlap between the vector and IFITM4P expressing Leuk-1cells and OL and OSCC; FIG. 3E and FIG. 3F, the induction of leukemia cells PD-L1 by IFITM4P was confirmed by qRT-PCR (E) and WB (F); fig. 3G and fig. 3H, confirmation of IFITM4P induction of PD-L1 by qRT-PCR (G) and WB (H) in HN4 cells; FIG. 3I, qRT-PCR analysis showed that PD-L1 was highest in the OSCC samples and lowest in the NM samples compared to the OL and NM samples; figure 3j, tcga data analysis showed that expression of PD-L1 was higher in HNSC tissue (n = 519) than in normal tissue (n = 44),/p <0.05; FIG. 3K, no PD-L1 and IFITM4P staining was observed in NM in samples from patients. However, PD-L1 and IFITM4P staining became stronger as OL progressed to early invasive OSCC (a-L, 200). (L and M) OL (. < 0.05) (L) and OSCC (. < 0.05); FIG. 3M, positive correlation between levels of IFITM4P and PL-D1 in samples. Wherein the data of fig. 3E, 3G and 3I are shown as mean ± SD of three independent experiments p <0.05.

FIG. 4A, 5x10 expressing IFITM4P or vector ⁵ B16F10 cells were implanted into C57BL/6J mice and received PD-1 monoclonal antibody or IgG isotype control (IgG 2 a) treatment, with the schedule of tumor induction and treatment shown; fig. 4B, showing tumor volume; FIG. 4C, introduction of IFITM4P into B16F10 cells significantly increased tumor volume in C57BL/6J mice, and PD-1 mAb significantly decreased tumor volume in C57BL/6J mice harboring B16F10 cells expressing IFITM4P; figure 4D, mouse body weights measured every 3 days, with no significant difference in the body weights of the groups; FIG. 4E, qRT-PCR shows a dose-dependent increase in LPS-induced IFITM4P transcription in leukemic cells; FIG. 4F, qRT-PCR shows that TLR4 shRNA has inhibitory effect on LPS-induced (100 mg/mL) expression of Leuk-1cell IFITM4P; FIG. 4G, qRT-PCR showed that TAK-242 (1 mM) inhibited LPS-induced (100 mg/mL) expression of IFITM4P in Leuk-1 cells; FIG. 4H, TAK-242 (1 mM) vs 293T cellsInhibition of luciferase activity driven by the IFITM4P promoter in response to LPS (100 mg/mL); FIG. 4I, time line schematic of a modified mouse leukoplakia linguistics/squamous cell carcinoma model; FIG. 4J, (a) Normal tongue (cage I) and (b-d) typical white tongue spots (cage II-IV), (c) and (d) large changes in white tongue spot, more rough texture (third and fourth cages), histopathological diagnosis: NM (e) (cage I), OL with moderate dysplasia (f) (cage II), OL with severe dysplasia and locally early invasive squamous cell carcinoma (g) and (h) (cage III and IV), (I-L) IHC staining of PD-L1, negative staining (I) (cage I), staining (k) and (L) (cage III and IV) of locally early invasive squamous cell carcinoma stronger than white tongue spot (j) (cage II), staining of early invasive squamous cell carcinoma areas stronger than adjacent OL areas (k). (m-P) fish staining of IFITM4P, negative staining (m) (cage I), staining of local early invasive squamous cell carcinoma (o) and (P) (cage III and IV) stronger than white tongue spot (n) (cage II), staining of early invasive squamous cell carcinoma region stronger than adjacent OL region (e-P, 200); FIG. 4K, qRT-PCR and WB confirmed that in the 4 NQO-induced tongue white spot/SCC mouse model, the expression of IFITM4P increased with the progression of the disease, and LPS significantly increased the expression of IFITM4P compared to ddH2O control group, promoting the canceration of tongue white spots; FIG. 4L, time representation intent of PD-1 mAb treatment of early stage leukoplakia mouse model; FIG. 4M, the PD-1 mAb was effective in the treatment of vitiligo, especially 4NQO and LPS-induced vitiligo. Macroscopic observation before and after PD-1 monoclonal antibody treatment; FIG. 4N, evaluation of the ratio of tongue lesion scores before and after PD-1 mab treatment; wherein the data of fig. 4E, 4F and 4G are shown as mean ± SD × p of three independent experiments<0.05. Data from (fig. 4B, fig. 4C, fig. 4D), (fig. 4H), (fig. 4J, fig. 4K) and (fig. 4M, fig. 4N) are shown as mean ± SD,. P from six independent experiments<0.05。

FIG. 5A, biotin-labeled IFITM4P pull-down and WB show that IFITM4P specifically co-precipitated with SASH1 in leukemia cells, with biotin HuR and antisense as positive and negative controls, respectively. FIG. 5B, RIP analysis, verifies the association of SASH1 with IFITM4P in Leuk-1cells, anti-GAPDH or control IgG antibodies as controls; FIGS. 5C and 5D, different truncated forms of SASH1 (FIG. 5C) and its binding to IFITM4P, RIP assay in Leuk-1cells (FIG. 5D); FIG. 5E,WB shows that the expression of PD-L1 was significantly reduced in cells overexpressing IFITM4P following SASH1 knock-out using ShSASH 1; FIG. 5F, detection of endogenous interaction between IFITM4P, TAK and SASH1 in Leuk-1cells by co-IP and qRT-PCR analysis; FIG. 5G, WB analysis showing that expression of pTAK1 (Thr 187) and pNF-kB P65 (Ser 536) increases with ectopic expression of IFITM4P in Leuk-1cells, but decreases by knock-out of IFITM4P; FIG. 5H, WB analysis shows that NF-kB, pNF-kB (Ser 536) and pTAK1 (Thr 187) levels were reduced following SASH1 deletion in control and IFITM 4P-expressing Leuk-1 cells; FIG. 5I, WB analysis, shows that control and IFITM4P expressing cells have reduced PD-L1 expression following BAY 11-7082 (10 mM) treatment; FIG. 5J, WB analysis showing that knockout of TAK1 gene for TAK1 inhibits PD-L1 transcription in Leuk-1cells expressing IFITM4P; FIG. 5K NF-kB-driven luciferase activity was enhanced in LPS-stimulated HN4 and Leuk-1 cells. Transfection of these cells with shTAK1, shSASH1 or shIFITM4P inhibited LPS-induced NF-kB signaling, pGL3.0 as a control; the data in fig. 5B and 5D are shown as mean ± SD + p <0.05 for three independent experiments, and the data in fig. 5K are shown as mean ± SD + p <0.05 for six independent experiments.

FIG. 6A, confocal microscopy, FISH analysis (400 fold) shows that IFITM4P is significantly transferred from the cytoplasm to the nucleus of Leuk-1cells under LPS stimulation (100 mg/mL), and assessment of nuclear morphology is performed by DAPI staining; FIG. 6B, biotin-labeled IFITM4P pull-down and WB show that after 12 hours of LPS stimulation (100 mg/mL), IFITM4P specifically co-precipitated with KDM5A in Leuk-1cells, magnetic beads as negative control. FIG. 6C, RIP analysis, verifies the correlation of KDM5A to IFITM4P in Leuk-1cells 12 hours after LPS stimulation (100 mg/mL) with IgG antibodies as controls; FIG. 6D, chip analysis of the Pten promoter of Leuk-1cells, KDM5A binding site in the upper panel of FIG. 6D, and the lower panel of FIG. 6D, showing that KDM5A specifically binds to P1 and P2; the combination of KDM5A and Pten can be obviously reduced by singly deleting IFITM4P, and the inhibition effect on the combination of KDM5A and Pten can be reduced by transfecting IFITM4MP-WT plasmid into stable knockout-IFITM 4P-Leuk-1; the chip detection takes an anti-KDM 5A antibody or IgG as a control; detecting enriched DNA fragments flanking P1 and P2 by RT-PCR using a specific primer set; FIG. 6E, WB analysis of the relative expression of Pten mRNA in Leuk-1cells overexpressing vector and KDM5A under LPS stimulation (100 mg/mL). FIG. 6F, IFITM4P-Leuk-1 and Vector-Leuk-1 cells transiently transfected with ShRNA into NF-kB P65 or KDM5A or disordered control (ShRNA NC), KDM5A or NF-kB P65 vectors treated with LPS (100 mg/mL), qRT-PCR showed a significant decrease in PD-L1 expression after KDM5A and NF-kB P65 were knocked out, while WT-KDM5A or WT-NF-kB P65 increased PD-L1 expression. FIG. 6G, a model depicting the role of IFITM4P as an oncogene by increasing PD-L1 abundance in IL; wherein the data in panels C, D and F are shown as mean ± SD × p <0.05 of three independent experiments; NS represents no significant difference and WL represents wild type.

FIG. 8A shows stable expression of IFITM4P and shPD-L1-treated PD-L1 in Leuk-1cells, FIG. 8B shows the effect of FITM4P and shPD-L1 on growth of Leuk-1cells overexpressing empty vector or IFITM4P-Leuk-1, cell proliferation was measured using the CCK-8 assay, FIG. 8C shows the effect of stable expression of PD-L1 in IFITM4P and shPD-L1-treated HN4 cells, FIG. 8D shows the effect of IFITM4P and shPD-L1 on growth of HN4 cells overexpressing empty vector or IFITM4P-Leuk-1, cell proliferation was measured by the CCK method, FIG. 8E and FIG. 8F show the results of T cell mediated cancer cell killing test, and Leuk-1 (E) or HN4 (F) cells co-cultured with activated T cells for 48 hours were subjected to crystal staining, and the ratio of Leuk-1 or SHPD-L1 to HN4 (8978);

in FIG. 9A, qRT-PCR showed a dose-dependent increase in LPS-induced apoptosis, and IFITM4P was transcribed in Leuk-1cells after 12 hours; in FIG. 9B, comparing the effect ng/mL of LPS (400 ng/mL) and LPS (400 ng/mL) alone) + PMB (10. Mu.g/mL) after 12 hours of incubation, the data are shown as the mean. + -. Standard deviation of three independent samples. NS = no difference significant, P <0.05.

Western blotting in FIG. 10 showed that shTLR4 was stably expressed in Leuk-1 cells.

In the upper panel of FIG. 11, images taken with a confocal microscope show localized FISH detection of IFITM4P expression in Leuk-1cells, in the lower panel of FIG. 11, and qRT-PCR shows IFITM4P expression in the nucleus and cytoplasm of Leuk-1cells, data derived from the mean of three independent values,. P <0.05.

RIP analysis in fig. 12 confirmed the correlation of HuR probes to HuR in Leuk-1cells as a positive control, (n = 3), data derived from the mean of three independent values,. P <0.05.

In fig. 13, qRT-PCR showed that PD-L1 expression was significantly reduced in IFITM4P overexpressing cells using ShSASH1 knock-out SASH1, data derived from the average of three independent values,. P <0.05.

The proteins relevant for the analysis of the biological information of the SASH1 interaction network are shown in FIG. 14.

From the results shown in fig. 15A and 15B, it can be seen that IFITM4P has no significant effect on the expression of TAK1 and SASH 1.

In FIG. 15C, IFITM4P significantly enhanced the phosphorylation of TAK1 (Thr 187) but not TAK1 (Thr 412) at doses of 0-4ng in Leuk-1 cells. In FIG. 15D, knockout of SASH1 in Leuk-1-IFITM4P inhibited mNAp 65 of NF-. Kappa.B. Data from A, B, D are shown as the mean ± standard deviation of three independent samples.

In FIG. 16A, ectopic expression of IFITM4P increased the expression level of NF- κ B P in Leuk-1cells, which was decreased by its knock-out. In FIG. 16B, qRT-PCR showed that PD-L1 transcription was inhibited in BAY 11-7082 (10. Mu.M) treated Leuk-1cells, expression vector or IFITM4P. In fig. 16C, qRT-PCR analysis showed that in IFITM4P expressing Leuk-1cells, shTAK1 knock-out TAK1 inhibited PD-L1 transcription, and data from A, B, D are shown as mean ± standard deviation of three independent samples.

FIG. 17A is a schematic representation of an RNA pull-down experiment for identifying IFITM 4P-related proteins; in FIG. 17B, leuk-1cells stably overexpressed by IFITM4P were treated with LPS for 12 hours and IFITM4P and its related complexes were enriched with streptavidin magnetic beads IP.

In FIG. 18, RT-PCR analysis of relative expression of Pten mRNA in vectors and KDM5A over-expressed Leuk-1cells under LPS (100. Mu.g/ml) treatment, data from A, B, D are shown as mean. + -. Standard deviation of three independent samples.

In FIG. 19, cells transfected with ShRNA from IFITM4P-HN4 cells and Vector-HN4 cells into NF-. Kappa. B P65 or KDM5A or disordered control (ShRNA NC), KDM5A or NF-. Kappa. B P65 Vector were treated with LPS (100. Mu.g/ml). qRT-PCR showed that PD-L1 expression was significantly reduced after KDM5A and NF- κ B p were knocked out, while KDM5A or NF- κ B p increased PD-L1 expression. Data are shown as mean ± standard deviation P <0.05.Ns = no significant difference of three independent samples. WT = wild type.

In fig. 20, data from cancer genomic map (TCGA) indicate that IFITM4P and PTEN (a) levels are negatively correlated (P < 0.05) in HNSC samples (n = 518), but IFITM4P levels are not correlated with SASH1 (B), NR2C2 (TAK 1) (C) and NFKB1 (D) levels.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.

Sequence listing

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Claims (6)

1. Use of a small interfering RNA targeting LncRNA IFITM4P for the treatment of oral leukoplakia and/or oral cancer, wherein the LncRNA IFITM4P nucleotide sequence is as defined in Seq ID NO:1 is shown.

2. The use of the LncRNA IFITM 4P-targeting small interfering RNA according to claim 1 in the treatment of oral leukoplakia and/or oral cancer, wherein the sequence of the small interfering RNA is at least one of the sequence shown as SEQ ID No.2 and the sequence shown as SEQ ID No. 3.

3. An RNA agent for the treatment of oral leukoplakia and/or oral cancer comprising a small interfering RNA targeting LncRNA IFITM4P, said LncRNA IFITM4P nucleotide sequence being as defined in Seq ID NO:1 is shown.

4. The drug for treating oral leukoplakia and/or oral cancer according to claim 3, wherein the sequence of the small interfering RNA is at least one of the sequence shown as SEQ ID No.2 and the sequence shown as SEQ ID No. 3.

5. A method for screening a drug for treating oral leukoplakia and/or an anticancer drug, comprising the steps of:

s1, determining the expression level of LncRNA IFITM4P in the oral tissue cells, wherein the nucleotide sequence of LncRNA IFITM4P is as defined in Seq ID NO:1 is shown in the specification;

s2, contacting the candidate medicine with the cell in the step S1;

s3, determining the expression level of LncRNA IFITM4P in the cells after the step S2;

s4, comparing the expression levels of LncRNA IFITM4P determined in step S1 and step S3, wherein a decreased expression level of LncRNA IFITM4P indicates that the drug candidate has the potential to treat vitiligo and/or to prevent cancer.

Use of a pd-1 mab in the manufacture of a medicament for treating a high expression LncRNA IFITM4P leukoplakia and/or oral cancer, wherein the LncRNA IFITM4P nucleotide sequence is as defined in Seq ID NO:1 is shown.

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